A novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in Staphylococcus aureus

A novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in Staphylococcus aureus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in Staphylococcus aureus Yun Liu, Zhen Wang, Jiamin Qiu, Haiming Wu, Jiayi Wu, Tairan Zhong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8837885/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Lysine 2-hydroxyisobutyrylation (Khib) controls important cellular physiological processes, but its functions in antibiotic resistance remain poorly understood. Here, we report that Khib positively regulates fluoroquinolone resistance in (). Quantitative proteomics revealed remodeling of Khib across multiple metabolic pathways in ciprofloxacin-resistant strains, and manipulation of Khib altered susceptibility to fluoroquinolones. We identified AcuA as a novel 2-hydroxyisobutyryltransferase in . AcuA deletion impaired biofilm formation, and lowered fluoroquinolone resistance. Mechanistically, AcuA enhances resistance to fluoroquinolones by 2-hydroxyisobutyrylating CodY, which alters the structure and electrostatic potential of DNA-binding domain, thereby weakening its binding to the promoter. Critically, mouse infection models also confirmed that strains with elevated CodY Khib levels exhibited even stronger resistance. Importantly, this signaling pathway was also validated in clinically multidrug-resistant (MDR) isolates. Thus, our study dissects a novel mechanism by which covalent modifications regulate antibiotic resistance in important human pathogens. Biological sciences/Biochemistry Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Microbiology 2-hydroxyisobutyrylation Staphylococcus aureus Antibiotic resistance Fluoroquinolones CodY Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The rapid emergence and global spread of antimicrobial resistance (AMR) represent one of the most critical challenges to modern public health 1 . Among the myriad of resistant pathogens, S. aureus stands out due to its high prevalence and its propensity to develop multidrug resistance, particularly to fluoroquinolones, a broad-spectrum antibiotic that have shown activity against S. aureus , including Methicillin-Resistant S. aureus (MRSA) 1 , 2 . Fluoroquinolone resistance in S. aureus is not only common but also often coupled with multidrug resistance, severely limiting therapeutic options and leading to adverse clinical outcomes 3 . Understanding the intricate mechanisms underpinning S. aureus resistance is thus imperative for devising effective therapeutic strategies. Traditionally, research on fluoroquinolone resistance has focused on genetic endpoints, the acquisition of mutations in drug target genes (e.g., gyrA , gyrB , grlA , grlB ) or the horizontal transfer of resistance determinants 4 , 5 . These alterations provide a stable, heritable advantage. However, the emergence of such mutations is a stochastic process that requires time and population expansion under selective pressure. A critical, yet less explored, question is: how bacterial populations survive the initial antibiotic assault to reach the necessary population size and time for favorable mutations to arise and become fixed. The answer likely lies in rapid, non-genetic adaptive strategies that act as a crucial first line of defense. This immediate survival response is orchestrated through dynamic metabolic reprogramming and fast-acting post-translational modifications (PTMs). Upon antibiotic exposure, bacteria must rewire their metabolism to allocate energy and resources toward repair, detoxification, and stress tolerance mechanisms. This metabolic shift is not merely a passive consequence but an active defense, and its precise execution is often regulated by PTMs. PTMs, such as phosphorylation, acetylation, succinylation, and lactylation, enable swift and reversible modulation of protein function, influencing enzyme activities, signaling pathways, and gene expression without altering the genome 6 , 7 , 8 , 9 . They serve as a real-time interface between the metabolic state and the functional output of the cell, allowing bacteria to fine-tune their physiology to withstand transient stress. For instance, it was discovered that host-derived lactate can enhance the virulence of human pathogen S. aureus by promoting the lactylation of alpha-toxin 8 . Concurrently, Yang Yi et al demonstrated that succinylation negatively regulates cell wall synthesis and vancomycin tolerance in vancomycin-intermediate S. aureus (VISA) 10 . Furthermore, a groundbreaking study identified a metabolism-dependent succinylation mechanism that governs resource allocation for antibiotic resistance 11 . It demonstrated that bacteria could undergo metabolic reprogramming to support resistance mechanisms, and that succinylation of triosephosphate isomerase (TPI) and transcription factors (CpxR, PdhR) could crucially modulate metabolic flux and eventually influence resistance phenotypes. Recently, our research group discovered that crotonoylation positively regulates polymyxin resistance in Escherichia coli ( E. coli ) while negatively regulating virulence in Streptococcus pneumoniae 12 , 13 . These findings compellingly suggest that PTMs serve as a sophisticated regulatory layer enabling bacterial adaptation. We hypothesize that these rapid, PTM-driven adaptations are essential for maintaining cell viability during the critical window before the consolidation of genetic resistance, thereby potentially facilitating the eventual emergence and selection of high-level, mutation-driven resistance. Lysine 2-hydroxyisobutyrylation (Khib) is an evolutionarily conserved PTM, first identified on lysine residues of human and mouse histones 14 . Khib plays critical regulatory roles across both eukaryotic and prokaryotic organisms. Functionally, Khib mediates chromatin remodeling, fine-tunes metabolic enzyme activity, and participates in stress response pathways 15 , 16 , 17 . Insights into the functions of PTMs often stem from the identification of catalytic enzymes responsible for the addition or removal of PTMs. Khib is dynamically controlled by specific “writer” and “eraser” enzymes. The enzyme responsible for adding the 2-hydroxyisobutyryl group, known as a 2-hydroxyisobutyryltransferase (“writer”), has been identified in eukaryotic cells as p300, Tip60, and Esa1p. Histone deacetylases (HDACs), specifically HDAC2 and HDAC3, were the major enzymes that remove the 2-hydroxyisobutyryl group (“eraser”) 18 . In prokaryotes, the tRNA(Met) cytidine acetyltransferase TmcA from E. coli has been shown to possess lysine 2-hydroxyisobutyryltransferase activity, while CobB functions as the core de-2-hydroxyisobutyrylase 19 , 20 . In microorganisms, Khib has emerged as a key modulator of virulence, stress adaptation, and host-pathogen interactions. TmcA-mediated Khib modification of the histone-like protein H-NS at Lys121 enhances acid resistance in E. coli by promoting the transcription of acid stress response genes 19 . Similarly, in fungal pathogens like Ustilaginoidea virens , Khib modification of the MAP kinase UvSlt2 strengthens its substrate-binding capacity and bacterial virulence 21 . Notably, CobB can precisely regulate the activity of enolase (Eno) by dual modulation of its K343hib and K326ac modification states, thereby directly influencing bacterial growth 20 . However, no studies have yet reported how Khib affects bacterial fluoroquinolone resistance in S. aureus , and the specific enzymatic machinery responsible for Khib in this pathogen has yet to be identified. In this study, we investigated the role of Khib in regulating fluoroquinolone resistance in S. aureus . We first identified a 2-hydroxyisobutyryltransferase, AcuA, in S. aureus and characterized its 2-hydroxyisobutyryltransferase properties and functions through a series of in vitro and in vivo experiments. We found that AcuA-mediated 2-hydroxyisobutyrylation promotes fluoroquinolone resistance in S. aureus by regulating the dimerization and DNA-binding activity of the transcription regulator CodY. Overall, this research reveals how S. aureus exploits Khib to survive antibiotic challenge and may open new avenues for developing novel adjuvant therapies targeting Khib machinery to counteract antibiotic resistance. Results 2-hydroxyisobutyrylation Enhances Fluoroquinolones Resistance in S. aureus The global spread of antibiotic resistance has become a major challenge in clinical anti-infective therapy. Recent studies indicate that Khib has emerged as a key regulator of bacterial physiology, but its role in regulating bacterial resistance is less characterized. We detected differences in PTMs (acetylation, crotonylation and Khib) between wild-type (WT) S. aureus NCTC 8325 and clinical multidrug-resistant (MDR) strains (Table S1). Western blot analysis with pan-antibodies for three PTMs revealed that Khib modification levels in MDR strains were significantly different from those in sensitive bacteria, encompassing proteins across a broad molecular weight range, while the other two modifications exhibited relatively minor changes (Fig. 1a). This finding suggests that Khib may be involved in regulating drug resistance in S. aureus . To investigate the contribution of Khib to bacterial resistance, we first profiled the global proteomic landscape of Khib in S. aureus across different growth phases (lag phase, logarithmic phase, and late logarithmic phase). Immunoblotting analysis showed that Khib modification levels increased progressively during bacterial growth, with the most pronounced modification observed in the late logarithmic phase (OD 600 = 1.0) (Fig. 1b). This dynamic change suggests a potential regulatory role for Khib in the transition between primary and secondary metabolism in S. aureus . Subsequent Khib proteomic analysis revealed that 3,485 lysine residues across 1,275 proteins in logarithmic-phase S. aureus were 2-hydroxyisobutyrylated (Fig. 1c). Gene Ontology (GO) analysis unveiled that these proteins are primarily enriched in intracellular, cytoplasm, and cell membrane, and are important for biological processes like translation, biosynthesis, gene expression, and stress response. Most molecular functions of these proteins are involved in transport and molecular bindings (Fig. S1a). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis linked these proteins to metabolic pathways, TCA cycle, aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis pathway, pentose phosphate pathway, and ribosome (Fig. 1d). Previous studies have demonstrated crosstalk between different PTMs, such as the ability of many lysines to undergo both crotonylation and acetylation. Considering that crosstalk between different PTMs may affect resistance, we compared Khib with previously reported PTMs in S. aureus 22 . We found that most 2-hydroxyisobutyrylated proteins were also subject to acetylation (Kac) and succinylation (Ksucc), which have been demonstrated to be involved in regulating S. aureus resistance to erythromycin and vancomycin, respectively 10,23 . However, approximately 57% of Khib protein did not overlap with Kac and Ksucc proteins (Fig. 1e). This indicates that Khib possesses a distinct regulatory mechanism independent of Kac and Ksucc, supporting the possibility of its specific involvement in regulating the resistance pathway. Early studies showed that succinylation of cell wall biosynthesis-related protein MurA at Lys69 and Lys191 significantly reduces its activity, thereby decreasing cell wall thickness and rendering the vancomycin-resistant strain more susceptible to antibiotic 10 . It is interesting to note that we also identified 2-hydroxyisobutyrylation at Lys69 of MurA, suggesting that Khib may play a regulatory role in S. aureus resistance. Therefore, we further investigated the relationship between Khib and bacterial resistance. To eliminate interference from genetic heterogeneity in clinical strains, we constructed a ciprofloxacin-resistant (CIP-R, MIC = 120 µg/mL) strain through continuous passage culture under pressure stress at 1/2 MIC concentration. Subsequently, we examined the difference in Khib modification levels between the WT and CIP-R. As shown in Fig. 1f, significantly elevated Khib modification levels were detected in CIP-R. To investigate whether this elevated Khib level is linked to antibiotic stress, we exposed the WT strain to sub-MIC concentrations of ciprofloxacin and monitored Khib dynamics. Notably, this treatment also led to a significant increase in global Khib modification levels (Fig. 1g). These results demonstrate that heightened Khib levels are a conserved feature associated with both acquired resistance (CIP-R strain) and acute antibiotic challenge. The rapid upregulation of Khib upon ciprofloxacin exposure suggests a close association between fluoroquinolone-induced stress and this modification, further implicating Khib in the bacterial adaptation to drug pressure. To further examine the impact of Khib on S. aureus growth and drug resistance, we supplemented the culture medium with 2-hydroxyisobutyrate (hib), a metabolic precursor of 2-hydroxyisobutyryl-CoA (Hib-CoA), which serves as the substrate for enzymatic 2-hydroxyisobutyrylation 14 . Western blot analysis showed that 2-hydroxyisobutyrate supplementation significantly increased Khib modification levels in S. aureus (Fig. 1h). Growth curve analysis revealed that treatment with 2-hydroxyisobutyrate resulted in a slight inhibition on bacterial growth (Fig. 1i). External supplementation with hib reduced ATP levels in both sensitive and resistant S. aureus , thereby markedly increasing their tolerance to fluoroquinolones (ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin) (Fig. 1j, k), indicating that bacteria lower energy expenditure to support resistance ability. The NAD + -dependent deacetylase CobB from Proteus mirabilis ( P. mirabilis ) and E. coli exhibits de-2-hydroxyisobutyrylation activity 20 . Through homology comparison analysis, we found that the CobB from S. aureus , P. mirabilis and E. coli , despite their pronounced sequence divergence (20% similarity), exhibit a conserved tertiary structure (Fig. S1b). Based on this structural conservation, we hypothesized that CobB might also mediate de-2-hydroxyisobutyrylation in S. aureus . To test this, we performed both in vitro and in vivo functional assays. In vitro experiments showed that purified CobB significantly reduced Khib modification levels when incubated with whole-cell protein extracts (Fig. 1l). Consistent with this phenomenon, overexpression of CobB in S. aureus significantly reduced global Khib modification levels, whereas deletion of the cobB gene resulted in their pronounced accumulation (Fig. 1m, n). Collectively, these results demonstrate that CobB functions as a de-2-hydroxyisobutyrylase in S. aureus . We next investigated the effect of CobB on drug resistance in S. aureus. Deletion of cobB did not affect the growth of the WT strain (data not shown). Unexpectedly, cobB deletion did not further enhance WT strain resistance to ciprofloxacin, maybe due to its ability to a deacetylase, such as deacetylation and desuccinylation. We then overexpressed cobB in CIP-R to further explore the role of CobB in antibiotic resistance. In comparison to the CIP-R, the cobB + strain showed significantly reduced resistance to ciprofloxacin, which was partially restored with the addition of additional 2-hydroxyisobutyrate (Fig. 1o). These combined findings highlight that Khib regulates various metabolic pathways in S. aureus and is closely associated with fluoroquinolone resistance. Comparative 2-hydroxyisobutyryl Proteomics Reveals Critical Proteins Linked to Fluoroquinolone Resistance To gain deeper insights into the connection between Khib and bacterial antibiotic resistance, we compared the differentially expressed 2-hydroxyisobutyrylated proteome between WT and CIP-R strains through antibody enrichment combined with DIA-MS analysis. In the CIP-R group, 1,339 Khib-modified proteins were identified across three biological replicates, with 9,366 Khib modification sites detected which is similar to the number in WT strain (Fig. 2a). Global profiling of all identified Khib sites revealed a higher overall modification level in the CIP-R strain compared to the WT (Fig. 2b). We collectively identified 461 differentially 2-hydroxyisobutyrylated proteins (DHPs), of which 299 were upregulated and 162 were downregulated in CIP-R (fold change > 1.5, p < 0.05), corresponding to 357 and 189 Khib sites, respectively (Fig. 2c). Furthermore, statistical analysis indicated that most proteins contained 1–10 2-hydroxyisobutyryl sites (Fig. 2d). To determine the amino acids preferences of Khib, the Motif-x tool was used to identify conserved amino acid sequence motifs within a ± 7-residue window surrounding each modified lysine. A total of 21 distinct motifs were identified (Fig. 2e, Fig. S2). Analysis of these motifs revealed a marked preference for positively charged side chain amino acids, arginine (R) and lysine (K) surrounding the modified lysine residues (Fig. 2e), which appears to be a unique feature of S. aureus 24 . This enrichment suggests that the change of local electrostatic environment may facilitate acyl group transfer by promoting the interaction between lysine residues and negatively charged acyl-CoA donors, thereby favoring 2-hydroxyisobutyrylation. Next, GO enrichment analysis was performed to determine the potential functions of all DHPs. In the molecular function category, DHPs were mainly associated with the catalytic activity, structural constituent of ribosome, RNA binding, and small molecule binding, indicating that Khib influences proteins translation and function (Fig. 2f). Notably, most of the 2-hydroxyisobutyrylated proteins were annotated to the ribosome ontology in the cellular component category (Fig. 2f). This observation is consistent with the previously characterized substrate preference of Khib modification: the consensus sequence flanking Khib sites is typically enriched in positively charged basic amino acids (e.g., arginine and lysine). As ribosomal proteins inherently contain a high abundance of these basic residues to maintain their structural stability and interaction with negatively charged rRNA, their enrichment as Khib targets is not surprising but rather reflects the intrinsic specificity of the Khib modification system. Furthermore, biological process classification revealed that the 2-hydroxyisobutyrylated proteins were mainly enriched in pathways of macromolecule/protein/peptide metabolic processes, gene expression, translation, and stress response (Fig. 2f). KEGG enrichment analysis showed the biosynthesis of secondary metabolites function exhibited the most significant alteration among 2-hydroxyisobutyrylated proteins, followed by biosynthesis of cofactor and carbon metabolisms. Notably, numerous known essential pathways involved in fluoroquinolone resistance are enriched in DHPs, including ABC transporters, two-component system, mismatch repair, quorum sensing, and oxidative phosphorylation (Fig. 2g). PTMs regulate energy-generating metabolic pathways and represent a common strategy for bacteria to develop resistance to multiple antibiotics. For instance, the primary enrichment pathway for differentially crotonylated proteins in polymyxin-resistant E. coli is considered to be metabolic processes 13 . A previous study on three kinds of antibiotic-resistant E. coli strains indicates that protein acetylation plays a common role in negatively regulating bacterial metabolism, thereby contributing to drug resistance 25 . Similarly, visualization of central carbon metabolism revealed an overall decline in 2-hydroxyisobutyrylation of proteins involved in bacterial energy metabolism (Fig. 2h). A decrease in ATP level is also observed in CIP-R (Fig. 2i). What is more, previous studies have demonstrated that restriction of arginine induces biofilm formation in S. aureus , thereby conferring resistance to delafloxacin 26 . Visualization of arginine metabolic flux also revealed decreased Khib modification levels in several other enzymes responsible for arginine metabolism, such as ArgG, ArgH, ArcA1, and ArcA2 (Fig. 2h). External supplementation with arginine reduced tolerance to ciprofloxacin in both sensitive and resistant S. aureus (Fig. 2j, k). These combined results further suggest that Khib may regulate antibiotic resistance by modifying substrate proteins. Bacteria sense environmental changes through transcription factors and respond by reprogramming gene expression 27 . Recent studies have revealed that Khib controls transcriptional activity through specific targeting of histones and chromatin-associated proteins 16,28 . In this study, we also identified 85 transcription factors modified by Khib in S. aureus . Among these proteins, 24 exhibited differential Khib modification levels in CIP-R, including key regulators such as CodY, BirA, NreC, SarA, and SarX (Fig. 2l, Table S2). It is worth noting that CodY is a global transcriptional regulator controlling metabolism and virulence of S. aureus 29 . These findings suggest the presence of a Khib-driven transcriptional network in S. aureus that mediates adaptive responses to antibiotic stress. AcuA Functions as a Novel 2-hydroxyisobutyryltransferase To further reveal the regulatory mechanism of Khib on bacterial resistance, it is crucial to identify the 2-hydroxyisobutyryltransferase(s) and elucidate their regulatory networks and key substates. At present, the tRNA (Met) cytidine acetyltransferase TmcA in E. coli , the histone acetyltransferase Ngg1 in Aspergillus flavus , and the histone acetyltransferase p300 in mammalian cells have been reported to act as 2-hydroxyisobutyryltransferases to perform a diverse range of cellular functions 19,30 . However, S. aureus lacks homologous proteins to these transferases. Given that GCN5-related N -acetyltransferases (GNATs) family proteins exhibit catalytic activities toward diverse lysine modifications (e.g., TmcA from E. coli ) 8,13 . So, we reasonably hypothesized that some GNAT proteins function as 2-hydroxyisobutyryltransferase mediating lysin 2-hydroxyisobutyrylation in S. aureus . To test this hypothesis, we screened the S. aureus genome for GNAT-encoding genes potentially involved in lysine 2-hydroxyisobutyrylation. Ten candidate genes were overexpressed in S. aureus , and their effects on global 2-hydroxyisobutyrylation patterns were subsequently analyzed. This analysis identified two genes, acuA and yncA , that influence 2-hydroxyisobutyrylation, with acuA showing the stronger effect, whereas overexpression of the other candidates caused no substantial changes (Fig. 3a, Fig. S3a). We then generated single-gene deletion mutants of both genes in wild-type S. aureus and analyzed their impact on 2-hydroxyisobutyrylation patterns. However, we did not detect significant such effects (Fig. S3b). We speculate that the failure to detect such changes in single gene knockout may be due to functional redundancy. Given the high level of Khib in drug-resistant bacteria, we hypothesize that transferases exert a significant influence on the modification of CIP-R. We then produced single-gene deletion mutants of both genes in ciprofloxacin-resistant S. aureus (CIP-R) and again analyzed 2-hydroxyisobutyrylation. Immunoblot assay showed reduced Khib modification levels in CIP-R Δ acuA , whereas no significant change was detected in CIP-R Δ yncA (Fig. 3b, c, Fig. S3c). Furthermore, incubating purified recombinant AcuA protein with 2-hydroxyisobutyryl coenzyme A (Hib-CoA) and a polyvinylidene fluoride (PVDF) membrane containing whole-cell proteins from S. aureus , we could show that AcuA was able to 2-hydroxyisobutyrylate many proteins on the membrane (Fig. 3c). Subsequent fluorescence titrations further showed that binding of Hib-CoA to AcuA resulted in a strong fluorescence quenching with a Kd value of 0.98 µM (Fig. 3d). Taken together, our results suggest that AcuA has a significant impact on global lysine 2-hydroxyisobutyrylation, and that AcuA is an important candidate for 2-hydroxyisobutyryltransferase in S. aureus . A previous study demonstrated that AcuA is a lysine acetyltransferase that reduces the activity of acetyl-coenzyme A synthetase (Acs) by acetylating residue Lys549 31 . We also analyzed the effect of CIP-R Δ acuA on acetylation patterns in S. aureus . As shown in Fig. 3e, single acuA gene knockout had no specific impact on global lysine acetylation levels in S. aureus . This finding is consistent with previous reports in Bacillus subtilis , where Δ acuA neither significantly increased nor decreased the overall acetylation level in the bacterium 32 . Therefore, this study focused on the contribution of AcuA as an S. aureus 2-hydroxyisobutyryltransferase to drug resistance. Next, to elucidate the key residue in AcuA involved in catalyzing the transfer of 2-hydroxyisobutyryl group, we generated the full-length structure of AcuA using the Swiss-Model server. We investigated the interaction between AcuA and Hib-CoA or Ac-CoA through molecular docking analysis. The results showed Hib-CoA and Ac-CoA could bind to AcuA, and Hib-CoA had slightly lower free binding energy (− 9.3 kcal/mol) than that to Ac-CoA (− 8.3 kcal/mol). Further structural analysis revealed that eight amino acid residues (Gln109, Arg108, His139, Ile105, Trp140, Tyr152, Val103, and Gly99) of AcuA were able to form hydrogen bonds with Hib-CoA (Fig. 3f). It was reported that His139 and Trp140 of AcuA in Bacillus subtilis are important sites for acetylation 33 . The His139 side chain can bind to Ac-CoA, acting as a general base during the catalytic process while coordinating with Ca²⁺ ions. On the other hand, the main chain amide of Trp140 is oriented toward the active center, plays a structural role in forming the hydrophobic cavity and facilitates the binding of acetyl-CoA 33 . Based on sequence alignment, we found that His139 and Trp140 of AcuA in Bacillus subtilis are conserved in S. aureus (His139 and Trp140) (Fig. 3g). Therefore, we predict that His139 and Trp140 of AcuA in S. aureus are likely to be the key sites for its 2-hydroxyisobutyrylase activity. To test this possibility, we expressed and purified WT and mutated AcuA (H139A and W140A) (Fig. 3h) and then performed fluorescence titration analysis using the Hib-CoA. Compared to wild-type AcuA, the W140A mutant exhibited relatively low Hib-CoA binding affinity, while the H139A mutant failed to bind to Hib-CoA (Fig. 3i, j). Structurally, both W140A and H139A mutations altered the overall conformation of AcuA. The W140A mutation resulted in a closed active center (Fig. 3k). Further molecular dynamics simulations indicated that the hydrophobic cavity housing Hib-CoA collapsed in the H139A mutant, as alanine substitution disrupted the binding pocket's conformation. This structural change is characterized by a significant reduction in the distances between the key structural residues Ser145 and Ile105, as well as Leu155 and Gln109 within the hydrophobic cavity (Fig. 3l). To functionally validate the critical role of residue His139 and Trp140 in mediating Khib catalysis in vivo , we introduced H139A and W140A mutations into AcuA in the CIP-R Δ acuA strain. As expected, both mutant strains exhibited significantly impaired Khib catalysis compared to the wild-type complement strain ( eWT ). The eH139A mutant strain nearly abolished Khib activity, and the eW140A mutant strain also showed a substantial defect, supporting the specificity of these residues for Khib catalysis (Fig. 3m). Collectively, these results confirm that His139 and Trp140 are essential for Hib-CoA binding in AcuA and substantiate its dual role in maintaining structural integrity and enabling Khib catalytic activity both in vitro and in vivo . 2-hydroxyisobutyryltransferase AcuA Induces Fluoroquinolone Resistance in S. aureus Prior studies have demonstrated that Khib plays a key role in regulating the survival of E. coli under extreme acid stress 19 . Moreover, our preliminary data also indicated that elevated Khib modification levels promote fluoroquinolone resistance in S. aureus (Fig. 1). Therefore, we further investigated the regulatory role of the 2-hydroxyisobutyryltransferase AcuA in S. aureus antibiotic resistance. Interestingly, deletion of the acuA gene significantly impaired bacterial growth, resulting in a significant growth defect compared with the CIP-R (Fig. 4a). AcuA is a component of the acuABC operon, which has been implicated in the catabolism of butanediol and acetoin (Fig. S4a). Thus, we speculate that the loss of acuA disrupts basal metabolic processes of S. aureus , thereby contributing to the observed growth impairment and antibiotic resistance. Then, we further assessed the contribution of AcuA to antibiotic resistance in S. aureus . The ciprofloxacin resistance decreased in CIP-R Δ acuA in which the Khib modification levels of whole proteome was reduced (Fig. 4b, Fig. 3b). Although no obvious change in drug resistance was observed in the WT Δ acuA strain (Fig. 4c), continuous passage under 1/2 MIC ciprofloxacin treatment resulted in the resistance development of WT-Δ acuA being obviously slower than that of WT strain (Fig. 4d). Compared to the empty vector, overexpression of the acuA gene increases Khib modification levels while simultaneously enhancing WT S. aureus resistance to ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin by 2-, 4-, 4-, and 4-fold, respectively (Fig. 4e). This result indicates that AcuA promotes bacterial resistance to fluoroquinolones by regulating Khib modification levels of specific substrate proteins. To further explore the regulatory networks of AcuA in S. aureus , we conducted a GST pull-down assay followed by mass spectrometry (MS) analysis (Fig. 4f). A total of 757 proteins were identified as potential interactors of AcuA. We mapped all identified proteins to KEGG pathways. These proteins are associated with several key metabolic pathways, including amino acid/carbon/nucleotide metabolism, as well as infection-related processes (Fig. 4g). It is worth noting that 30 transcription factors were identified, and the Khib modification levels of 7 of these proteins exhibited significant differences in CIP-R strains (Fig. 4h, Table S2). These findings indicate that AcuA-mediated Khib modification affects numerous key metabolic pathways in S. aureus , thereby altering bacterial drug resistance through metabolic reprogramming. Subsequently, to further elucidate the molecular mechanism by which Khib promotes antibiotic resistance, we conducted DIA-based quantitative proteomics on CIP-R and CIP-R Δ acuA strains. This analysis totally identified 465 differentially expressed proteins (DEPs) were identified, including 180 upregulated proteins and 285 downregulated proteins (fold change > 1.2, p < 0.05) (Fig. S4b). Upregulated proteins were primarily enriched in pathways related to biosynthesis of nucleotide sugars, pyruvate metabolism, riboflavin metabolism, and glycolysis/gluconeogenesis (Fig. S4c). Conversely, downregulated proteins were associated with pathways such as secondary metabolite biosynthesis, ribosome, biosynthesis of various amino acids, and fatty acid degradation (Fig. S4d). Notably, compared to the control strain CIP-R, CIP-R Δ acuA exhibits downregulation in the expression levels of several known essential proteins involved in biofilm formation, including FnbA, FnbB, SraP, IsaA, Spa, SasG, SdrD, and SdrC (Fig. 4i). Furthermore, qPCR analysis revealed that the expression level of the icaADBC operon, a key operon involved in S. aureus biofilm formation, was also significantly decreased (Fig. 4j). Prior research reported that AcuA regulates biofilm formation and swarming of B. subtilis by acetylating YmcA and GtaB 32 . Consistent with these findings, bacterial biofilm analysis showed that overexpression of acuA led to increased biofilm formation in S. aureus , whereas knockout of acuA reduced biofilm formation (Fig. 4k). It is also noteworthy that the addition of hib to CD medium led to increased expression of the icaADBC operon genes and decreased expression of the repressor icaR , ultimately leading to enhanced biofilm formation (Fig. 4l, m). To further validate the association between AcuA, biofilm formation, and drug resistance in clinical isolates, we examined the expression of the icaADBC operon and biofilm-forming capacity in MDR strains. The results indicated that, compared with the WT strain, the transcriptional levels of icaA and icaB genes were significantly increased in MDR strains, accompanied by enhanced biofilm formation (Fig. 4n, o). Western blot analysis further confirmed significantly higher protein levels of AcuA in clinically multidrug-resistant bacteria and CIP-R relative to the WT strain (Fig. 4p), suggesting that S. aureus may upregulate AcuA expression as an adaptive response to adverse environments. Collectively, these findings suggest that AcuA may regulate S. aureus resistance to fluoroquinolone antibiotics by upregulating the Khib modification levels of biofilm-associated proteins. AcuA Influences Bacterial Resistance to Fluoroquinolones through 2-hydroxyisobutyrylation of CodY Increasing evidence indicates that post-translational modifications (PTMs) can rapidly reprogram bacterial metabolic networks by directly regulating the activity of key transcription factors (TFs), thereby promoting the development of antibiotic resistance 13,27 . Notably, in our constructed ciprofloxacin-resistant S. aureus model, we found that the global transcription factor CodY serves as a prominent example of this regulatory mechanism. In ciprofloxacin-resistant S. aureus , the Khib-modified lysine residue at positions 6, 16, and 223 in CodY were differentially modified sites vs. WT, increasing by 1.97-fold, 1.50-fold, and 2.21-fold, respectively (P < 0.05) (Fig. 5a, Fig. S5a). Notably, CodY protein expression levels remained unchanged between WT and CIP-R as well as MDR strains (Fig. 5b). In S. aureus , CodY is a central transcriptional repressor that integrates nutrient availability with pathogenicity. It directly controls the expression of critical targets, including virulence genes (e.g., hla), biofilm-forming components, and metabolic enzymes to coordinate adaptation and pathogenicity 29,34 . Therefore, we hypothesize that AcuA modulates fluoroquinolone resistance not by altering CodY abundance, but by regulating its Khib modification levels, thereby influencing its transcriptional regulatory activity. Acyltransferases function by binding both an acyl-CoA donor and a protein substrate 35 . To determine whether AcuA catalyzes the 2-hydroxyisobutyrylation of CodY, we first assessed their physical interaction. Glutathione S-transferase (GST)-tagged AcuA and polyhistidine (His)-tagged CodY were incubated together, and western blot analysis confirmed that AcuA directly binds to CodY (Fig. 5c). We next performed in vitro Khib assays by incubating recombinant CodY with Hib-CoA in the presence or absence of purified AcuA. The results showed that CodY undergoes non-enzymatic Khib modification when incubated with Hib-CoA alone. Moreover, the addition of AcuA significantly enhanced the Khib modification level of CodY (Fig. 5d, e). These findings indicate that AcuA functions as an acyltransferase that promotes CodY 2-hydroxyisobutyrylation. It is worth noting that 2-hydroxyisobutyrylation of CodY can occur through both enzymatic and non-enzymatic mechanisms. While enzymatic acylation has been reported in several contexts, such as modifications mediated by AcuA or other acyltransferases, non-enzymatic 2-hydroxyisobutyrylation of bacteria itself also plays a regulated and biologically significant role 36 . To validate the lysine residues of AcuA-catalyzed 2-hydroxyisobutyrylation on CodY, we performed in vitro 2-hydroxyisobutyrylation assays using purified wild-type CodY and mutant proteins in which specific lysine residues were replaced with arginine (K6R, K16R, and K223R) to mimic the de-2-hydroxyisobutyrylated state. Western blot analysis showed significantly reduced Khib modification levels at the K16R and K223R mutants, whereas the K6R mutant exhibited a modification level comparable to that of the wild-type protein (Fig. 5f). Consistently, when the mutants were incubated with AcuA and Hib-CoA, Khib modification levels remained low at the K16R and K223R sites, while the K6R mutant again showed no significant difference from wild-type CodY (Fig. 5g). These results indicate that Lys16 and Lys223 of CodY are critical residues for AcuA-mediated 2-hydroxyisobutyrylation. We then looked at how these changes in Khib modification levels affect in vivo bacterial resistance to fluoroquinolones. Four plasmid point mutants were constructed by individually mutating Lys16 and Lys223 of CodY to glutamine (Q) or arginine (R). The lysine-to-glutamine (K to Q) substitution mimics the 2-hydroxyisobutyrylated state. These plasmids were introduced into the Δ codY strain (Fig. S5b) (the generated strains were named eK16Q , eK16R , eK223Q , and eK223R ). We measured the growth curves of the K-to-Q and K-to-R strains. The growth rate of K-to-R strains ( eK16R and eK223R ) reaching the plateau phase 8–9 h after inoculation was significantly slower than that of the K-to-Q strains ( eK16Q and eK223Q ) (Fig. S5c). Meanwhile, MIC assays showed that eK16Q and eK223Q strains exhibited elevated resistance to multiple fluoroquinolones compared to the eK16R and eK223R strains. Specifically, the eK16Q mutant led to 4-, 2-, 1-, and 1-fold increases in MIC for ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin, respectively, while the eK223Q mutant resulted in 8-, 8-, 4-, and 16-fold increases (Fig. 5h, i). These results indicate that AcuA enhances bacterial resistance to fluoroquinolones through 2-hydroxyisobutyrylation of CodY. Notably, the K223Q mutation contributed significantly more to resistance than K16Q, suggesting that AcuA primarily promotes S. aureus fluoroquinolone resistance by upregulating 2-hydroxyisobutyrylation at the CodY Lys223 site. Importantly, the Lys223 site is highly conserved in Gram-positive bacteria (Fig. S5d), further suggesting that 2-hydroxyisobutyrylation at this site plays an important biological role in regulating antibiotic resistance. To further validate these results, we established mouse pneumonia infection models using eK223Q and eK223R mutant strains. In the absence of antibiotic treatment, mice infected with the eK223R strain exhibited significantly higher mortality (90%, 9/10), whereas mice infected with the eK223Q strain showed lower mortality (50%, 5/10) (Fig. 5j). Subsequently, we evaluated the response of the strains to ciprofloxacin treatment. Compared to the untreated groups, ciprofloxacin administration effectively protected mice infected with the eK223R strain, reducing mortality to 0% (0/10) and resulting in very low post-treatment bacterial burden in the lungs. In contrast, although ciprofloxacin treatment also reduced the mortality of mice infected with the eK223Q strain to 20% (2/10), the bacterial load in their lung tissue post-treatment was significantly higher than that in the treated eK223R infection group (Fig. 5j, k). This key finding indicates that Lys223 2-hydroxyisobutyrylation of CodY, while attenuating acute pathogenicity, significantly enhances bacterial resistance to ciprofloxacin, enabling persistent colonization within the host under antibiotic pressure. Given the established role of CodY in regulating biofilm-associated operons and the significant contribution of biofilm formation to antibiotic tolerance, we hypothesized that 2-hydroxyisobutyrylation of CodY may regulate resistance by influencing biofilm formation 29 . To test this hypothesis, we evaluated the biofilm-forming capacity of the mutant strains. qPCR analysis revealed that the expression of key biofilm-forming genes, icaA and icaB , was significantly upregulated in the eK16Q and eK223Q strains compared to the eK16R and eK223R mutants (Fig. 5l). Subsequent biofilm assays further confirmed that the eK16Q and eK223Q mutants formed significantly more biofilm than the eK16R and eK223R mutants (Fig. 5m), indicating that 2-hydroxyisobutyrylation of CodY enhances fluoroquinolone resistance, at least in part, by promoting biofilm production. 2-hydroxyisobutyrylation Regulates Dimerization and DNA-binding Ability of CodY To assess whether Khib modification influences the structural integrity of CodY, we evaluated its structure and thermal stability through circular dichroism CD spectroscopy. CD analysis revealed no significant differences in the secondary structure composition of wild-type CodY and its mutants (Fig. S6a, b). Although the secondary structure is largely unaffected, thermal denaturation experiments revealed reduced melting temperatures (Tm) for K16Q (50.9°C) and K223Q (51.5°C) compared to wild-type CodY (54.1°C) (Fig. 6a), indicating diminished protein stability of mutant proteins. This decrease in thermal stability can be attributed to the disruption of key intramolecular interactions that were originally mediated by the wild-type lysine residues. The K-to-Q mutations, by removing positive charges and altering side-chain chemistry, compromise the intricate network of non-covalent interactions that lock the protein into its stable, native conformation. CodY functions as a homodimeric transcription factor, and its DNA-binding capacity is closely linked to its oligomeric state 37,38 . CodY consists of an N-terminal ligand-binding domain GAF and a C-terminal DNA-binding domain (DBD), connected by an extended helical linker (LHL), which incorporates a winged helix-turn-helix (wHTH) motif. Structural analysis revealed that in the CodY dimer, two monomers (designated here as protomer A and protomer B) interact via an interface involving both the GAF and DBD domains (Fig. S6c). Intriguingly, Lys16 in the GAF domain of monomer and Lys223 in the DBD domain were both 2-hydroxyisobutyrylated, and both residues are located at the dimer interface (Fig. 6b, Fig. S6c), suggesting that their modification may influence the self-assembly of the two monomers. To test this hypothesis, we performed formaldehyde crosslinking followed by immunoblotting, which confirmed the presence of CodY dimers (Fig. 6c). Notably, the K16Q and K223Q mutants exhibited enhanced dimerization relative to wild-type CodY, whereas charge-conservative arginine substitutions (K16R and K223R) showed no such effect. These results suggest that Khib modification at Lys16 and Lys223 strengthens intermolecular interactions and promotes CodY dimerization. The GAF domain plays a crucial role in CodY dimer assembly. Hainzl et al 37 found that, in the absence of ligands, residues Gln15-Lys18 sterically clash with the linker helix and the S3–S4 loop of protomer B, thereby preventing dimer formation (Fig. 6d). However, ligand-binding induces the formation of an α-helix involving Leu14, Gln15, and Lys16, resulting in the extension of helix H1, enabling hydrogen bonding with residues in protomer B, thereby moving residues Gln15-Lys18 to allow dimer formation (Fig. S6d). In the K16Q mutant, this steric hindrance is alleviated even in the absence of ligand, effectively mimicking a pre-activated state, which would facilitate dimer formation (Fig. 6e). Lys223 is critical for stabilizing the dimer interface 37 . Structural analysis showed that Lys223 forms a bulge within the DNA binding groove, which facilitates dimer formation (Fig. S6c). Analysis of the dimer interface indicates that the K223Q mutant forms more hydrogen bonds compared to WT CodY (Fig. 6f, g), and the distance between the two bulge is significantly reduced, causing protomer A and B to bind more tightly (Fig. S6e, f). We next ask whether this increase in dimerization translates to enhanced DNA binding. Using electrophoretic mobility shift assays (EMSAs), we evaluated the interaction of CodY variants with the target regulatory regions of two downstream genes (P icaB and P cap ). icaB is part of the biofilm synthesis operon icaADBC , while cap is the promoter of the capsular polysaccharide (CP) operon in S. aureus . Surprisingly, despite their increased propensity to dimerize, the K223Q mutant displayed reduced binding to both the icaB and cap promoters (Fig. 6h). Moreover, the K16Q mutant exhibited promoter-specific differences: K16Q showed stronger binding to the cap promoter, while its binding to the icaB promoter is lower than that of the wild type (Fig. 6i). These findings indicate that Khib-induced dimerization does not uniformly enhance DNA binding. Instead, it appears to modulate promoter affinity in a site- and context-dependent manner, likely by allosterically tuning the CodY–DNA interface. Since dimerization is thought to facilitate transcription factor binding to DNA, the distinct binding patterns of CodY mutants observed in EMSA analyses were unexpected. In the previously characterized binding modes of CodY with ligands, CodY dimers exhibit distinct conformational arrangements (PDB ID: 5eyo and 5ey1). Thus, although both K16Q and K223Q facilitate dimerization, they may perturb allosteric coupling between ligand sensing and DNA recognition. Consequently, we employed AlphaFold 3 to simulate the structures of K16Q and K223Q dimers in GTP-bound states 39 . Structural analyses revealed that 2-hydroxyisobutyrylation at either Lys16 or Lys223 site leads to significant changes in the conformation and spatial organization of DBD (Fig. 6j). Specifically, the K-to-Q mutation at the Lys223 site results in a markedly shallower DNA-binding groove, potentially hindering DNA binding. Furthermore, we observed distinct differences in the electrostatic potential of the DNA-binding groove between the K16Q and K223Q mutants and the wild-type CodY (Fig. 6k). The K16Q mutant exhibits altered local electrostatic potential, while the K223Q mutant increases the net negative electrostatic potential of the binding interface. Given that the DNA backbone is highly negatively charged, this alteration would introduce electrostatic repulsion and diminish the favorable electrostatic complementarity that is essential for high-affinity binding. Thus, while the two mutants stabilize the dimeric form, they concomitantly compromise the electrostatics of DNA recognition, leading to an overall decrease in binding affinity. This may account for the binding states of CodY to different promoters despite enhanced dimerization capacity. Discussion The escalating challenge of antimicrobial resistance in S. aureus , particularly its high-level resistance to critical antibiotics like fluoroquinolones, poses a severe threat to modern medicine. While mutations in target enzymes such as DNA gyrase are established primary drivers of clinical fluoroquinolone resistance, there is growing recognition that non-mutational, adaptive mechanisms significantly contribute to the resistance phenotype and treatment failure. In-depth research into these complementary mechanisms will help address this global challenge.Emerging evidence emphasizes that post-translational modifications (PTMs) are pivotal regulators of bacterial virulence and antibiotic resistance 12 , 13 . Among these, Khib is a recently discovered metabolite-derived PTM. Structurally analogous to-but bulkier than-acetylation, Khib can induce more profound alterations in protein function and interactions 40 , 41 . Although Khib has been shown to dynamically reprogram metabolic pathways in pathogens like Ustilaginoidea virens and regulate critical processes in mammalian cells, its role in S. aureus , particularly its connection to antibiotic resistance and pathogenicity, remains largely unexplored 15 , 21 . Our study reveals that Khib serves as a critical adaptive regulator that potentiates fluoroquinolone resistance in S. aureus . At the global level, we found that Khib is widely distributed across the S. aureus proteome and is markedly remodeled in both clinical MDR isolates and laboratory-evolved ciprofloxacin-resistant (CIP-R) strains. Further quantitative Khib proteomics was performed to compare the DHPs between CIP-R and WT S. aureus. CIP-R cells displayed substantial changes in 2-hydroxyisobutyrylation compared with susceptible strains, with hundreds of proteins showing increased or decreased modification. These differentially 2-hydroxyisobutyrylated proteins were significantly enriched in metabolic pathways, particularly those involved in carbon and energy metabolism, as well as in pathways previously implicated in quinolone resistance. One striking pattern was the overall decrease in 2-hydroxyisobutyrylation of enzymes associated with carbon metabolism in CIP-R strains. Given that Khib adds a bulky, polar group to lysine residues, loss of 2-hydroxyisobutyrylation at key metabolic nodes is expected to alter enzyme activity, complex assembly, or cofactor affinity. Such changes could reprogram central metabolism to favor energy-efficient pathways, thereby influencing the bactericidal activity of fluoroquinolones. Importantly, pharmacologic manipulation of global Khib modification levels by adding 2-hydroxyisobutyrate or overexpressing the de-2-hydroxyisobutyrylase CobB bidirectionally changed susceptibility to fluoroquinolones. Together, these findings argue that 2-hydroxyisobutyrylation is not a passive bystander but an active determinant of drug response that fine-tunes metabolic fluxes under antibiotic stress. Protein 2-hydroxyisobutyrylation and de-2-hydroxyisobutyrylation are reversibly catalyzed by two classes of enzymes, 2-hydroxyisobutyryltransferases (writers) and de-2-hydroxyisobutyrylases (erasers) 40 . To date, the only 2-hydroxyisobutyryl transferase identified in prokaryotes is TmcA from E. coli 19 . TmcA exhibits tRNA(Met) cytidine acetyltransferase activity, demonstrating the complexity of bacterial Khib regulatory factor classification. The regulatory function of Khib in Staphylococcus resistance, and its role as both “writers” and “erasers”, remains elusive. To understand how 2-hydroxyisobutyrylation is written onto the proteome, we systematically screened GNAT-family acyltransferases and identified AcuA as a 2-hydroxyisobutyryl transferase in S. aureus . Biochemical assays confirmed that AcuA catalyzes 2-hydroxyisobutyrylation in vitro and in vivo, and mutational analysis of the predicted Hib-CoA binding pocket demonstrated that residues such as His139 and Trp140 are essential for activity. Beyond its enzymatic function, AcuA plays a broad physiological role. Deletion of acuA impaired bacterial growth, reduced global Khib modification levels and significantly lowered resistance to ciprofloxacin and other fluoroquinolones in CIP-R strains, whereas its overexpression had opposite effects. Proteomic profiling of the acuA mutant revealed perturbation of amino acid, carbon, and nucleotide metabolism and, importantly, down-regulation of multiple biofilm-associated proteins, including FnbA, SraP, and the icaADBC operon. Consistent with these molecular changes, AcuA was required for robust biofilm formation. Notably, AcuA protein expression is significantly upregulated in clinically isolated multidrug-resistant strains, suggesting that upregulation of this writer enzyme is part of the adaptive program that enables S. aureus to persist under antibiotic pressure. These observations prompted us to search for regulatory substrates that connect AcuA-dependent 2-hydroxyisobutyrylation to biofilm-mediated resistance. We focused on CodY, a highly conserved global transcription regulator in Gram-positive bacteria that senses intracellular GTP and branched-chain amino acids to adjust gene expression according to nutritional status. CodY controls a wide regulatory network encompassing metabolic genes, virulence factors, and stress-response pathways 29 , 34 . We observed higher levels of Khib at CodY Lys6, Lys16, and Lys223 in CIP-R strains than in susceptible strains and showed that AcuA physically interacts with CodY and catalyzes 2-hydroxyisobutyrylation at Lys16 and Lys223. Among these sites, Lys223 emerged as the most critical for fluoroquinolone resistance. Critically, in a mouse infection model, strains harboring high Khib modification levels at CodY Lys223 exhibited significantly enhanced survival upon ciprofloxacin challenge, indicative of increased fluoroquinolone resistance. Notably, our in vivo experiments also revealed that Khib modification at CodY Lys223 exerts a negatively regulatory effect on acute bacterial virulence (Fig. 5 j). This observation indicated that AcuA-mediated modification may coordinately regulate multiple aspects of S. aureus adaptation, including both antibiotic resistance and pathogenic potential. Elucidating the precise role of this PTM in pathogenicity is a key direction of our future research. Functional studies using CodY mutants that mimic de-2-hydroxyisobutyrylated (K→R) or 2-hydroxyisobutyrylated (K→Q) states revealed a clear regulatory pattern. CodY K16R/K223R displayed enhanced binding to the icaADBC promoter, stronger repression of biofilm-associated genes, and reduced fluoroquinolone resistance. In contrast, CodY K16Q/K223Q weakened promoter binding, derepressed icaADBC expression, promoted biofilm formation, and increased resistance. Given that biofilms are a well-characterized niche for antibiotic tolerance and resistance development, our findings delineate a novel Khib-dependent signaling pathway that enhances bacterial survival under antibiotic stress by fostering a protective biofilm lifestyle. Because Lys16 and Lys223 lie within the oligomerization domain of CodY, we propose that 2-hydroxyisobutyrylation at these positions stabilizes CodY dimerization and alters the conformation and charge distribution of the DNA-binding groove, thereby decreasing affinity for target promoters. In nutrient-replete conditions, CodY is already prone to dissociation upon changes in GTP or amino acid levels; 2-hydroxyisobutyrylation could serve as an additional signal that accelerates its release from DNA when cells experience antibiotic-induced metabolic stress. Given the high conservation of Lys223 in CodY homologs from other Gram-positive pathogens, this PTM site may represent a broadly relevant node where metabolic cues, PTMs, and antimicrobial resistance intersect. Integrating these findings, we propose a model in which 2-hydroxyisobutyrylation drives metabolic reprogramming in S. aureus to survive fluoroquinolone stress. Up-regulation of AcuA increases 2-hydroxyisobutyrylation of specific metabolic enzymes and the global regulator CodY. At the metabolic level, changes in Khib promote rerouting of carbon flux and energy production toward states that favor survival under antibiotic stress. At the regulatory level, AcuA-dependent 2-hydroxyisobutyrylation of CodY, particularly at Lys223, diminishes repression of the icaADBC operon and other biofilm-associated genes, leading to enhanced biofilm formation. Biofilms, in turn, provide a protective niche with reduced antibiotic penetration, altered microenvironment, and increased tolerance. In summary, we identified a novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in S. aureus . Specifically, AcuA-mediated 2-hydroxyisobutyrylation of CodY at Lys16/Lys223 alleviates its transcriptional repression of the icaADBC operon, thereby promoting biofilm-associated antibiotic resistance. This work contributes to the field of drug-resistant bacterial control by revealing the promising role of protein 2-hydroxyisobutyrylation, thereby opening a new avenue for combating these infections. Limitations of this study Lysine modification in bacteria can occur through both enzymatic and non-enzymatic mechanisms. We focus solely on the role of enzyme-mediated Khib in regulating bacterial physiological functions, but non-enzyme-mediated Khib may contribute to the development of bacterial fluoroquinolone resistance. Furthermore, we concentrate only on proteins exhibiting elevated Khib modification levels in CIP-R, but those with downregulated Khib modification levels may also play roles in the emergence of bacterial fluoroquinolone resistance. Methods Bacterial strains and cell culture Bacterial strains, plasmids, and primers used in this study are listed in Table S3 and Table S4. Unless otherwise noted, all E. coli strains were grown in Luria Bertani (LB) broth, S. aureus strains and their derivatives were cultured in tryptic soy broth (TSB) or in chemically defined (CD) medium at 37°C 42 . For plasmid maintenance, antibiotics were used at the following concentrations: ampicillin, 100 µg/mL; kanamycin, 50 µg/mL; chloramphenicol, 10 µg/mL; erythromycin, 25 µg/mL. Construction of plasmids for gene expression To construct overexpression plasmids for the enzymes that responsible for 2-hydroxyisobutyrylation, the PCR-amplified DNA fragment and plasmid pCN51 were subjected to digestion with restriction enzymes BamH Ι and EcoR Ι. Then, the digested fragment was ligated into pCN51 and transformed into E. coli DC10B. The resulting plasmids were subsequently transformed into S. aureus by electroporation. Of note, these genes were positioned downstream of the cadmium resistance transcriptional regulatory CadC in pCN51, thereby enabling its induction by Cadmium Chloride (0.25 µM). To construct the codY expression plasmids for genetic complementation, the codY gene was amplified by PCR, incorporating homologous fragments from the pWWW412. The pWWW412 plasmid was digested with the restriction enzyme Nde I. The amplified fragment and digested pWWW412 were ligated via homologous recombination and transformed into E. coli DC10B. Subsequently, the resulting pWWW412 -codY plasmids were electroporated into S. aureus . Recombinant proteins were expressed and purified according to established protocols. Genes were amplified by PCR and cloned into either the PGEX-4T-1 or pET-28a vectors, and transformed into E. coli BL21 (λDE3) for protein expression. A final concentration of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression. Proteins containing His-tags or GST-tags were purified using nickel or GST affinity chromatography columns, respectively. Construction of gene mutants The gene knockout was constructed using the temperature-sensitive plasmid pKOR1 via allelic replacement. Briefly, upstream and downstream fragments flanking the target gene were fused by PCR and cloned into pKOR1. The resulting plasmid was electroporated into S. aureus , and integrants were selected at 43°C under chloramphenicol selection. Excision and loss of the plasmid were subsequently promoted by culturing at 30°C without antibiotics, followed by counter-selection on anhydrotetracycline. Mutants were screened for chloramphenicol sensitivity and the deletion was confirmed by PCR and sequencing. Metabolite analysis ATP was determined with the ATP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Determination of MICs The minimum inhibitory concentration (MIC) of fluoroquinolone antibiotics (ciprofloxacin, levofloxacin, norfloxacin and moxifloxacin) against S. aureus isolates was determined by the broth microdilution method in CD medium according to the guidelines established by the Clinical and Laboratory Standards Institute (CLSI M07). Briefly, bacterial suspensions were adjusted to a density of 5×10 5 CFU/mL and incubated with serial two-fold dilutions of antibiotics for 24 h at 37°C. Bacterial growth was quantified by measuring the OD 600 with a microplate reader (BioTek Epoch, USA). The MIC value is the antibiotic concentration under which the OD 600 of cultures is lower than 0.1. Whole protein extraction and western blot Total protein was prepared from S. aureus cultures harvested during the late-logarithmic growth phase (OD 600 = 1.0). Bacterial cells were digested with lysostaphin (10 µg/mL) for 30 min at 37°C to weaken the cell wall, followed by sonication in SDS lysis buffer containing protease and deacetylase inhibitors. The crude lysate was clarified by centrifugation at 12,000 g for 15 min at 4°C. Protein concentration was determined using the Bradford assay with BSA as a standard. For immunoblotting, 20 µg of total protein per sample was separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4°C with the primary antibody (diluted 1:1,000). After washing, the membrane was probed with an HRP-conjugated secondary antibody (1:5,000 dilution) for 1 h at room temperature. Signal was developed using an enhanced chemiluminescence substrate and imaged with a chemiluminescence detection system. Coomassie blue staining Following SDS-PAGE, proteins were stained with 0.1% Coomassie Brilliant Blue R-250 (in 40% methanol and 10% acetic acid) for 1 h. Subsequently, the gels were destained in a solution of 5% ethanol and 10% acetic acid with multiple changes of the destaining solution until clear protein bands were visible against a transparent background. The stained gels were imaged using a standard white light scanner. The raw image was changed to grayscale, and the brightness and contrast were adjusted to better visualize protein bands. In vitro de-2‐hydroxyisobutyrylation and 2‐hydroxyisobutyrylation assay For in vitro de-2-hydroxyisobutyrylation assays, total protein lysates (20 µg) or purified proteins (1 µg) were incubated with recombinant CobB (2 µg) in reaction buffer (50 mM Tris‐HCl (pH 8.5), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol, 1 mM NAD⁺) at 37°C for 2 h. For in vitro 2‐hydroxyisobutyrylation assays, proteins were incubated with purified AcuA at 25°C for 16 h in reaction buffer (20 mM HEPES, 20 mM MgCl 2 , 20 mM KCl, 200 mM NaCl, pH 7.5). Control reactions were performed in the absence of enzyme or 2-hydroxyisobutyryl-CoA (Hib-CoA) (50 µM). All reactions were terminated by adding 5×Laemmli buffer and boiling. The modification states were subsequently analyzed by western blotting using a specific anti-2-hydroxyisobutyryllysine antibody. Molecular docking and dynamics simulation analysis The initial structure of AcuA was retrieved from the AlphaFold Database using Uniprot accession ID Q2G293 ( https://alphafold.ebi.ac.uk/entry/AF-Q2G293-F1 ). Structures for Acetyl-CoA (Ac-CoA) and 2-hydroxyisobutyryl-CoA (Hib-CoA) were obtained from the PubChem database. Wild-type AcuA and site-directed AcuA H139A mutant, by substituting the amino acid using PyMOL 3.1, were prepared in GROMACS 2025. Each system was solvated in a TIP3P water box (10 Å buffer) and neutralized with Na⁺/Cl⁻ to 0.15 M. Charmm36 force field was used for protein parameterization. Molecular dynamics simulations followed a standardized protocol: steepest-descent minimization (50,000 steps) to resolve steric clashes, 100 ps NVT equilibration (300 K, velocity-rescaling thermostat) for thermal stabilization, and 100 ps NPT equilibration (1 atm, Parrinello-Rahman barostat) for volume adjustment. Production MD ran 100 ns (NPT, 2 fs step) with LINCS (hydrogen bond constraints) and PME (long-range electrostatics). The major post-molecular dynamics conformations of proteins were analyzed in the 20-100ns molecular dynamics trajectory of wild-type AcuA and site-directed AcuA by using the gmx cluster tool. The structure for receptors (AcuA, AcuA W140A) was prepared with AutoDockTools 1.5.7 with the default setting. The conformation of Hib-CoA was prepared using Scrubber ( https://github.com/forlilab/molscrub ). Molecular docking was performed with AutoDock Vina using the following parameters: Grid center: X = -1, Y = -7, Z = 7, Grid box size: X = 29, Y = 29, Z = 29. The top ten lowest-binding-free-energy complexes were analyzed via PyMOL 3.1, with intermolecular interactions (hydrogen bonds, hydrophobic contacts) quantified to delineate critical AcuA-CoA binding residues and the top one lowest-binding-free-energy complexes was picked for visualization. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from 3mL S. aureus cultures using the Bacterial RNA Extraction Kit (R403-01) (Vazyme, Nanjing, China) following the manufacturer's instructions. RNA quality and concentration were determined by spectrophotometry. cDNA was synthesized from 1 µg of total RNA with the Hifair II 1st strand kit (Yeasen, Shanghai, China). Quantitative PCR was performed on Mini Option real-time PCR system (Bio-Rad) using the Hieff ® qPCR SYBR Green Master Mix (No Rox) (Yeasen, Shanghai, China). Relative expression levels were calculated using the 2^ (−ΔΔCt) method with 16S rRNA as the internal reference gene. All reactions were performed in triplicate. GST pull-down assay The GST pull-down assay was performed to investigate protein-protein interactions in vitro . The bait protein (GST-tagged AcuA) was incubated with purified prey protein or total protein in binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM DTT) for 2 h at 4°C with gentle rotation. Beads were washed five times with ice-cold binding buffer to remove non-specifically bound proteins. Bound proteins were eluted by boiling in 5× SDS-PAGE loading buffer and analyzed by immunoblotting using antibodies against the tags (anti-GST and anti-His antibodies) or separated by SDS-PAGE, or identified using mass spectrometry. Biofilm assay Biofilm formation was quantified using a standard crystal violet staining assay. S. aureus strains were cultured in CD medium in 24-well polystyrene plates at 37°C for 24 h. After incubation, the planktonic cells were removed, and the adhered biofilms were gently washed with phosphate-buffered saline (PBS), fixed with 99% methanol, and stained with 0.1% crystal violet for 15 min. The excess stain was rinsed off, and the bound dye was solubilized with 33% glacial acetic acid. The absorbance of the solubilized crystal violet was measured at 570 nm using a microplate reader. Each assay was performed with at least three biological replicates. Protein extraction and trypsin digestion S. aureus and its derivative strains were inoculated in 200 mL of CD medium in a five-hundred mL Erlenmeyer baffled flask under shaking at 200 rpm at 37°C. Cells at late logarithmic phase (OD 600 = 1.0) were harvested by centrifugation at 4°C at 12,000 g for 5 min. The cell pellets were washed twice with cold PBS and subsequently treated with proper lysostaphin for 30 min at 37°C to facilitate cell wall digestion. The harvested cells were resuspended in lysis buffer (8 M urea, 1% Triton X-100, 10 mM dithiothreitol (DTT), 1% protease inhibitor, 3 mM TSA, 50 mM NAM, and 2 mM EDTA) and sonicated on ice for 10 min. The cell debris was removed by centrifugation at 12,000 g and 4°C for 30 min, and the proteins were precipitated with 20% cold TCA at 4°C for 2 h. After centrifugation at 4°C for 20 min, the supernatant was discarded. The remaining protein was resuspended and washed three times with cold acetone. Finally, the target protein was redissolved in 8 M Urea, and the protein concentration was quantified with a BCA protein assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. For trypsin digestion, protein samples were reduced with 5 mM dithiothreitol for 1 h at 37°C and alkylated with 100 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. Proteins were washed five times with 100 mM TEAB to remove urea using ultrafiltration tubes. Trypsin was added at a 1:50 trypsin-to-protein mass ratio for digestion overnight, followed by another 4-h digestion at a 1:100 ratio. Immunoaffinity enrichment of Khib peptides For Khib-modified peptides enrichment, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 0.5% NP-40) were incubated overnight with drained pre-washed anti-2-hydroxyisobutyryllysine antibody-conjugated agarose beads (PTM Bio, China) at 4°C, with gentle rotation. After gently washing four times with NETN buffer and twice with double-distilled water, peptides bound to the beads were eluted with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined, vacuum-dried, and cleaned with C18 ZipTips (Millipore, Bedford, MA, USA) before LC-MS/MS analysis. HPLC-MS/MS analysis Lyophilized peptides were reconstituted in 0.1% formic acid and spiked with iRT calibration peptides (Biognosys, USA). LC-MS/MS analysis was performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific). MS Data acquisition parameters: MS1 Spectra: Acquired in the Orbitrap at a resolution of 60,000, with a scan range of 400–1500 m/z. Automatic gain control (AGC) target was set to 4 × 10 5 , and maximum injection time was 50 ms. MS2 Spectra (DIA): A total of 40 variable-width isolation windows were used to cover the MS1 mass range. MS2 spectra were acquired at a resolution of 30,000, with an AGC target of 5 × 10 5 , maximum injection time of 56 ms, and higher-energy collisional dissociation (HCD) collision energy set to 32%. Database search and data filtering criteria The raw MS/MS data acquired in data-independent acquisition (DIA) mode were analyzed using Spectronaut 19 (Biognosys) against the Staphylococcus aureus NCTC 8325 reference proteome (UniProt Proteome ID: UP000008816). Trypsin/P was specified as the protease, allowing up to two missed cleavages. The minimum peptide length was set to seven amino acids. Carbamidomethylation (C) was set as a fixed modification. Methionine oxidation, protein N-terminal acetylation, and 2-hydroxyisobutyrylation (Khib) were set as variable modifications. The mass tolerance was set to ± 10 ppm for precursor ions and ± 0.02 Da for fragment ions. The false discovery rate (FDR) for peptide-spectrum matches was estimated at < 1% using a target-decoy approach. To ensure high-confidence identification of Khib-modified sites, we applied additional filtering criteria: only peptides with a Spectronaut cross-run normalized score > 40 and a modification site localization probability > 0.99 were retained. The abundance of quantified Khib peptides was normalized to the total intensity of their corresponding proteins derived from the global proteomic profile. Bioinformatics Analysis KEGG and GO enrichment analyses were performed using the online service tool ( http://www.omicsolution.org/wu-kong-beta-linux/main/ ) and Cytoscape software (version 1.5.1). Using the online tool ( https://hiplot.com.cn/home/index.html ) for KEGG visualization analysis. Protein sequence alignment was conducted using the Clustal W server. Amino acid sequence motifs (seven amino acids upstream and downstream of the 2-hydroxyisobutyrylated lysine) were analyzed using pLogo 43 . The protein structures were visualized with PyMOL (v. 3.1) 44 . The structures of the AcuA and CodY mutants were simulated using Alphafold3 39 . Mouse pneumonia infection model 6-weeks-old BALB/c female mice were obtained from the Experimental Animal Department of Guangzhou Southern Medical University (Guangzhou, China) and housed for one week before experiments. All experiments adhered to institutional guidelines and were approved by the Animal Experiment Ethics Committee of Jinan University. For the pneumonia infection model, mice were anesthetized with a mixture of 2,2,2-tribromoethanol and 2-methyl-2-butanol. Cultured S. aureus (OD 600 = 0.8) strains were dissolved in 20 µL PBS solution and administered intranasally; mice receiving PBS alone served as negative controls. Twelve hours post-infection, treatment was initiated with intraperitoneal injections of ciprofloxacin (30 mg/kg) administered twice daily. Forty-eight hours later, mice were euthanized by cervical dislocation, and lung tissue homogenates were serially diluted with PBS and spread onto TSB plates to assess the bacterial load in the lungs. Electrophoresis migration shift assays (EMSA) The target regulatory regions of cap/ica operons were amplified by PCR. Then purified CodY variants were incubated with 30 ng DNA fragments in 20 µL of binding buffer containing 20 mM Tris-Cl [pH 8.0], 50 mM KCl, 2 mM MgCl₂, 5% [v/v] glycerol, 10 mM DTP, 10 mM each of valine, leucine, and isoleucine at room temperature for 30 min. Protein-DNA complexes were resolved on 8% native polyacrylamide gel in 0.5×TBE buffer at 100 V for 1 h in ice bath and transferred to a nylon membrane. Biotin-labeled DNA was detected using chemiluminescent substrate. Formaldehyde cross-linking Incubate wild-type or mutant CodY protein (1 µg) in 1× cross-linking buffer (100 mM KCl, 15 mM Tris-HCl, pH 7.5) at room temperature for 15 min, in a total volume of 20 µL. Then add formaldehyde to the reaction mixture to a final concentration of 1% (v/v), and incubate at room temperature for 20 min. Quench the formaldehyde-treated samples with 125 mM glycine and incubate at room temperature for 15 min. Then heat each sample for 10 min at 65°C in a reducing buffer containing 50 mM DTT, followed by western blot analysis using SDS-PAGE and specific antibodies. Statistical analysis All in vitro experiments were performed with at least two independent replicates to ensure reproducibility. Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were carried out using appropriate tests (e.g., Student’s t-test or one-way ANOVA), with significance levels defined as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****); “ns” denotes not significant. Declarations Data availability The raw proteomic data and search results have been deposited to the ProteomeXchange Consortium via the PRIDE 45 partner repository on November 14, 2025, with the dataset identifier PXD070748, and can be accessed with the reviewer account at https://www.ebi.ac.uk/pride using the username: [email protected] and the password: DGf5EliZ919c. Ethics statement All experimental protocols involving animals were reviewed, evaluated, and formally approved by the Ethics Committee for Animal Experiments of Jinan University (IACUC Approval No. 20260112-02). The entire study was strictly performed in accordance with the institutional guidelines for the care and use of laboratory animals, and all efforts were made to minimize the suffering of experimental animals. Acknowledgements We thank Professor Xue Ting (Anhui Agricultural University) for providing the knockout strains. This work was supported by the National Natural Science Foundation of China (22377035 and 21977037 to X.S), Guangdong National Science Foundation (2022A1515010674 and 2023A1515011750 to X.S.). International Atomic Energy Agency Coordinated Research Project (25076 to X.S.) Author information Authors are ranked in descending order of their contribution Authors and Affiliations MOE Key Laboratory of Tumor Molecular Biology and State Key Laboratory of Bioactive Molecules and Druggability Assessment, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China Yun Liu, Zhen Wang, Jiamin Qiu, Haiming Wu, Jiayi Wu, Tairan Zhong, Yundan Zheng, Nan Li, Yunpeng Yang, Zhenghua Sun, Qing-Yu He, Xuesong Sun Contributions The experiment was designed by Y.L and X.S., and performed by Y.L., Z.W., J.Q., H.W., J.W., T.Z., and Y.Z.; Clinical multidrug-resistant strains were collected by N.L. and Y.Y.; LC-MS analysis was conducted by Z.S.; The draft manuscript was written by Y.L. and critically revised by X.S., and Q.H. All authors have read and approved the final manuscript. Corresponding authors Correspondence to Qing-Yu He or Xuesong Sun. 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Staphylococcus aureus CodY negatively regulates virulence gene expression. J. Bacteriol. 190, 2257–2265 (2008). De Carvalho, C. C., Murray, I. P., Nguyen, H., Nguyen, T. & Cantu, D. C. Acyltransferase families that act on thioesters: Sequences, structures, and mechanisms. Proteins 92, 157–169 (2024). Li, J., Wang, T., Xia, J., Yao, W. & Huang, F. Enzymatic and nonenzymatic protein acetylations control glycolysis process in liver diseases. The FASEB J 33, 11640–11654 (2019). Hainzl, T., Bonde, M., Almqvist, F., Johansson, J. & Sauer-Eriksson, A. E. Structural insights into CodY activation and DNA recognition. Nucleic Acids Res. 51, 7631–7648 (2023). Levdikov, V. M. et al. Structure of the Branched-chain amino Acid and GTP-sensing global regulator, CodY, from Bacillus subtilis . J. Biol. Chem. 292, 2714–2728 (2017). Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024). Huang, S., Tang, D. & Dai, Y. Metabolic functions of lysine 2-hydroxyisobutyrylation. Cureus 12, e965 (2020). Huang, H. et al. p300-Mediated lysine 2-hydroxyisobutyrylation regulates glycolysis. Mol. Cell 70, 663–678.e6 (2018). Pohl, K. et al. CodY in Staphylococcus aureus : a regulatory link between metabolism and virulence gene expression. J. Bacteriol. 191, 2953–2963 (2009). O’Shea, J. P. et al. pLogo: a probabilistic approach to visualizing sequence motifs. Nat. Methods 10, 1211–1212 (2013). Seeliger, D. & De Groot, B. L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J. Comput.-Aided Mol. Des. 24, 417–422 (2010). Vizcaíno, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationnpj.docx GA.png Graphical abstract The AcuA–CodY 2-hydroxyisobutyrylation axis promotes biofilm-dependent fluoroquinolone resistance in S. aureus . AcuA, a newly identified 2-hydroxyisobutyrylation transferase, drives a CodY-centered regulatory axis that promotes biofilm-based fluoroquinolone resistance in S. aureus . AcuA-mediated 2-hydroxyisobutyrylation releases CodY repression of the icaADBC operon, stimulating biofilm formation and generating a dense matrix that limits antibiotic penetration. Loss of this modification restores CodY repression, reduces biofilm production, and diminishes fluoroquinolone resistance, revealing a key mechanism underlying antimicrobial evasion. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 Mar, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers invited by journal 21 Feb, 2026 Editor assigned by journal 16 Feb, 2026 Submission checks completed at journal 12 Feb, 2026 First submitted to journal 10 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8837885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":632089054,"identity":"d14de697-b56c-480e-9160-13b270cb0481","order_by":0,"name":"Yun Liu","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Liu","suffix":""},{"id":632089055,"identity":"a647eff4-6fa4-437c-9bbd-e12dcb327584","order_by":1,"name":"Zhen Wang","email":"","orcid":"","institution":"Jinan 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09:35:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2-hydroxyisobutyrylation positively regulates fluoroquinolone resistance in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Western blot analysis of lysine acylation levels (including acetylation, crotonylation and Khib) in whole-cell lysates of\u0026nbsp;\u003cem\u003eS. aureus\u003c/em\u003e NCTC 8325 and clinical strains. \u003cstrong\u003eb.\u003c/strong\u003e Detection of Khib modification levels in\u0026nbsp;\u003cem\u003eS. aureus\u003c/em\u003e NCTC 8325 at different growth phases (OD\u003csub\u003e600\u003c/sub\u003e = 0.4, 0.8, and 1.0) by western blot. \u003cstrong\u003ec.\u003c/strong\u003e Number of identified 2-hydroxyisobutyrylated proteins and sites in\u0026nbsp;\u003cem\u003eS. aureus\u003c/em\u003e. \u003cstrong\u003ed.\u003c/strong\u003e KEGG pathway enrichment analysis of identified proteins. The value of -log10 (Fisher’s test Q value) is shown. \u003cstrong\u003ee.\u003c/strong\u003e Intersections of acetylated, succinylated and 2-hydroxyisobutyrylated proteins in \u003cem\u003eS. aureus\u003c/em\u003e by Venn diagram. \u003cstrong\u003ef.\u003c/strong\u003e Comparison of Khib modification levels between WT and CIP-R strains. \u003cstrong\u003eg.\u003c/strong\u003e Enhancement of bacterial Khib modification levels by sub-MIC ciprofloxacin treatment (1/4 and 1/2 MIC). \u003cstrong\u003eh.\u003c/strong\u003e Effect of exogenous 2-hydroxyisobutyrate (hib) supplementation on Khib modification levels. \u003cstrong\u003ei.\u003c/strong\u003e Bacterial growth curves with or without hib supplementation. \u003cstrong\u003ej.\u003c/strong\u003e Bacterial ATP content following hib treatment. \u003cstrong\u003ek.\u003c/strong\u003e Time-Kill curve after external supplementation with hib. \u003cstrong\u003el.\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e de-2-hydroxyisobutyrylase activity of CobB assessed by western blot. \u003cstrong\u003em.\u003c/strong\u003e Western blot analysis of Khib modification levels in whole-cell lysates from \u003cem\u003ecobB\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e.\u0026nbsp; \u003cstrong\u003en.\u003c/strong\u003e Western blot analysis of Khib modification levels in whole-cell lysates from Δ\u003cem\u003ecobB\u003c/em\u003e. \u003cstrong\u003eo.\u003c/strong\u003e Effect of\u0026nbsp;\u003cem\u003ecobB\u003c/em\u003e\u0026nbsp;overexpression and exogenous hib on ciprofloxacin resistance in the CIP-R strain. In Figure j and k, n = 3, and Data are mean ± standard deviation (s. d), Unpaired student’s two-tailed t-test was applied to compare two experimental groups.\u0026nbsp; In Figure o, data shown are from one representative experiment (n = 3 technical replicates) out of two independent experiments performed. Statistical significance was defined as P\u0026nbsp;\u0026lt; 0.05 (*),\u0026nbsp;P\u0026nbsp;\u0026lt; 0.01 (**),\u0026nbsp;P\u0026nbsp;\u0026lt; 0.001 (***),\u0026nbsp;P\u0026nbsp;\u0026lt; 0.0001 (****).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/62b43455b8b0724b5cf0aee5.png"},{"id":108188620,"identity":"4eee0056-812e-4bbc-8c84-d3b93b66a54e","added_by":"auto","created_at":"2026-04-30 09:35:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous substrates for Khib in ciprofloxacin-resistant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eNumber of DHPs and sites in CIP-R versus WT strains. \u003cstrong\u003eb.\u003c/strong\u003e Scatter plot comparing the intensity of Khib sites between CIP-R and WT strains. \u003cstrong\u003ec.\u003c/strong\u003eVolcano plots for the statistical analysis of Khib proteins between WT and CIP-R strains. Differentially expressed proteins are defined as those with p \u0026lt; 0.05, and absolute fold change greater than 1.5 or less than 0.667. \u003cstrong\u003ed.\u003c/strong\u003e Statistical analysis of Khib sites per protein. \u0026nbsp;\u003cstrong\u003ee.\u003c/strong\u003e Column Chart showing the features of different amino acid residues around Khib sites in identified peptides. \u003cstrong\u003ef.\u003c/strong\u003e GO analysis for molecular function, cellular component and the biological processes of DHPs. \u003cstrong\u003eg.\u003c/strong\u003e KEGG categories of DHPs. Pathways associated with fluoroquinolone resistance are highlighted in blue. \u003cstrong\u003eh.\u003c/strong\u003e Visualization of the central carbon metabolism pathway in DHPs. Up-regulated and down-regulated DHPs are shown in red and blue, respectively. \u003cstrong\u003ei.\u003c/strong\u003e Comparison of intracellular ATP levels between CIP-R and WT strains. \u003cstrong\u003ej-k.\u003c/strong\u003e Relative MIC of WT (j) and CIP-R (k) strains with or without exogenous addition of arginine. \u003cstrong\u003el.\u003c/strong\u003e Protein-protein interaction network of 2-hydroxyisobutyrylated transcription factors (TFs). TFs with differential Khib modification levels are marked with orange stars. Gene names are according to \u003cem\u003eS. aureus\u003c/em\u003e NCTC 8325 strain (“00679” for \u003cem\u003eSAOUHSC_00679\u003c/em\u003e, etc.). In Figure i, n = 3, and Data are mean ± standard deviation (s. d), Unpaired student’s two-tailed t-test was applied to compare two experimental groups. In Figure j and k, Data shown are from one representative experiment (n = 3 technical replicates) out of two independent experiments performed. Statistical significance was defined as P \u0026lt; 0.05 (*), P \u0026lt; 0.01 (**), P \u0026lt; 0.001 (***), P \u0026lt; 0.0001 (****).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/89fa74323a376f1a719faeea.png"},{"id":108491386,"identity":"5ed8bca0-6ac2-411e-907a-c5aade67b2c9","added_by":"auto","created_at":"2026-05-05 09:53:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":356508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of AcuA as a lysine 2-hydroxyisobutyryltransferase in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eWestern blot analysis of Khib modification levels in cell lysates of \u003cem\u003eS. aureus\u003c/em\u003eoverexpressing specific potential 2-hydroxyisobutyryltransferase. Gene names are according to \u003cem\u003eS. aureus\u003c/em\u003e NCTC 8325 strain (“02653” for \u003cem\u003eSAOUHSC_02653\u003c/em\u003e, etc.). The WT strain harboring an empty plasmid (\u003cem\u003epCN51\u003c/em\u003e) served as a control. \u003cstrong\u003eb. \u003c/strong\u003eWestern blot analysis of Khib modification levels in whole-cell lysates of the CIP-R knockout strains (CIP-R Δ\u003cem\u003eyncA\u003c/em\u003e and CIP-R Δ\u003cem\u003eacuA\u003c/em\u003e) compared with those of the CIP-R strain. \u003cstrong\u003ec. \u003c/strong\u003eIn vitro 2-hydroxyisobutyrylation of \u003cem\u003eS. aureus\u003c/em\u003e proteins by purified AcuA. The whole proteins in \u003cem\u003eS. aureus \u003c/em\u003ewere\u003cem\u003e \u003c/em\u003etransferred to the PVDF membrane and incubated with purified AcuA. \u003cstrong\u003ed.\u003c/strong\u003e The binding affinity of AcuA for Hib-CoA was measured by fluorescence titration. The binding constant of AcuA with Hib-CoA was fitted using the Hill equation. \u003cstrong\u003ee. \u003c/strong\u003eWestern blot analysis of total protein acetylation in the CIP-R Δ\u003cem\u003eacuA\u003c/em\u003e strain. \u003cstrong\u003ef.\u003c/strong\u003e Molecular docking of Hib-CoA and Ac-CoA to the AcuA structure. The hydrogen bonds between Hib-CoA and AcuA were visualized using LigPlot+. \u003cstrong\u003eg. \u003c/strong\u003eMultiple sequence alignment of AcuA from different species by Clustal W and visualized using ESPript3. \u003cstrong\u003eh. \u003c/strong\u003eSDS-PAGE analysis of purified AcuA, H139A, and W140A. \u003cstrong\u003ei-j.\u003c/strong\u003e Binding affinity of AcuA H139A and W140A for Hib-CoA measured by fluorescence titration. \u003cstrong\u003ek.\u003c/strong\u003e Structures of AcuA and its mutants (H139A and W140A) obtained through Alphafold3 modeling. The amino acid residues at positions 139 and 140 are labeled in yellow and blue, respectively. \u003cstrong\u003el. \u003c/strong\u003eStructural features of AcuA and H139A obtained by MD simulations. Arrows highlight the hydrophobic pocket. The distances between Ser140 and Ile105, and between Leu155 and Gln109 in WT AcuA are shown with green dashed lines, while the distances between S140 and I105, and between L155 and Q109 in mutant H139A are shown with yellow dashed lines. \u003cstrong\u003em. \u003c/strong\u003eWestern blot detection of Khib modification levels in whole-cell lysates from strains \u003cem\u003eeWT\u003c/em\u003e, \u003cem\u003eeH139A\u003c/em\u003e, and \u003cem\u003eeW140A\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/847374394abf901a6146f254.png"},{"id":108491522,"identity":"77d15eeb-e20c-42e9-91fd-ba97a4a3d036","added_by":"auto","created_at":"2026-05-05 09:54:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":203788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcuA promotes bacterial growth and antibiotic resistance\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eGrowth curves of CIP-R and CIP-R Δ\u003cem\u003eacuA\u003c/em\u003e strains in CD medium. Optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) indicates bacterial growth. \u003cstrong\u003eb. \u003c/strong\u003eMICs of ciprofloxacin for CIP-R and CIP-R Δ\u003cem\u003eacuA\u003c/em\u003e strains. \u003cstrong\u003ec.\u003c/strong\u003e MICs of ciprofloxacin for WT and WT Δ\u003cem\u003eacuA\u003c/em\u003e strains. \u003cstrong\u003ed.\u003c/strong\u003e MICs of ciprofloxacin for WT and WT Δ\u003cem\u003eacuA \u003c/em\u003estrains during experimental evolution. \u003cstrong\u003ee.\u003c/strong\u003e MICs of fluoroquinolones for vector and\u003cem\u003e acuA\u003c/em\u003e\u0026nbsp;overexpression strains. \u003cstrong\u003ef.\u003c/strong\u003e Coomassie brilliant blue staining of GST-AcuA pull-down assay showing potential interacting proteins. \u003cstrong\u003eg.\u003c/strong\u003e KEGG enrichment analysis of proteins identified in the GST-AcuA pull-down experiment. \u003cstrong\u003eh.\u003c/strong\u003e Intersections of pull-down proteins, transcription factors (TFs) and differentially 2-hydroxyisobutyrylated transcription factors (DH-TFs) by Venn diagram. \u003cstrong\u003ei.\u003c/strong\u003e Downregulated proteins related to biofilm formation in CIP-R Δ\u003cem\u003eacuA\u003c/em\u003e strains. \u003cstrong\u003ej. \u003c/strong\u003eThe transcript levels of \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaB\u003c/em\u003e in the CIP-R and CIP-R Δ\u003cem\u003eacuA\u003c/em\u003e strains. \u003cstrong\u003ek.\u003c/strong\u003e Quantification of biofilm formation in \u003cem\u003eacuA\u003c/em\u003e knockout or overexpression strains using 1 % crystal violet staining. Absorbance at 570 nm was measured to assess biofilm density. \u003cstrong\u003el.\u003c/strong\u003e The transcript levels of \u003cem\u003eicaA\u003c/em\u003e,\u003cem\u003e icaB \u003c/em\u003eand \u003cem\u003eicaR\u003c/em\u003e in the WT strain cultured in CD medium or CD medium supplemented with hib. \u003cstrong\u003em.\u003c/strong\u003e Quantification of biofilm formation in the WT strain cultured in CD medium or CD medium supplemented with hib. \u003cstrong\u003en.\u003c/strong\u003e The transcript levels of \u003cem\u003eicaA\u003c/em\u003e and\u003cem\u003e icaB\u003c/em\u003e in WT and MDR strains. \u003cstrong\u003eo.\u003c/strong\u003e Quantification of biofilm formation in WT and MDR strains.\u003cstrong\u003e p.\u003c/strong\u003e Western blot analysis showing the AcuA contents in CIP-R and MDR strains. In Figure n and o, n = 4, and Data are mean ± standard deviation (s. d). One-way ANOVA with Dunnett's multiple comparisons test. In Figure j, k, l, and m, Unpaired student’s two-tailed t-test was applied to compare two experimental groups. In Figure b, c, and e, Data shown are from one representative experiment (n = 3 technical replicates) out of two independent experiments performed. Statistical significance was defined as P\u0026nbsp;\u0026lt; 0.05 (*),\u0026nbsp;P\u0026nbsp;\u0026lt; 0.01 (**),\u0026nbsp;P\u0026nbsp;\u0026lt; 0.001 (***),\u0026nbsp;P\u0026nbsp;\u0026lt; 0.0001 (****).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/008b5c49e22a390eae199f0e.png"},{"id":108188621,"identity":"31c7f593-921e-43be-89e3-d292985aaa9b","added_by":"auto","created_at":"2026-04-30 09:35:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcuA enhances fluoroquinolone resistance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by regulating Khib at Lys16 and Lys223 of CodY.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eIncreased Khib modification levels at sites Lys6, Lys16, and Lys223 of the transcription factor CodY in the CIP-R strain. \u003cstrong\u003eb.\u003c/strong\u003e Western blot analysis of CodY protein levels in WT, CIP-R, and MDR strains. \u003cstrong\u003ec. \u003c/strong\u003eDirect AcuA-CodY interaction demonstrated by GST pull-down and western blot analysis. \u003cstrong\u003ed.\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e 2-hydroxyisobutyrylation of CodY by AcuA. \u0026nbsp;\u003cstrong\u003ee.\u003c/strong\u003eQuantification of the grayscale values from panel (d) using ImageJ. \u003cstrong\u003ef-g.\u003c/strong\u003eWestern blot analysis of Khib modification levels in WT and mutant CodY catalyzed by AcuA. \u003cstrong\u003eh-i.\u003c/strong\u003e MICs of fluoroquinolones for \u003cem\u003eeK16Q\u003c/em\u003e, \u003cem\u003eeK16R\u003c/em\u003e, \u003cem\u003eeK223Q,\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e mutant strains. \u003cstrong\u003ej.\u003c/strong\u003e Kaplan–Meier survival rate of mice infected with \u003cem\u003eeK223Q\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e strains after treatment with ciprofloxacin (30 mg/kg) (n = 10). \u003cstrong\u003ek.\u003c/strong\u003e Pulmonary bacterial load in mice infected with \u003cem\u003eeK223Q\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e strains after treatment with ciprofloxacin (30 mg/kg) (n = 3). \u003cstrong\u003el.\u003c/strong\u003e The transcript levels of \u003cem\u003eicaA \u003c/em\u003eand \u003cem\u003eicaB\u003c/em\u003e between the mutant strains. \u003cstrong\u003em.\u003c/strong\u003e The biofilm-forming ability of \u003cem\u003eeK16Q\u003c/em\u003e, \u003cem\u003eeK16R\u003c/em\u003e, \u003cem\u003eeK223Q\u003c/em\u003e, and \u003cem\u003eeK223R\u003c/em\u003emutant strains. In Figure e, n = 3, and Data are mean ± standard deviation (s. d). One-way ANOVA with Dunnett's multiple comparisons test. In Figure k, l and m, Unpaired student’s two-tailed t-test was applied to compare two experimental groups. In Figure h and i, Data shown are from one representative experiment (n = 3 technical replicates) out of two independent experiments performed. Statistical significance was defined as P \u0026lt; 0.05 (*), P \u0026lt; 0.01 (**), P \u0026lt; 0.001 (***), P \u0026lt; 0.0001 (****).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/f4e81b4833cfa8c205f1f203.png"},{"id":108491318,"identity":"0b9151f5-e039-46cd-bf71-c6644a05edea","added_by":"auto","created_at":"2026-05-05 09:53:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":392259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2-hydroxyisobutyrylationregulates the DNA-binding ability of CodY.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eThermal stability of CodY and mutants (K16Q and K223Q) analyzed via circular dichroism (CD). \u003cstrong\u003eb. \u003c/strong\u003eLocation of 2-hydroxyisobutyrylated residues Lys16 and Lys223 at the CodY dimer interface. \u003cstrong\u003ec.\u003c/strong\u003e\u003cem\u003e In vitro\u003c/em\u003e cross-linking and immunoblot analyses to monitor dimerization of CodY and mutant proteins. \u003cstrong\u003ed. \u003c/strong\u003eSteric clashes restrict the conversion of ligand-free monomeric into dimeric structures. The conformation of the H1-H2 loop in the monomer structure (green) causes steric conflict with both the linker helix (LH) and the S3-S4 loop of protomer B within the dimeric structure (yellow). \u003cstrong\u003ee.\u003c/strong\u003e Shows how the sterically clash was disrupted by the K16Q mutation. Lys16 is labeled with red. \u003cstrong\u003ef-g.\u003c/strong\u003e Comparison of hydrogen-bonding interactions at the CodY dimer interface. (f) Wild-type CodY. (g) K223Q mutant. The mutation to glutamine reshapes the interface, resulting in a strengthened hydrogen-bonding network and enhanced dimer stability. \u003cstrong\u003eh-i. \u003c/strong\u003eEMSA experiments showed the impact of Khib on CodY binding to P\u003cem\u003eicaB\u003c/em\u003e/P\u003cem\u003ecap\u003c/em\u003e (‘P’ represents promoter). Different concentrations of purified CodY/CodY mutants were incubated with P\u003cem\u003eicaB\u003c/em\u003e/P\u003cem\u003ecap\u003c/em\u003e, and CodY variants differed greatly in the binding affinity with P\u003cem\u003eicaB\u003c/em\u003e/P\u003cem\u003ecap\u003c/em\u003eat the same concentration. \u003cstrong\u003ej. \u003c/strong\u003eConformational changes in the DBD domain of CodY caused by K16Q and K223Q. \u003cstrong\u003ek. \u003c/strong\u003eThe surface electrostatic potential of CodY, K16Q, and K223Q were analyzed using PyMOL.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/f07a4947c11821c2847d3b27.png"},{"id":108495645,"identity":"52a81715-4ab0-41d2-8d29-82676cf80bc9","added_by":"auto","created_at":"2026-05-05 10:10:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2360163,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/a49ab44e-31c3-450f-97f6-783c39ec3393.pdf"},{"id":108188618,"identity":"ab55ce91-78c2-49c6-ae8f-c68aa46deb18","added_by":"auto","created_at":"2026-04-30 09:35:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6910082,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationnpj.docx","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/a2e22b4a0b359f69b0090b54.docx"},{"id":108491339,"identity":"3603cb60-7101-4d3a-bcfe-2fc3f163a930","added_by":"auto","created_at":"2026-05-05 09:53:22","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":185297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe AcuA–CodY 2-hydroxyisobutyrylation axis promotes biofilm-dependent fluoroquinolone resistance in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e AcuA, a newly identified 2-hydroxyisobutyrylation transferase, drives a CodY-centered regulatory axis that promotes biofilm-based fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e. AcuA-mediated 2-hydroxyisobutyrylation releases CodY repression of the \u003cem\u003eicaADBC \u003c/em\u003eoperon, stimulating biofilm formation and generating a dense matrix that limits antibiotic penetration. Loss of this modification restores CodY repression, reduces biofilm production, and diminishes fluoroquinolone resistance, revealing a key mechanism underlying antimicrobial evasion.\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-8837885/v1/cad728987073a4df3f054f39.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in Staphylococcus aureus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe rapid emergence and global spread of antimicrobial resistance (AMR) represent one of the most critical challenges to modern public health\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the myriad of resistant pathogens, \u003cem\u003eS. aureus\u003c/em\u003e stands out due to its high prevalence and its propensity to develop multidrug resistance, particularly to fluoroquinolones, a broad-spectrum antibiotic that have shown activity against \u003cem\u003eS. aureus\u003c/em\u003e, including Methicillin-Resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e is not only common but also often coupled with multidrug resistance, severely limiting therapeutic options and leading to adverse clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Understanding the intricate mechanisms underpinning \u003cem\u003eS. aureus\u003c/em\u003e resistance is thus imperative for devising effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eTraditionally, research on fluoroquinolone resistance has focused on genetic endpoints, the acquisition of mutations in drug target genes (e.g., \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e, \u003cem\u003egrlA\u003c/em\u003e, \u003cem\u003egrlB\u003c/em\u003e) or the horizontal transfer of resistance determinants\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These alterations provide a stable, heritable advantage. However, the emergence of such mutations is a stochastic process that requires time and population expansion under selective pressure. A critical, yet less explored, question is: how bacterial populations survive the initial antibiotic assault to reach the necessary population size and time for favorable mutations to arise and become fixed. The answer likely lies in rapid, non-genetic adaptive strategies that act as a crucial first line of defense.\u003c/p\u003e \u003cp\u003eThis immediate survival response is orchestrated through dynamic metabolic reprogramming and fast-acting post-translational modifications (PTMs). Upon antibiotic exposure, bacteria must rewire their metabolism to allocate energy and resources toward repair, detoxification, and stress tolerance mechanisms. This metabolic shift is not merely a passive consequence but an active defense, and its precise execution is often regulated by PTMs. PTMs, such as phosphorylation, acetylation, succinylation, and lactylation, enable swift and reversible modulation of protein function, influencing enzyme activities, signaling pathways, and gene expression without altering the genome\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. They serve as a real-time interface between the metabolic state and the functional output of the cell, allowing bacteria to fine-tune their physiology to withstand transient stress. For instance, it was discovered that host-derived lactate can enhance the virulence of human pathogen \u003cem\u003eS. aureus\u003c/em\u003e by promoting the lactylation of alpha-toxin\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Concurrently, Yang Yi et al demonstrated that succinylation negatively regulates cell wall synthesis and vancomycin tolerance in vancomycin-intermediate \u003cem\u003eS. aureus\u003c/em\u003e (VISA)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, a groundbreaking study identified a metabolism-dependent succinylation mechanism that governs resource allocation for antibiotic resistance\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It demonstrated that bacteria could undergo metabolic reprogramming to support resistance mechanisms, and that succinylation of triosephosphate isomerase (TPI) and transcription factors (CpxR, PdhR) could crucially modulate metabolic flux and eventually influence resistance phenotypes. Recently, our research group discovered that crotonoylation positively regulates polymyxin resistance in \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) while negatively regulating virulence in \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These findings compellingly suggest that PTMs serve as a sophisticated regulatory layer enabling bacterial adaptation. We hypothesize that these rapid, PTM-driven adaptations are essential for maintaining cell viability during the critical window before the consolidation of genetic resistance, thereby potentially facilitating the eventual emergence and selection of high-level, mutation-driven resistance.\u003c/p\u003e \u003cp\u003eLysine 2-hydroxyisobutyrylation (Khib) is an evolutionarily conserved PTM, first identified on lysine residues of human and mouse histones\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Khib plays critical regulatory roles across both eukaryotic and prokaryotic organisms. Functionally, Khib mediates chromatin remodeling, fine-tunes metabolic enzyme activity, and participates in stress response pathways\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Insights into the functions of PTMs often stem from the identification of catalytic enzymes responsible for the addition or removal of PTMs. Khib is dynamically controlled by specific \u0026ldquo;writer\u0026rdquo; and \u0026ldquo;eraser\u0026rdquo; enzymes. The enzyme responsible for adding the 2-hydroxyisobutyryl group, known as a 2-hydroxyisobutyryltransferase (\u0026ldquo;writer\u0026rdquo;), has been identified in eukaryotic cells as p300, Tip60, and Esa1p. Histone deacetylases (HDACs), specifically HDAC2 and HDAC3, were the major enzymes that remove the 2-hydroxyisobutyryl group (\u0026ldquo;eraser\u0026rdquo;) \u003csup\u003e18\u003c/sup\u003e. In prokaryotes, the tRNA(Met) cytidine acetyltransferase TmcA from \u003cem\u003eE. coli\u003c/em\u003e has been shown to possess lysine 2-hydroxyisobutyryltransferase activity, while CobB functions as the core de-2-hydroxyisobutyrylase\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In microorganisms, Khib has emerged as a key modulator of virulence, stress adaptation, and host-pathogen interactions. TmcA-mediated Khib modification of the histone-like protein H-NS at Lys121 enhances acid resistance in \u003cem\u003eE. coli\u003c/em\u003e by promoting the transcription of acid stress response genes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Similarly, in fungal pathogens like \u003cem\u003eUstilaginoidea virens\u003c/em\u003e, Khib modification of the MAP kinase UvSlt2 strengthens its substrate-binding capacity and bacterial virulence\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Notably, CobB can precisely regulate the activity of enolase (Eno) by dual modulation of its K343hib and K326ac modification states, thereby directly influencing bacterial growth\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, no studies have yet reported how Khib affects bacterial fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e, and the specific enzymatic machinery responsible for Khib in this pathogen has yet to be identified.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of Khib in regulating fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e. We first identified a 2-hydroxyisobutyryltransferase, AcuA, in \u003cem\u003eS. aureus\u003c/em\u003e and characterized its 2-hydroxyisobutyryltransferase properties and functions through a series of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. We found that AcuA-mediated 2-hydroxyisobutyrylation promotes fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e by regulating the dimerization and DNA-binding activity of the transcription regulator CodY. Overall, this research reveals how \u003cem\u003eS. aureus\u003c/em\u003e exploits Khib to survive antibiotic challenge and may open new avenues for developing novel adjuvant therapies targeting Khib machinery to counteract antibiotic resistance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2-hydroxyisobutyrylation Enhances Fluoroquinolones Resistance in\u003c/strong\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe global spread of antibiotic resistance has become a major challenge in clinical anti-infective therapy. Recent studies indicate that Khib has emerged as a key regulator of bacterial physiology, but its role in regulating bacterial resistance is less characterized. We detected differences in PTMs (acetylation, crotonylation and Khib) between wild-type (WT) \u003cem\u003eS. aureus\u003c/em\u003e NCTC 8325 and clinical multidrug-resistant (MDR) strains (Table S1). Western blot analysis with pan-antibodies for three PTMs revealed that Khib modification levels in MDR strains were significantly different from those in sensitive bacteria, encompassing proteins across a broad molecular weight range, while the other two modifications exhibited relatively minor changes (Fig.\u0026nbsp;1a). This finding suggests that Khib may be involved in regulating drug resistance in \u003cem\u003eS. aureus\u003c/em\u003e. To investigate the contribution of Khib to bacterial resistance, we first profiled the global proteomic landscape of Khib in \u003cem\u003eS. aureus\u003c/em\u003e across different growth phases (lag phase, logarithmic phase, and late logarithmic phase). Immunoblotting analysis showed that Khib modification levels increased progressively during bacterial growth, with the most pronounced modification observed in the late logarithmic phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0) (Fig.\u0026nbsp;1b). This dynamic change suggests a potential regulatory role for Khib in the transition between primary and secondary metabolism in \u003cem\u003eS. aureus\u003c/em\u003e. Subsequent Khib proteomic analysis revealed that 3,485 lysine residues across 1,275 proteins in logarithmic-phase \u003cem\u003eS. aureus\u003c/em\u003e were 2-hydroxyisobutyrylated (Fig.\u0026nbsp;1c). Gene Ontology (GO) analysis unveiled that these proteins are primarily enriched in intracellular, cytoplasm, and cell membrane, and are important for biological processes like translation, biosynthesis, gene expression, and stress response. Most molecular functions of these proteins are involved in transport and molecular bindings (Fig. S1a). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis linked these proteins to metabolic pathways, TCA cycle, aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis pathway, pentose phosphate pathway, and ribosome (Fig.\u0026nbsp;1d). Previous studies have demonstrated crosstalk between different PTMs, such as the ability of many lysines to undergo both crotonylation and acetylation. Considering that crosstalk between different PTMs may affect resistance, we compared Khib with previously reported PTMs in \u003cem\u003eS. aureus\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e. We found that most 2-hydroxyisobutyrylated proteins were also subject to acetylation (Kac) and succinylation (Ksucc), which have been demonstrated to be involved in regulating \u003cem\u003eS. aureus\u003c/em\u003e resistance to erythromycin and vancomycin, respectively\u003csup\u003e10,23\u003c/sup\u003e. However, approximately 57% of Khib protein did not overlap with Kac and Ksucc proteins (Fig.\u0026nbsp;1e). This indicates that Khib possesses a distinct regulatory mechanism independent of Kac and Ksucc, supporting the possibility of its specific involvement in regulating the resistance pathway.\u003c/p\u003e\n\u003cp\u003eEarly studies showed that succinylation of cell wall biosynthesis-related protein MurA at Lys69 and Lys191 significantly reduces its activity, thereby decreasing cell wall thickness and rendering the vancomycin-resistant strain more susceptible to antibiotic\u003csup\u003e10\u003c/sup\u003e. It is interesting to note that we also identified 2-hydroxyisobutyrylation at Lys69 of MurA, suggesting that Khib may play a regulatory role in \u003cem\u003eS. aureus\u003c/em\u003e resistance. Therefore, we further investigated the relationship between Khib and bacterial resistance. To eliminate interference from genetic heterogeneity in clinical strains, we constructed a ciprofloxacin-resistant (CIP-R, MIC\u0026thinsp;=\u0026thinsp;120 \u0026micro;g/mL) strain through continuous passage culture under pressure stress at 1/2 MIC concentration. Subsequently, we examined the difference in Khib modification levels between the WT and CIP-R. As shown in Fig.\u0026nbsp;1f, significantly elevated Khib modification levels were detected in CIP-R. To investigate whether this elevated Khib level is linked to antibiotic stress, we exposed the WT strain to sub-MIC concentrations of ciprofloxacin and monitored Khib dynamics. Notably, this treatment also led to a significant increase in global Khib modification levels (Fig.\u0026nbsp;1g). These results demonstrate that heightened Khib levels are a conserved feature associated with both acquired resistance (CIP-R strain) and acute antibiotic challenge. The rapid upregulation of Khib upon ciprofloxacin exposure suggests a close association between fluoroquinolone-induced stress and this modification, further implicating Khib in the bacterial adaptation to drug pressure.\u003c/p\u003e\n\u003cp\u003eTo further examine the impact of Khib on \u003cem\u003eS. aureus\u003c/em\u003e growth and drug resistance, we supplemented the culture medium with 2-hydroxyisobutyrate (hib), a metabolic precursor of 2-hydroxyisobutyryl-CoA (Hib-CoA), which serves as the substrate for enzymatic 2-hydroxyisobutyrylation\u003csup\u003e14\u003c/sup\u003e. Western blot analysis showed that 2-hydroxyisobutyrate supplementation significantly increased Khib modification levels in \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;1h). Growth curve analysis revealed that treatment with 2-hydroxyisobutyrate resulted in a slight inhibition on bacterial growth (Fig.\u0026nbsp;1i). External supplementation with hib reduced ATP levels in both sensitive and resistant \u003cem\u003eS. aureus\u003c/em\u003e, thereby markedly increasing their tolerance to fluoroquinolones (ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin) (Fig.\u0026nbsp;1j, k), indicating that bacteria lower energy expenditure to support resistance ability.\u003c/p\u003e\n\u003cp\u003eThe NAD\u003csup\u003e+\u003c/sup\u003e-dependent deacetylase CobB from \u003cem\u003eProteus mirabilis\u003c/em\u003e (\u003cem\u003eP. mirabilis\u003c/em\u003e) and \u003cem\u003eE. coli\u003c/em\u003e exhibits de-2-hydroxyisobutyrylation activity\u003csup\u003e20\u003c/sup\u003e. Through homology comparison analysis, we found that the CobB from \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eP. mirabilis\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, despite their pronounced sequence divergence (20% similarity), exhibit a conserved tertiary structure (Fig. S1b). Based on this structural conservation, we hypothesized that CobB might also mediate de-2-hydroxyisobutyrylation in \u003cem\u003eS. aureus\u003c/em\u003e. To test this, we performed both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e functional assays. \u003cem\u003eIn vitro\u003c/em\u003e experiments showed that purified CobB significantly reduced Khib modification levels when incubated with whole-cell protein extracts (Fig.\u0026nbsp;1l). Consistent with this phenomenon, overexpression of CobB in \u003cem\u003eS. aureus\u003c/em\u003e significantly reduced global Khib modification levels, whereas deletion of the \u003cem\u003ecobB\u003c/em\u003e gene resulted in their pronounced accumulation (Fig.\u0026nbsp;1m, n). Collectively, these results demonstrate that CobB functions as a de-2-hydroxyisobutyrylase in \u003cem\u003eS. aureus\u003c/em\u003e. We next investigated the effect of CobB on drug resistance in \u003cem\u003eS. aureus.\u003c/em\u003e Deletion of \u003cem\u003ecobB\u003c/em\u003e did not affect the growth of the WT strain (data not shown). Unexpectedly, \u003cem\u003ecobB\u003c/em\u003e deletion did not further enhance WT strain resistance to ciprofloxacin, maybe due to its ability to a deacetylase, such as deacetylation and desuccinylation. We then overexpressed \u003cem\u003ecobB\u003c/em\u003e in CIP-R to further explore the role of CobB in antibiotic resistance. In comparison to the CIP-R, the \u003cem\u003ecobB\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e strain showed significantly reduced resistance to ciprofloxacin, which was partially restored with the addition of additional 2-hydroxyisobutyrate (Fig.\u0026nbsp;1o). These combined findings highlight that Khib regulates various metabolic pathways in \u003cem\u003eS. aureus\u003c/em\u003e and is closely associated with fluoroquinolone resistance.\u003c/p\u003e\n\u003cdiv\u003e\n\u003ch2\u003eComparative 2-hydroxyisobutyryl Proteomics Reveals Critical Proteins Linked to Fluoroquinolone Resistance\u003c/h2\u003e\n\u003cp\u003eTo gain deeper insights into the connection between Khib and bacterial antibiotic resistance, we compared the differentially expressed 2-hydroxyisobutyrylated proteome between WT and CIP-R strains through antibody enrichment combined with DIA-MS analysis. In the CIP-R group, 1,339 Khib-modified proteins were identified across three biological replicates, with 9,366 Khib modification sites detected which is similar to the number in WT strain (Fig.\u0026nbsp;2a). Global profiling of all identified Khib sites revealed a higher overall modification level in the CIP-R strain compared to the WT (Fig.\u0026nbsp;2b). We collectively identified 461 differentially 2-hydroxyisobutyrylated proteins (DHPs), of which 299 were upregulated and 162 were downregulated in CIP-R (fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), corresponding to 357 and 189 Khib sites, respectively (Fig.\u0026nbsp;2c). Furthermore, statistical analysis indicated that most proteins contained 1\u0026ndash;10 2-hydroxyisobutyryl sites (Fig.\u0026nbsp;2d). To determine the amino acids preferences of Khib, the Motif-x tool was used to identify conserved amino acid sequence motifs within a\u0026thinsp;\u0026plusmn;\u0026thinsp;7-residue window surrounding each modified lysine. A total of 21 distinct motifs were identified (Fig.\u0026nbsp;2e, Fig. S2). Analysis of these motifs revealed a marked preference for positively charged side chain amino acids, arginine (R) and lysine (K) surrounding the modified lysine residues (Fig.\u0026nbsp;2e), which appears to be a unique feature of \u003cem\u003eS. aureus\u003c/em\u003e\u003csup\u003e24\u003c/sup\u003e. This enrichment suggests that the change of local electrostatic environment may facilitate acyl group transfer by promoting the interaction between lysine residues and negatively charged acyl-CoA donors, thereby favoring 2-hydroxyisobutyrylation.\u003c/p\u003e\n\u003cp\u003eNext, GO enrichment analysis was performed to determine the potential functions of all DHPs. In the molecular function category, DHPs were mainly associated with the catalytic activity, structural constituent of ribosome, RNA binding, and small molecule binding, indicating that Khib influences proteins translation and function (Fig.\u0026nbsp;2f). Notably, most of the 2-hydroxyisobutyrylated proteins were annotated to the ribosome ontology in the cellular component category (Fig.\u0026nbsp;2f). This observation is consistent with the previously characterized substrate preference of Khib modification: the consensus sequence flanking Khib sites is typically enriched in positively charged basic amino acids (e.g., arginine and lysine). As ribosomal proteins inherently contain a high abundance of these basic residues to maintain their structural stability and interaction with negatively charged rRNA, their enrichment as Khib targets is not surprising but rather reflects the intrinsic specificity of the Khib modification system. Furthermore, biological process classification revealed that the 2-hydroxyisobutyrylated proteins were mainly enriched in pathways of macromolecule/protein/peptide metabolic processes, gene expression, translation, and stress response (Fig.\u0026nbsp;2f). KEGG enrichment analysis showed the biosynthesis of secondary metabolites function exhibited the most significant alteration among 2-hydroxyisobutyrylated proteins, followed by biosynthesis of cofactor and carbon metabolisms. Notably, numerous known essential pathways involved in fluoroquinolone resistance are enriched in DHPs, including ABC transporters, two-component system, mismatch repair, quorum sensing, and oxidative phosphorylation (Fig.\u0026nbsp;2g). PTMs regulate energy-generating metabolic pathways and represent a common strategy for bacteria to develop resistance to multiple antibiotics. For instance, the primary enrichment pathway for differentially crotonylated proteins in polymyxin-resistant \u003cem\u003eE. coli\u003c/em\u003e is considered to be metabolic processes\u003csup\u003e13\u003c/sup\u003e. A previous study on three kinds of antibiotic-resistant \u003cem\u003eE. coli\u003c/em\u003e strains indicates that protein acetylation plays a common role in negatively regulating bacterial metabolism, thereby contributing to drug resistance\u003csup\u003e25\u003c/sup\u003e. Similarly, visualization of central carbon metabolism revealed an overall decline in 2-hydroxyisobutyrylation of proteins involved in bacterial energy metabolism (Fig.\u0026nbsp;2h). A decrease in ATP level is also observed in CIP-R (Fig.\u0026nbsp;2i). What is more, previous studies have demonstrated that restriction of arginine induces biofilm formation in \u003cem\u003eS. aureus\u003c/em\u003e, thereby conferring resistance to delafloxacin\u003csup\u003e26\u003c/sup\u003e. Visualization of arginine metabolic flux also revealed decreased Khib modification levels in several other enzymes responsible for arginine metabolism, such as ArgG, ArgH, ArcA1, and ArcA2 (Fig.\u0026nbsp;2h). External supplementation with arginine reduced tolerance to ciprofloxacin in both sensitive and resistant \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;2j, k). These combined results further suggest that Khib may regulate antibiotic resistance by modifying substrate proteins.\u003c/p\u003e\n\u003cp\u003eBacteria sense environmental changes through transcription factors and respond by reprogramming gene expression\u003csup\u003e27\u003c/sup\u003e. Recent studies have revealed that Khib controls transcriptional activity through specific targeting of histones and chromatin-associated proteins\u003csup\u003e16,28\u003c/sup\u003e. In this study, we also identified 85 transcription factors modified by Khib in \u003cem\u003eS. aureus\u003c/em\u003e. Among these proteins, 24 exhibited differential Khib modification levels in CIP-R, including key regulators such as CodY, BirA, NreC, SarA, and SarX (Fig.\u0026nbsp;2l, Table S2). It is worth noting that CodY is a global transcriptional regulator controlling metabolism and virulence of \u003cem\u003eS. aureus\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e. These findings suggest the presence of a Khib-driven transcriptional network in \u003cem\u003eS. aureus\u003c/em\u003e that mediates adaptive responses to antibiotic stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAcuA Functions as a Novel 2-hydroxyisobutyryltransferase\u003c/h3\u003e\n\u003cp\u003eTo further reveal the regulatory mechanism of Khib on bacterial resistance, it is crucial to identify the 2-hydroxyisobutyryltransferase(s) and elucidate their regulatory networks and key substates. At present, the tRNA (Met) cytidine acetyltransferase TmcA in \u003cem\u003eE. coli\u003c/em\u003e, the histone acetyltransferase Ngg1 in \u003cem\u003eAspergillus flavus\u003c/em\u003e, and the histone acetyltransferase p300 in mammalian cells have been reported to act as 2-hydroxyisobutyryltransferases to perform a diverse range of cellular functions\u003csup\u003e19,30\u003c/sup\u003e. However, \u003cem\u003eS. aureus\u003c/em\u003e lacks homologous proteins to these transferases. Given that GCN5-related \u003cem\u003eN\u003c/em\u003e-acetyltransferases (GNATs) family proteins exhibit catalytic activities toward diverse lysine modifications (e.g., TmcA from \u003cem\u003eE. coli\u003c/em\u003e)\u003csup\u003e8,13\u003c/sup\u003e. So, we reasonably hypothesized that some GNAT proteins function as 2-hydroxyisobutyryltransferase mediating lysin 2-hydroxyisobutyrylation in \u003cem\u003eS. aureus\u003c/em\u003e. To test this hypothesis, we screened the \u003cem\u003eS. aureus\u003c/em\u003e genome for GNAT-encoding genes potentially involved in lysine 2-hydroxyisobutyrylation. Ten candidate genes were overexpressed in \u003cem\u003eS. aureus\u003c/em\u003e, and their effects on global 2-hydroxyisobutyrylation patterns were subsequently analyzed. This analysis identified two genes, \u003cem\u003eacuA\u003c/em\u003e and \u003cem\u003eyncA\u003c/em\u003e, that influence 2-hydroxyisobutyrylation, with \u003cem\u003eacuA\u003c/em\u003e showing the stronger effect, whereas overexpression of the other candidates caused no substantial changes (Fig.\u0026nbsp;3a, Fig. S3a).\u003c/p\u003e\n\u003cp\u003eWe then generated single-gene deletion mutants of both genes in wild-type \u003cem\u003eS. aureus\u003c/em\u003e and analyzed their impact on 2-hydroxyisobutyrylation patterns. However, we did not detect significant such effects (Fig. S3b). We speculate that the failure to detect such changes in single gene knockout may be due to functional redundancy. Given the high level of Khib in drug-resistant bacteria, we hypothesize that transferases exert a significant influence on the modification of CIP-R. We then produced single-gene deletion mutants of both genes in ciprofloxacin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (CIP-R) and again analyzed 2-hydroxyisobutyrylation. Immunoblot assay showed reduced Khib modification levels in CIP-R \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e, whereas no significant change was detected in CIP-R \u0026Delta;\u003cem\u003eyncA\u003c/em\u003e (Fig.\u0026nbsp;3b, c, Fig. S3c). Furthermore, incubating purified recombinant AcuA protein with 2-hydroxyisobutyryl coenzyme A (Hib-CoA) and a polyvinylidene fluoride (PVDF) membrane containing whole-cell proteins from \u003cem\u003eS. aureus\u003c/em\u003e, we could show that AcuA was able to 2-hydroxyisobutyrylate many proteins on the membrane (Fig.\u0026nbsp;3c). Subsequent fluorescence titrations further showed that binding of Hib-CoA to AcuA resulted in a strong fluorescence quenching with a Kd value of 0.98 \u0026micro;M (Fig.\u0026nbsp;3d). Taken together, our results suggest that AcuA has a significant impact on global lysine 2-hydroxyisobutyrylation, and that AcuA is an important candidate for 2-hydroxyisobutyryltransferase in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eA previous study demonstrated that AcuA is a lysine acetyltransferase that reduces the activity of acetyl-coenzyme A synthetase (Acs) by acetylating residue Lys549\u003csup\u003e31\u003c/sup\u003e. We also analyzed the effect of CIP-R \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e on acetylation patterns in \u003cem\u003eS. aureus\u003c/em\u003e. As shown in Fig.\u0026nbsp;3e, single \u003cem\u003eacuA\u003c/em\u003e gene knockout had no specific impact on global lysine acetylation levels in \u003cem\u003eS. aureus\u003c/em\u003e. This finding is consistent with previous reports in \u003cem\u003eBacillus subtilis\u003c/em\u003e, where \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e neither significantly increased nor decreased the overall acetylation level in the bacterium\u003csup\u003e32\u003c/sup\u003e. Therefore, this study focused on the contribution of AcuA as an \u003cem\u003eS. aureus\u003c/em\u003e 2-hydroxyisobutyryltransferase to drug resistance.\u003c/p\u003e\n\u003cp\u003eNext, to elucidate the key residue in AcuA involved in catalyzing the transfer of 2-hydroxyisobutyryl group, we generated the full-length structure of AcuA using the Swiss-Model server. We investigated the interaction between AcuA and Hib-CoA or Ac-CoA through molecular docking analysis. The results showed Hib-CoA and Ac-CoA could bind to AcuA, and Hib-CoA had slightly lower free binding energy (\u0026minus;\u0026thinsp;9.3 kcal/mol) than that to Ac-CoA (\u0026minus;\u0026thinsp;8.3 kcal/mol). Further structural analysis revealed that eight amino acid residues (Gln109, Arg108, His139, Ile105, Trp140, Tyr152, Val103, and Gly99) of AcuA were able to form hydrogen bonds with Hib-CoA (Fig.\u0026nbsp;3f). It was reported that His139 and Trp140 of AcuA in \u003cem\u003eBacillus subtilis\u003c/em\u003e are important sites for acetylation\u003csup\u003e33\u003c/sup\u003e. The His139 side chain can bind to Ac-CoA, acting as a general base during the catalytic process while coordinating with Ca\u0026sup2;⁺ ions. On the other hand, the main chain amide of Trp140 is oriented toward the active center, plays a structural role in forming the hydrophobic cavity and facilitates the binding of acetyl-CoA\u003csup\u003e33\u003c/sup\u003e. Based on sequence alignment, we found that His139 and Trp140 of AcuA in \u003cem\u003eBacillus subtilis\u003c/em\u003e are conserved in \u003cem\u003eS. aureus\u003c/em\u003e (His139 and Trp140) (Fig.\u0026nbsp;3g). Therefore, we predict that His139 and Trp140 of AcuA in \u003cem\u003eS. aureus\u003c/em\u003e are likely to be the key sites for its 2-hydroxyisobutyrylase activity. To test this possibility, we expressed and purified WT and mutated AcuA (H139A and W140A) (Fig.\u0026nbsp;3h) and then performed fluorescence titration analysis using the Hib-CoA. Compared to wild-type AcuA, the W140A mutant exhibited relatively low Hib-CoA binding affinity, while the H139A mutant failed to bind to Hib-CoA (Fig.\u0026nbsp;3i, j). Structurally, both W140A and H139A mutations altered the overall conformation of AcuA. The W140A mutation resulted in a closed active center (Fig.\u0026nbsp;3k). Further molecular dynamics simulations indicated that the hydrophobic cavity housing Hib-CoA collapsed in the H139A mutant, as alanine substitution disrupted the binding pocket's conformation. This structural change is characterized by a significant reduction in the distances between the key structural residues Ser145 and Ile105, as well as Leu155 and Gln109 within the hydrophobic cavity (Fig.\u0026nbsp;3l).\u003c/p\u003e\n\u003cp\u003eTo functionally validate the critical role of residue His139 and Trp140 in mediating Khib catalysis \u003cem\u003ein vivo\u003c/em\u003e, we introduced H139A and W140A mutations into AcuA in the CIP-R \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e strain. As expected, both mutant strains exhibited significantly impaired Khib catalysis compared to the wild-type complement strain (\u003cem\u003eeWT\u003c/em\u003e). The \u003cem\u003eeH139A\u003c/em\u003e mutant strain nearly abolished Khib activity, and the \u003cem\u003eeW140A\u003c/em\u003e mutant strain also showed a substantial defect, supporting the specificity of these residues for Khib catalysis (Fig.\u0026nbsp;3m). Collectively, these results confirm that His139 and Trp140 are essential for Hib-CoA binding in AcuA and substantiate its dual role in maintaining structural integrity and enabling Khib catalytic activity both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyisobutyryltransferase AcuA Induces Fluoroquinolone Resistance in\u003c/strong\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior studies have demonstrated that Khib plays a key role in regulating the survival of \u003cem\u003eE. coli\u003c/em\u003e under extreme acid stress\u003csup\u003e19\u003c/sup\u003e. Moreover, our preliminary data also indicated that elevated Khib modification levels promote fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;1). Therefore, we further investigated the regulatory role of the 2-hydroxyisobutyryltransferase AcuA in \u003cem\u003eS. aureus\u003c/em\u003e antibiotic resistance. Interestingly, deletion of the \u003cem\u003eacuA\u003c/em\u003e gene significantly impaired bacterial growth, resulting in a significant growth defect compared with the CIP-R (Fig.\u0026nbsp;4a). AcuA is a component of the \u003cem\u003eacuABC\u003c/em\u003e operon, which has been implicated in the catabolism of butanediol and acetoin (Fig. S4a). Thus, we speculate that the loss of \u003cem\u003eacuA\u003c/em\u003e disrupts basal metabolic processes of \u003cem\u003eS. aureus\u003c/em\u003e, thereby contributing to the observed growth impairment and antibiotic resistance. Then, we further assessed the contribution of AcuA to antibiotic resistance in \u003cem\u003eS. aureus\u003c/em\u003e. The ciprofloxacin resistance decreased in CIP-R \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e in which the Khib modification levels of whole proteome was reduced (Fig.\u0026nbsp;4b, Fig.\u0026nbsp;3b). Although no obvious change in drug resistance was observed in the WT \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e strain (Fig.\u0026nbsp;4c), continuous passage under 1/2 MIC ciprofloxacin treatment resulted in the resistance development of WT-\u0026Delta;\u003cem\u003eacuA\u003c/em\u003e being obviously slower than that of WT strain (Fig.\u0026nbsp;4d). Compared to the empty vector, overexpression of the \u003cem\u003eacuA\u003c/em\u003e gene increases Khib modification levels while simultaneously enhancing WT \u003cem\u003eS. aureus\u003c/em\u003e resistance to ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin by 2-, 4-, 4-, and 4-fold, respectively (Fig.\u0026nbsp;4e). This result indicates that AcuA promotes bacterial resistance to fluoroquinolones by regulating Khib modification levels of specific substrate proteins.\u003c/p\u003e\n\u003cp\u003eTo further explore the regulatory networks of AcuA in \u003cem\u003eS. aureus\u003c/em\u003e, we conducted a GST pull-down assay followed by mass spectrometry (MS) analysis (Fig.\u0026nbsp;4f). A total of 757 proteins were identified as potential interactors of AcuA. We mapped all identified proteins to KEGG pathways. These proteins are associated with several key metabolic pathways, including amino acid/carbon/nucleotide metabolism, as well as infection-related processes (Fig.\u0026nbsp;4g). It is worth noting that 30 transcription factors were identified, and the Khib modification levels of 7 of these proteins exhibited significant differences in CIP-R strains (Fig.\u0026nbsp;4h, Table S2). These findings indicate that AcuA-mediated Khib modification affects numerous key metabolic pathways in \u003cem\u003eS. aureus\u003c/em\u003e, thereby altering bacterial drug resistance through metabolic reprogramming.\u003c/p\u003e\n\u003cp\u003eSubsequently, to further elucidate the molecular mechanism by which Khib promotes antibiotic resistance, we conducted DIA-based quantitative proteomics on CIP-R and CIP-R \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e strains. This analysis totally identified 465 differentially expressed proteins (DEPs) were identified, including 180 upregulated proteins and 285 downregulated proteins (fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. S4b). Upregulated proteins were primarily enriched in pathways related to biosynthesis of nucleotide sugars, pyruvate metabolism, riboflavin metabolism, and glycolysis/gluconeogenesis (Fig. S4c). Conversely, downregulated proteins were associated with pathways such as secondary metabolite biosynthesis, ribosome, biosynthesis of various amino acids, and fatty acid degradation (Fig. S4d). Notably, compared to the control strain CIP-R, CIP-R \u0026Delta;\u003cem\u003eacuA\u003c/em\u003e exhibits downregulation in the expression levels of several known essential proteins involved in biofilm formation, including FnbA, FnbB, SraP, IsaA, Spa, SasG, SdrD, and SdrC (Fig.\u0026nbsp;4i). Furthermore, qPCR analysis revealed that the expression level of the \u003cem\u003eicaADBC\u003c/em\u003e operon, a key operon involved in \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation, was also significantly decreased (Fig.\u0026nbsp;4j). Prior research reported that AcuA regulates biofilm formation and swarming of \u003cem\u003eB. subtilis\u003c/em\u003e by acetylating YmcA and GtaB\u003csup\u003e32\u003c/sup\u003e. Consistent with these findings, bacterial biofilm analysis showed that overexpression of \u003cem\u003eacuA\u003c/em\u003e led to increased biofilm formation in \u003cem\u003eS. aureus\u003c/em\u003e, whereas knockout of \u003cem\u003eacuA\u003c/em\u003e reduced biofilm formation (Fig.\u0026nbsp;4k). It is also noteworthy that the addition of hib to CD medium led to increased expression of the \u003cem\u003eicaADBC\u003c/em\u003e operon genes and decreased expression of the repressor \u003cem\u003eicaR\u003c/em\u003e, ultimately leading to enhanced biofilm formation (Fig.\u0026nbsp;4l, m). To further validate the association between AcuA, biofilm formation, and drug resistance in clinical isolates, we examined the expression of the \u003cem\u003eicaADBC\u003c/em\u003e operon and biofilm-forming capacity in MDR strains. The results indicated that, compared with the WT strain, the transcriptional levels of \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaB\u003c/em\u003e genes were significantly increased in MDR strains, accompanied by enhanced biofilm formation (Fig.\u0026nbsp;4n, o). Western blot analysis further confirmed significantly higher protein levels of AcuA in clinically multidrug-resistant bacteria and CIP-R relative to the WT strain (Fig.\u0026nbsp;4p), suggesting that \u003cem\u003eS. aureus\u003c/em\u003e may upregulate AcuA expression as an adaptive response to adverse environments. Collectively, these findings suggest that AcuA may regulate \u003cem\u003eS. aureus\u003c/em\u003e resistance to fluoroquinolone antibiotics by upregulating the Khib modification levels of biofilm-associated proteins.\u003c/p\u003e\n\u003ch3\u003eAcuA Influences Bacterial Resistance to Fluoroquinolones through 2-hydroxyisobutyrylation of CodY\u003c/h3\u003e\n\u003cp\u003eIncreasing evidence indicates that post-translational modifications (PTMs) can rapidly reprogram bacterial metabolic networks by directly regulating the activity of key transcription factors (TFs), thereby promoting the development of antibiotic resistance\u003csup\u003e13,27\u003c/sup\u003e. Notably, in our constructed ciprofloxacin-resistant \u003cem\u003eS. aureus\u003c/em\u003e model, we found that the global transcription factor CodY serves as a prominent example of this regulatory mechanism. In ciprofloxacin-resistant \u003cem\u003eS. aureus\u003c/em\u003e, the Khib-modified lysine residue at positions 6, 16, and 223 in CodY were differentially modified sites vs. WT, increasing by 1.97-fold, 1.50-fold, and 2.21-fold, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;5a, Fig. S5a). Notably, CodY protein expression levels remained unchanged between WT and CIP-R as well as MDR strains (Fig.\u0026nbsp;5b). In \u003cem\u003eS. aureus\u003c/em\u003e, CodY is a central transcriptional repressor that integrates nutrient availability with pathogenicity. It directly controls the expression of critical targets, including virulence genes (e.g., hla), biofilm-forming components, and metabolic enzymes to coordinate adaptation and pathogenicity\u003csup\u003e29,34\u003c/sup\u003e. Therefore, we hypothesize that AcuA modulates fluoroquinolone resistance not by altering CodY abundance, but by regulating its Khib modification levels, thereby influencing its transcriptional regulatory activity.\u003c/p\u003e\n\u003cp\u003eAcyltransferases function by binding both an acyl-CoA donor and a protein substrate\u003csup\u003e35\u003c/sup\u003e. To determine whether AcuA catalyzes the 2-hydroxyisobutyrylation of CodY, we first assessed their physical interaction. Glutathione S-transferase (GST)-tagged AcuA and polyhistidine (His)-tagged CodY were incubated together, and western blot analysis confirmed that AcuA directly binds to CodY (Fig.\u0026nbsp;5c). We next performed in vitro Khib assays by incubating recombinant CodY with Hib-CoA in the presence or absence of purified AcuA. The results showed that CodY undergoes non-enzymatic Khib modification when incubated with Hib-CoA alone. Moreover, the addition of AcuA significantly enhanced the Khib modification level of CodY (Fig.\u0026nbsp;5d, e). These findings indicate that AcuA functions as an acyltransferase that promotes CodY 2-hydroxyisobutyrylation. It is worth noting that 2-hydroxyisobutyrylation of CodY can occur through both enzymatic and non-enzymatic mechanisms. While enzymatic acylation has been reported in several contexts, such as modifications mediated by AcuA or other acyltransferases, non-enzymatic 2-hydroxyisobutyrylation of bacteria itself also plays a regulated and biologically significant role\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo validate the lysine residues of AcuA-catalyzed 2-hydroxyisobutyrylation on CodY, we performed in vitro 2-hydroxyisobutyrylation assays using purified wild-type CodY and mutant proteins in which specific lysine residues were replaced with arginine (K6R, K16R, and K223R) to mimic the de-2-hydroxyisobutyrylated state. Western blot analysis showed significantly reduced Khib modification levels at the K16R and K223R mutants, whereas the K6R mutant exhibited a modification level comparable to that of the wild-type protein (Fig.\u0026nbsp;5f). Consistently, when the mutants were incubated with AcuA and Hib-CoA, Khib modification levels remained low at the K16R and K223R sites, while the K6R mutant again showed no significant difference from wild-type CodY (Fig.\u0026nbsp;5g). These results indicate that Lys16 and Lys223 of CodY are critical residues for AcuA-mediated 2-hydroxyisobutyrylation.\u003c/p\u003e\n\u003cp\u003eWe then looked at how these changes in Khib modification levels affect in vivo bacterial resistance to fluoroquinolones. Four plasmid point mutants were constructed by individually mutating Lys16 and Lys223 of CodY to glutamine (Q) or arginine (R). The lysine-to-glutamine (K to Q) substitution mimics the 2-hydroxyisobutyrylated state. These plasmids were introduced into the \u0026Delta;\u003cem\u003ecodY\u003c/em\u003e strain (Fig. S5b) (the generated strains were named \u003cem\u003eeK16Q\u003c/em\u003e, \u003cem\u003eeK16R\u003c/em\u003e, \u003cem\u003eeK223Q\u003c/em\u003e, and \u003cem\u003eeK223R\u003c/em\u003e). We measured the growth curves of the K-to-Q and K-to-R strains. The growth rate of K-to-R strains (\u003cem\u003eeK16R\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e) reaching the plateau phase 8\u0026ndash;9 h after inoculation was significantly slower than that of the K-to-Q strains (\u003cem\u003eeK16Q\u003c/em\u003e and \u003cem\u003eeK223Q\u003c/em\u003e) (Fig. S5c). Meanwhile, MIC assays showed that \u003cem\u003eeK16Q\u003c/em\u003e and \u003cem\u003eeK223Q\u003c/em\u003e strains exhibited elevated resistance to multiple fluoroquinolones compared to the \u003cem\u003eeK16R\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e strains. Specifically, the \u003cem\u003eeK16Q\u003c/em\u003e mutant led to 4-, 2-, 1-, and 1-fold increases in MIC for ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin, respectively, while the \u003cem\u003eeK223Q\u003c/em\u003e mutant resulted in 8-, 8-, 4-, and 16-fold increases (Fig.\u0026nbsp;5h, i). These results indicate that AcuA enhances bacterial resistance to fluoroquinolones through 2-hydroxyisobutyrylation of CodY. Notably, the K223Q mutation contributed significantly more to resistance than K16Q, suggesting that AcuA primarily promotes \u003cem\u003eS. aureus\u003c/em\u003e fluoroquinolone resistance by upregulating 2-hydroxyisobutyrylation at the CodY Lys223 site. Importantly, the Lys223 site is highly conserved in Gram-positive bacteria (Fig. S5d), further suggesting that 2-hydroxyisobutyrylation at this site plays an important biological role in regulating antibiotic resistance.\u003c/p\u003e\n\u003cp\u003eTo further validate these results, we established mouse pneumonia infection models using \u003cem\u003eeK223Q\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e mutant strains. In the absence of antibiotic treatment, mice infected with the \u003cem\u003eeK223R\u003c/em\u003e strain exhibited significantly higher mortality (90%, 9/10), whereas mice infected with the \u003cem\u003eeK223Q\u003c/em\u003e strain showed lower mortality (50%, 5/10) (Fig.\u0026nbsp;5j). Subsequently, we evaluated the response of the strains to ciprofloxacin treatment. Compared to the untreated groups, ciprofloxacin administration effectively protected mice infected with the \u003cem\u003eeK223R\u003c/em\u003e strain, reducing mortality to 0% (0/10) and resulting in very low post-treatment bacterial burden in the lungs. In contrast, although ciprofloxacin treatment also reduced the mortality of mice infected with the \u003cem\u003eeK223Q\u003c/em\u003e strain to 20% (2/10), the bacterial load in their lung tissue post-treatment was significantly higher than that in the treated \u003cem\u003eeK223R\u003c/em\u003e infection group (Fig.\u0026nbsp;5j, k). This key finding indicates that Lys223 2-hydroxyisobutyrylation of CodY, while attenuating acute pathogenicity, significantly enhances bacterial resistance to ciprofloxacin, enabling persistent colonization within the host under antibiotic pressure.\u003c/p\u003e\n\u003cp\u003eGiven the established role of CodY in regulating biofilm-associated operons and the significant contribution of biofilm formation to antibiotic tolerance, we hypothesized that 2-hydroxyisobutyrylation of CodY may regulate resistance by influencing biofilm formation\u003csup\u003e29\u003c/sup\u003e. To test this hypothesis, we evaluated the biofilm-forming capacity of the mutant strains. qPCR analysis revealed that the expression of key biofilm-forming genes, \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaB\u003c/em\u003e, was significantly upregulated in the \u003cem\u003eeK16Q\u003c/em\u003e and \u003cem\u003eeK223Q\u003c/em\u003e strains compared to the \u003cem\u003eeK16R\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e mutants (Fig.\u0026nbsp;5l). Subsequent biofilm assays further confirmed that the \u003cem\u003eeK16Q\u003c/em\u003e and \u003cem\u003eeK223Q\u003c/em\u003e mutants formed significantly more biofilm than the \u003cem\u003eeK16R\u003c/em\u003e and \u003cem\u003eeK223R\u003c/em\u003e mutants (Fig.\u0026nbsp;5m), indicating that 2-hydroxyisobutyrylation of CodY enhances fluoroquinolone resistance, at least in part, by promoting biofilm production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyisobutyrylation Regulates Dimerization and DNA-binding Ability of CodY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess whether Khib modification influences the structural integrity of CodY, we evaluated its structure and thermal stability through circular dichroism CD spectroscopy. CD analysis revealed no significant differences in the secondary structure composition of wild-type CodY and its mutants (Fig. S6a, b). Although the secondary structure is largely unaffected, thermal denaturation experiments revealed reduced melting temperatures (Tm) for K16Q (50.9\u0026deg;C) and K223Q (51.5\u0026deg;C) compared to wild-type CodY (54.1\u0026deg;C) (Fig.\u0026nbsp;6a), indicating diminished protein stability of mutant proteins. This decrease in thermal stability can be attributed to the disruption of key intramolecular interactions that were originally mediated by the wild-type lysine residues. The K-to-Q mutations, by removing positive charges and altering side-chain chemistry, compromise the intricate network of non-covalent interactions that lock the protein into its stable, native conformation.\u003c/p\u003e\n\u003cp\u003eCodY functions as a homodimeric transcription factor, and its DNA-binding capacity is closely linked to its oligomeric state\u003csup\u003e37,38\u003c/sup\u003e. CodY consists of an N-terminal ligand-binding domain GAF and a C-terminal DNA-binding domain (DBD), connected by an extended helical linker (LHL), which incorporates a winged helix-turn-helix (wHTH) motif. Structural analysis revealed that in the CodY dimer, two monomers (designated here as protomer A and protomer B) interact via an interface involving both the GAF and DBD domains (Fig. S6c). Intriguingly, Lys16 in the GAF domain of monomer and Lys223 in the DBD domain were both 2-hydroxyisobutyrylated, and both residues are located at the dimer interface (Fig.\u0026nbsp;6b, Fig. S6c), suggesting that their modification may influence the self-assembly of the two monomers. To test this hypothesis, we performed formaldehyde crosslinking followed by immunoblotting, which confirmed the presence of CodY dimers (Fig.\u0026nbsp;6c). Notably, the K16Q and K223Q mutants exhibited enhanced dimerization relative to wild-type CodY, whereas charge-conservative arginine substitutions (K16R and K223R) showed no such effect. These results suggest that Khib modification at Lys16 and Lys223 strengthens intermolecular interactions and promotes CodY dimerization.\u003c/p\u003e\n\u003cp\u003eThe GAF domain plays a crucial role in CodY dimer assembly. Hainzl et al\u003csup\u003e37\u003c/sup\u003e found that, in the absence of ligands, residues Gln15-Lys18 sterically clash with the linker helix and the S3\u0026ndash;S4 loop of protomer B, thereby preventing dimer formation (Fig.\u0026nbsp;6d). However, ligand-binding induces the formation of an \u0026alpha;-helix involving Leu14, Gln15, and Lys16, resulting in the extension of helix H1, enabling hydrogen bonding with residues in protomer B, thereby moving residues Gln15-Lys18 to allow dimer formation (Fig. S6d). In the K16Q mutant, this steric hindrance is alleviated even in the absence of ligand, effectively mimicking a pre-activated state, which would facilitate dimer formation (Fig.\u0026nbsp;6e). Lys223 is critical for stabilizing the dimer interface\u003csup\u003e37\u003c/sup\u003e. Structural analysis showed that Lys223 forms a bulge within the DNA binding groove, which facilitates dimer formation (Fig. S6c). Analysis of the dimer interface indicates that the K223Q mutant forms more hydrogen bonds compared to WT CodY (Fig.\u0026nbsp;6f, g), and the distance between the two bulge is significantly reduced, causing protomer A and B to bind more tightly (Fig. S6e, f).\u003c/p\u003e\n\u003cp\u003eWe next ask whether this increase in dimerization translates to enhanced DNA binding. Using electrophoretic mobility shift assays (EMSAs), we evaluated the interaction of CodY variants with the target regulatory regions of two downstream genes (P\u003cem\u003eicaB\u003c/em\u003e and P\u003cem\u003ecap\u003c/em\u003e). \u003cem\u003eicaB\u003c/em\u003e is part of the biofilm synthesis operon \u003cem\u003eicaADBC\u003c/em\u003e, while \u003cem\u003ecap\u003c/em\u003e is the promoter of the capsular polysaccharide (CP) operon in \u003cem\u003eS. aureus\u003c/em\u003e. Surprisingly, despite their increased propensity to dimerize, the K223Q mutant displayed reduced binding to both the \u003cem\u003eicaB\u003c/em\u003e and \u003cem\u003ecap\u003c/em\u003e promoters (Fig.\u0026nbsp;6h). Moreover, the K16Q mutant exhibited promoter-specific differences: K16Q showed stronger binding to the \u003cem\u003ecap\u003c/em\u003e promoter, while its binding to the \u003cem\u003eicaB\u003c/em\u003e promoter is lower than that of the wild type (Fig.\u0026nbsp;6i). These findings indicate that Khib-induced dimerization does not uniformly enhance DNA binding. Instead, it appears to modulate promoter affinity in a site- and context-dependent manner, likely by allosterically tuning the CodY\u0026ndash;DNA interface.\u003c/p\u003e\n\u003cp\u003eSince dimerization is thought to facilitate transcription factor binding to DNA, the distinct binding patterns of CodY mutants observed in EMSA analyses were unexpected. In the previously characterized binding modes of CodY with ligands, CodY dimers exhibit distinct conformational arrangements (PDB ID: 5eyo and 5ey1). Thus, although both K16Q and K223Q facilitate dimerization, they may perturb allosteric coupling between ligand sensing and DNA recognition. Consequently, we employed AlphaFold 3 to simulate the structures of K16Q and K223Q dimers in GTP-bound states\u003csup\u003e39\u003c/sup\u003e. Structural analyses revealed that 2-hydroxyisobutyrylation at either Lys16 or Lys223 site leads to significant changes in the conformation and spatial organization of DBD (Fig.\u0026nbsp;6j). Specifically, the K-to-Q mutation at the Lys223 site results in a markedly shallower DNA-binding groove, potentially hindering DNA binding. Furthermore, we observed distinct differences in the electrostatic potential of the DNA-binding groove between the K16Q and K223Q mutants and the wild-type CodY (Fig.\u0026nbsp;6k). The K16Q mutant exhibits altered local electrostatic potential, while the K223Q mutant increases the net negative electrostatic potential of the binding interface. Given that the DNA backbone is highly negatively charged, this alteration would introduce electrostatic repulsion and diminish the favorable electrostatic complementarity that is essential for high-affinity binding. Thus, while the two mutants stabilize the dimeric form, they concomitantly compromise the electrostatics of DNA recognition, leading to an overall decrease in binding affinity. This may account for the binding states of CodY to different promoters despite enhanced dimerization capacity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe escalating challenge of antimicrobial resistance in \u003cem\u003eS. aureus\u003c/em\u003e, particularly its high-level resistance to critical antibiotics like fluoroquinolones, poses a severe threat to modern medicine. While mutations in target enzymes such as DNA gyrase are established primary drivers of clinical fluoroquinolone resistance, there is growing recognition that non-mutational, adaptive mechanisms significantly contribute to the resistance phenotype and treatment failure. In-depth research into these complementary mechanisms will help address this global challenge.Emerging evidence emphasizes that post-translational modifications (PTMs) are pivotal regulators of bacterial virulence and antibiotic resistance\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Among these, Khib is a recently discovered metabolite-derived PTM. Structurally analogous to-but bulkier than-acetylation, Khib can induce more profound alterations in protein function and interactions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Although Khib has been shown to dynamically reprogram metabolic pathways in pathogens like \u003cem\u003eUstilaginoidea virens\u003c/em\u003e and regulate critical processes in mammalian cells, its role in \u003cem\u003eS. aureus\u003c/em\u003e, particularly its connection to antibiotic resistance and pathogenicity, remains largely unexplored\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study reveals that Khib serves as a critical adaptive regulator that potentiates fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e. At the global level, we found that Khib is widely distributed across the \u003cem\u003eS. aureus\u003c/em\u003e proteome and is markedly remodeled in both clinical MDR isolates and laboratory-evolved ciprofloxacin-resistant (CIP-R) strains. Further quantitative Khib proteomics was performed to compare the DHPs between CIP-R and WT \u003cem\u003eS. aureus.\u003c/em\u003e CIP-R cells displayed substantial changes in 2-hydroxyisobutyrylation compared with susceptible strains, with hundreds of proteins showing increased or decreased modification. These differentially 2-hydroxyisobutyrylated proteins were significantly enriched in metabolic pathways, particularly those involved in carbon and energy metabolism, as well as in pathways previously implicated in quinolone resistance. One striking pattern was the overall decrease in 2-hydroxyisobutyrylation of enzymes associated with carbon metabolism in CIP-R strains. Given that Khib adds a bulky, polar group to lysine residues, loss of 2-hydroxyisobutyrylation at key metabolic nodes is expected to alter enzyme activity, complex assembly, or cofactor affinity. Such changes could reprogram central metabolism to favor energy-efficient pathways, thereby influencing the bactericidal activity of fluoroquinolones. Importantly, pharmacologic manipulation of global Khib modification levels by adding 2-hydroxyisobutyrate or overexpressing the de-2-hydroxyisobutyrylase CobB bidirectionally changed susceptibility to fluoroquinolones. Together, these findings argue that 2-hydroxyisobutyrylation is not a passive bystander but an active determinant of drug response that fine-tunes metabolic fluxes under antibiotic stress.\u003c/p\u003e \u003cp\u003eProtein 2-hydroxyisobutyrylation and de-2-hydroxyisobutyrylation are reversibly catalyzed by two classes of enzymes, 2-hydroxyisobutyryltransferases (writers) and de-2-hydroxyisobutyrylases (erasers) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To date, the only 2-hydroxyisobutyryl transferase identified in prokaryotes is TmcA from \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. TmcA exhibits tRNA(Met) cytidine acetyltransferase activity, demonstrating the complexity of bacterial Khib regulatory factor classification. The regulatory function of Khib in \u003cem\u003eStaphylococcus\u003c/em\u003e resistance, and its role as both “writers” and “erasers”, remains elusive. To understand how 2-hydroxyisobutyrylation is written onto the proteome, we systematically screened GNAT-family acyltransferases and identified AcuA as a 2-hydroxyisobutyryl transferase in \u003cem\u003eS. aureus\u003c/em\u003e. Biochemical assays confirmed that AcuA catalyzes 2-hydroxyisobutyrylation in vitro and in vivo, and mutational analysis of the predicted Hib-CoA binding pocket demonstrated that residues such as His139 and Trp140 are essential for activity. Beyond its enzymatic function, AcuA plays a broad physiological role. Deletion of \u003cem\u003eacuA\u003c/em\u003e impaired bacterial growth, reduced global Khib modification levels and significantly lowered resistance to ciprofloxacin and other fluoroquinolones in CIP-R strains, whereas its overexpression had opposite effects. Proteomic profiling of the \u003cem\u003eacuA\u003c/em\u003e mutant revealed perturbation of amino acid, carbon, and nucleotide metabolism and, importantly, down-regulation of multiple biofilm-associated proteins, including FnbA, SraP, and the \u003cem\u003eicaADBC\u003c/em\u003e operon. Consistent with these molecular changes, AcuA was required for robust biofilm formation. Notably, AcuA protein expression is significantly upregulated in clinically isolated multidrug-resistant strains, suggesting that upregulation of this writer enzyme is part of the adaptive program that enables \u003cem\u003eS. aureus\u003c/em\u003e to persist under antibiotic pressure.\u003c/p\u003e \u003cp\u003eThese observations prompted us to search for regulatory substrates that connect AcuA-dependent 2-hydroxyisobutyrylation to biofilm-mediated resistance. We focused on CodY, a highly conserved global transcription regulator in Gram-positive bacteria that senses intracellular GTP and branched-chain amino acids to adjust gene expression according to nutritional status. CodY controls a wide regulatory network encompassing metabolic genes, virulence factors, and stress-response pathways\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We observed higher levels of Khib at CodY Lys6, Lys16, and Lys223 in CIP-R strains than in susceptible strains and showed that AcuA physically interacts with CodY and catalyzes 2-hydroxyisobutyrylation at Lys16 and Lys223. Among these sites, Lys223 emerged as the most critical for fluoroquinolone resistance. Critically, in a mouse infection model, strains harboring high Khib modification levels at CodY Lys223 exhibited significantly enhanced survival upon ciprofloxacin challenge, indicative of increased fluoroquinolone resistance. Notably, our in vivo experiments also revealed that Khib modification at CodY Lys223 exerts a negatively regulatory effect on acute bacterial virulence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ej). This observation indicated that AcuA-mediated modification may coordinately regulate multiple aspects of \u003cem\u003eS. aureus\u003c/em\u003e adaptation, including both antibiotic resistance and pathogenic potential. Elucidating the precise role of this PTM in pathogenicity is a key direction of our future research.\u003c/p\u003e \u003cp\u003eFunctional studies using CodY mutants that mimic de-2-hydroxyisobutyrylated (K→R) or 2-hydroxyisobutyrylated (K→Q) states revealed a clear regulatory pattern. CodY K16R/K223R displayed enhanced binding to the \u003cem\u003eicaADBC\u003c/em\u003e promoter, stronger repression of biofilm-associated genes, and reduced fluoroquinolone resistance. In contrast, CodY K16Q/K223Q weakened promoter binding, derepressed \u003cem\u003eicaADBC\u003c/em\u003e expression, promoted biofilm formation, and increased resistance. Given that biofilms are a well-characterized niche for antibiotic tolerance and resistance development, our findings delineate a novel Khib-dependent signaling pathway that enhances bacterial survival under antibiotic stress by fostering a protective biofilm lifestyle. Because Lys16 and Lys223 lie within the oligomerization domain of CodY, we propose that 2-hydroxyisobutyrylation at these positions stabilizes CodY dimerization and alters the conformation and charge distribution of the DNA-binding groove, thereby decreasing affinity for target promoters. In nutrient-replete conditions, CodY is already prone to dissociation upon changes in GTP or amino acid levels; 2-hydroxyisobutyrylation could serve as an additional signal that accelerates its release from DNA when cells experience antibiotic-induced metabolic stress. Given the high conservation of Lys223 in CodY homologs from other Gram-positive pathogens, this PTM site may represent a broadly relevant node where metabolic cues, PTMs, and antimicrobial resistance intersect.\u003c/p\u003e \u003cp\u003eIntegrating these findings, we propose a model in which 2-hydroxyisobutyrylation drives metabolic reprogramming in \u003cem\u003eS. aureus\u003c/em\u003e to survive fluoroquinolone stress. Up-regulation of AcuA increases 2-hydroxyisobutyrylation of specific metabolic enzymes and the global regulator CodY. At the metabolic level, changes in Khib promote rerouting of carbon flux and energy production toward states that favor survival under antibiotic stress. At the regulatory level, AcuA-dependent 2-hydroxyisobutyrylation of CodY, particularly at Lys223, diminishes repression of the \u003cem\u003eicaADBC\u003c/em\u003e operon and other biofilm-associated genes, leading to enhanced biofilm formation. Biofilms, in turn, provide a protective niche with reduced antibiotic penetration, altered microenvironment, and increased tolerance.\u003c/p\u003e \u003cp\u003eIn summary, we identified a novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in \u003cem\u003eS. aureus\u003c/em\u003e. Specifically, AcuA-mediated 2-hydroxyisobutyrylation of CodY at Lys16/Lys223 alleviates its transcriptional repression of the \u003cem\u003eicaADBC\u003c/em\u003e operon, thereby promoting biofilm-associated antibiotic resistance. This work contributes to the field of drug-resistant bacterial control by revealing the promising role of protein 2-hydroxyisobutyrylation, thereby opening a new avenue for combating these infections.\u003c/p\u003e\n\u003ch3\u003eLimitations of this study\u003c/h3\u003e\n\u003cp\u003eLysine modification in bacteria can occur through both enzymatic and non-enzymatic mechanisms. We focus solely on the role of enzyme-mediated Khib in regulating bacterial physiological functions, but non-enzyme-mediated Khib may contribute to the development of bacterial fluoroquinolone resistance. Furthermore, we concentrate only on proteins exhibiting elevated Khib modification levels in CIP-R, but those with downregulated Khib modification levels may also play roles in the emergence of bacterial fluoroquinolone resistance.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e\n\n \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003ch2\u003eBacterial strains and cell culture\u003c/h2\u003e\u003cp\u003eBacterial strains, plasmids, and primers used in this study are listed in Table S3 and Table S4. Unless otherwise noted, all \u003cem\u003eE. coli\u003c/em\u003e strains were grown in Luria Bertani (LB) broth, \u003cem\u003eS. aureus\u003c/em\u003e strains and their derivatives were cultured in tryptic soy broth (TSB) or in chemically defined (CD) medium at 37°C\u003csup\u003e42\u003c/sup\u003e. For plasmid maintenance, antibiotics were used at the following concentrations: ampicillin, 100 µg/mL; kanamycin, 50 µg/mL; chloramphenicol, 10 µg/mL; erythromycin, 25 µg/mL.\u003c/p\u003e\u003ch3\u003eConstruction of plasmids for gene expression\u003c/h3\u003e\u003cp\u003eTo construct overexpression plasmids for the enzymes that responsible for 2-hydroxyisobutyrylation, the PCR-amplified DNA fragment and plasmid pCN51 were subjected to digestion with restriction enzymes \u003cem\u003eBamH\u003c/em\u003eΙ and \u003cem\u003eEcoR\u003c/em\u003eΙ. Then, the digested fragment was ligated into pCN51 and transformed into \u003cem\u003eE. coli\u003c/em\u003e DC10B. The resulting plasmids were subsequently transformed into \u003cem\u003eS. aureus\u003c/em\u003e by electroporation. Of note, these genes were positioned downstream of the cadmium resistance transcriptional regulatory CadC in pCN51, thereby enabling its induction by Cadmium Chloride (0.25 µM). To construct the \u003cem\u003ecodY\u003c/em\u003e expression plasmids for genetic complementation, the \u003cem\u003ecodY\u003c/em\u003e gene was amplified by PCR, incorporating homologous fragments from the pWWW412. The pWWW412 plasmid was digested with the restriction enzyme \u003cem\u003eNde\u003c/em\u003eI. The amplified fragment and digested pWWW412 were ligated via homologous recombination and transformed into \u003cem\u003eE. coli\u003c/em\u003e DC10B. Subsequently, the resulting pWWW412\u003cem\u003e-codY\u003c/em\u003e plasmids were electroporated into \u003cem\u003eS. aureus\u003c/em\u003e. Recombinant proteins were expressed and purified according to established protocols. Genes were amplified by PCR and cloned into either the PGEX-4T-1 or pET-28a vectors, and transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (λDE3) for protein expression. A final concentration of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression. Proteins containing His-tags or GST-tags were purified using nickel or GST affinity chromatography columns, respectively.\u003c/p\u003e\u003ch2\u003eConstruction of gene mutants\u003c/h2\u003e\u003cp\u003eThe gene knockout was constructed using the temperature-sensitive plasmid pKOR1 via allelic replacement. Briefly, upstream and downstream fragments flanking the target gene were fused by PCR and cloned into pKOR1. The resulting plasmid was electroporated into \u003cem\u003eS. aureus\u003c/em\u003e, and integrants were selected at 43°C under chloramphenicol selection. Excision and loss of the plasmid were subsequently promoted by culturing at 30°C without antibiotics, followed by counter-selection on anhydrotetracycline. Mutants were screened for chloramphenicol sensitivity and the deletion was confirmed by PCR and sequencing.\u003c/p\u003e\u003ch2\u003eMetabolite analysis\u003c/h2\u003e\u003cp\u003eATP was determined with the ATP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.\u003c/p\u003e\u003ch2\u003eDetermination of MICs\u003c/h2\u003e\u003cp\u003eThe minimum inhibitory concentration (MIC) of fluoroquinolone antibiotics (ciprofloxacin, levofloxacin, norfloxacin and moxifloxacin) against \u003cem\u003eS. aureus\u003c/em\u003e isolates was determined by the broth microdilution method in CD medium according to the guidelines established by the Clinical and Laboratory Standards Institute (CLSI M07). Briefly, bacterial suspensions were adjusted to a density of 5×10\u003csup\u003e5\u003c/sup\u003e CFU/mL and incubated with serial two-fold dilutions of antibiotics for 24 h at 37°C. Bacterial growth was quantified by measuring the OD\u003csub\u003e600\u003c/sub\u003e with a microplate reader (BioTek Epoch, USA). The MIC value is the antibiotic concentration under which the OD\u003csub\u003e600\u003c/sub\u003e of cultures is lower than 0.1.\u003c/p\u003e\u003ch2\u003eWhole protein extraction and western blot\u003c/h2\u003e\u003cp\u003eTotal protein was prepared from \u003cem\u003eS. aureus\u003c/em\u003e cultures harvested during the late-logarithmic growth phase (OD\u003csub\u003e600\u003c/sub\u003e = 1.0). Bacterial cells were digested with lysostaphin (10 µg/mL) for 30 min at 37°C to weaken the cell wall, followed by sonication in SDS lysis buffer containing protease and deacetylase inhibitors. The crude lysate was clarified by centrifugation at 12,000 \u003cem\u003eg\u003c/em\u003e for 15 min at 4°C. Protein concentration was determined using the Bradford assay with BSA as a standard. For immunoblotting, 20 µg of total protein per sample was separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4°C with the primary antibody (diluted 1:1,000). After washing, the membrane was probed with an HRP-conjugated secondary antibody (1:5,000 dilution) for 1 h at room temperature. Signal was developed using an enhanced chemiluminescence substrate and imaged with a chemiluminescence detection system.\u003c/p\u003e\u003ch2\u003eCoomassie blue staining\u003c/h2\u003e\u003cp\u003eFollowing SDS-PAGE, proteins were stained with 0.1% Coomassie Brilliant Blue R-250 (in 40% methanol and 10% acetic acid) for 1 h. Subsequently, the gels were destained in a solution of 5% ethanol and 10% acetic acid with multiple changes of the destaining solution until clear protein bands were visible against a transparent background. The stained gels were imaged using a standard white light scanner. The raw image was changed to grayscale, and the brightness and contrast were adjusted to better visualize protein bands.\u003c/p\u003e\u003ch2\u003eIn vitro de-2‐hydroxyisobutyrylation and 2‐hydroxyisobutyrylation assay\u003c/h2\u003e\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e de-2-hydroxyisobutyrylation assays, total protein lysates (20 µg) or purified proteins (1 µg) were incubated with recombinant CobB (2 µg) in reaction buffer (50 mM Tris‐HCl (pH 8.5), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol, 1 mM NAD⁺) at 37°C for 2 h. For \u003cem\u003ein vitro\u003c/em\u003e 2‐hydroxyisobutyrylation assays, proteins were incubated with purified AcuA at 25°C for 16 h in reaction buffer (20 mM HEPES, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM KCl, 200 mM NaCl, pH 7.5). Control reactions were performed in the absence of enzyme or 2-hydroxyisobutyryl-CoA (Hib-CoA) (50 µM). All reactions were terminated by adding 5×Laemmli buffer and boiling. The modification states were subsequently analyzed by western blotting using a specific anti-2-hydroxyisobutyryllysine antibody.\u003c/p\u003e\u003ch2\u003eMolecular docking and dynamics simulation analysis\u003c/h2\u003e\u003cp\u003eThe initial structure of AcuA was retrieved from the AlphaFold Database using Uniprot accession ID Q2G293 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/entry/AF-Q2G293-F1\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Structures for Acetyl-CoA (Ac-CoA) and 2-hydroxyisobutyryl-CoA (Hib-CoA) were obtained from the PubChem database. Wild-type AcuA and site-directed AcuA H139A mutant, by substituting the amino acid using PyMOL 3.1, were prepared in GROMACS 2025. Each system was solvated in a TIP3P water box (10 Å buffer) and neutralized with Na⁺/Cl⁻ to 0.15 M. Charmm36 force field was used for protein parameterization. Molecular dynamics simulations followed a standardized protocol: steepest-descent minimization (50,000 steps) to resolve steric clashes, 100 ps NVT equilibration (300 K, velocity-rescaling thermostat) for thermal stabilization, and 100 ps NPT equilibration (1 atm, Parrinello-Rahman barostat) for volume adjustment. Production MD ran 100 ns (NPT, 2 fs step) with LINCS (hydrogen bond constraints) and PME (long-range electrostatics). The major post-molecular dynamics conformations of proteins were analyzed in the 20-100ns molecular dynamics trajectory of wild-type AcuA and site-directed AcuA by using the gmx cluster tool. The structure for receptors (AcuA, AcuA W140A) was prepared with AutoDockTools 1.5.7 with the default setting. The conformation of Hib-CoA was prepared using Scrubber (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/forlilab/molscrub\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Molecular docking was performed with AutoDock Vina using the following parameters: Grid center: X = -1, Y = -7, Z = 7, Grid box size: X = 29, Y = 29, Z = 29. The top ten lowest-binding-free-energy complexes were analyzed via PyMOL 3.1, with intermolecular interactions (hydrogen bonds, hydrophobic contacts) quantified to delineate critical AcuA-CoA binding residues and the top one lowest-binding-free-energy complexes was picked for visualization.\u003c/p\u003e\u003ch2\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from 3mL \u003cem\u003eS. aureus\u003c/em\u003e cultures using the Bacterial RNA Extraction Kit (R403-01) (Vazyme, Nanjing, China) following the manufacturer's instructions. RNA quality and concentration were determined by spectrophotometry. cDNA was synthesized from 1 µg of total RNA with the Hifair II 1st strand kit (Yeasen, Shanghai, China). Quantitative PCR was performed on Mini Option real-time PCR system (Bio-Rad) using the Hieff ® qPCR SYBR Green Master Mix (No Rox) (Yeasen, Shanghai, China). Relative expression levels were calculated using the 2^\u003csup\u003e(−ΔΔCt)\u003c/sup\u003e method with 16S rRNA as the internal reference gene. All reactions were performed in triplicate.\u003c/p\u003e\u003ch2\u003eGST pull-down assay\u003c/h2\u003e\u003cp\u003eThe GST pull-down assay was performed to investigate protein-protein interactions \u003cem\u003ein vitro\u003c/em\u003e. The bait protein (GST-tagged AcuA) was incubated with purified prey protein or total protein in binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM DTT) for 2 h at 4°C with gentle rotation. Beads were washed five times with ice-cold binding buffer to remove non-specifically bound proteins. Bound proteins were eluted by boiling in 5× SDS-PAGE loading buffer and analyzed by immunoblotting using antibodies against the tags (anti-GST and anti-His antibodies) or separated by SDS-PAGE, or identified using mass spectrometry.\u003c/p\u003e\u003ch2\u003eBiofilm assay\u003c/h2\u003e\u003cp\u003eBiofilm formation was quantified using a standard crystal violet staining assay. \u003cem\u003eS. aureus\u003c/em\u003e strains were cultured in CD medium in 24-well polystyrene plates at 37°C for 24 h. After incubation, the planktonic cells were removed, and the adhered biofilms were gently washed with phosphate-buffered saline (PBS), fixed with 99% methanol, and stained with 0.1% crystal violet for 15 min. The excess stain was rinsed off, and the bound dye was solubilized with 33% glacial acetic acid. The absorbance of the solubilized crystal violet was measured at 570 nm using a microplate reader. Each assay was performed with at least three biological replicates.\u003c/p\u003e\u003ch2\u003eProtein extraction and trypsin digestion\u003c/h2\u003e\u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e and its derivative strains were inoculated in 200 mL of CD medium in a five-hundred mL Erlenmeyer baffled flask under shaking at 200 rpm at 37°C. Cells at late logarithmic phase (OD\u003csub\u003e600\u003c/sub\u003e = 1.0) were harvested by centrifugation at 4°C at 12,000 \u003cem\u003eg\u003c/em\u003e for 5 min. The cell pellets were washed twice with cold PBS and subsequently treated with proper lysostaphin for 30 min at 37°C to facilitate cell wall digestion. The harvested cells were resuspended in lysis buffer (8 M urea, 1% Triton X-100, 10 mM dithiothreitol (DTT), 1% protease inhibitor, 3 mM TSA, 50 mM NAM, and 2 mM EDTA) and sonicated on ice for 10 min. The cell debris was removed by centrifugation at 12,000 \u003cem\u003eg\u003c/em\u003e and 4°C for 30 min, and the proteins were precipitated with 20% cold TCA at 4°C for 2 h. After centrifugation at 4°C for 20 min, the supernatant was discarded. The remaining protein was resuspended and washed three times with cold acetone. Finally, the target protein was redissolved in 8 M Urea, and the protein concentration was quantified with a BCA protein assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. For trypsin digestion, protein samples were reduced with 5 mM dithiothreitol for 1 h at 37°C and alkylated with 100 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. Proteins were washed five times with 100 mM TEAB to remove urea using ultrafiltration tubes. Trypsin was added at a 1:50 trypsin-to-protein mass ratio for digestion overnight, followed by another 4-h digestion at a 1:100 ratio.\u003c/p\u003e\u003ch2\u003eImmunoaffinity enrichment of Khib peptides\u003c/h2\u003e\u003cp\u003eFor Khib-modified peptides enrichment, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 0.5% NP-40) were incubated overnight with drained pre-washed anti-2-hydroxyisobutyryllysine antibody-conjugated agarose beads (PTM Bio, China) at 4°C, with gentle rotation. After gently washing four times with NETN buffer and twice with double-distilled water, peptides bound to the beads were eluted with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined, vacuum-dried, and cleaned with C18 ZipTips (Millipore, Bedford, MA, USA) before LC-MS/MS analysis.\u003c/p\u003e\u003ch2\u003eHPLC-MS/MS analysis\u003c/h2\u003e\u003cp\u003eLyophilized peptides were reconstituted in 0.1% formic acid and spiked with iRT calibration peptides (Biognosys, USA). LC-MS/MS analysis was performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific). MS Data acquisition parameters: MS1 Spectra: Acquired in the Orbitrap at a resolution of 60,000, with a scan range of 400–1500 m/z. Automatic gain control (AGC) target was set to 4 × 10\u003csup\u003e5\u003c/sup\u003e, and maximum injection time was 50 ms. MS2 Spectra (DIA): A total of 40 variable-width isolation windows were used to cover the MS1 mass range. MS2 spectra were acquired at a resolution of 30,000, with an AGC target of 5 × 10\u003csup\u003e5\u003c/sup\u003e, maximum injection time of 56 ms, and higher-energy collisional dissociation (HCD) collision energy set to 32%.\u003c/p\u003e\u003ch2\u003eDatabase search and data filtering criteria\u003c/h2\u003e\u003cp\u003eThe raw MS/MS data acquired in data-independent acquisition (DIA) mode were analyzed using Spectronaut 19 (Biognosys) against the \u003cem\u003eStaphylococcus aureus\u003c/em\u003e NCTC 8325 reference proteome (UniProt Proteome ID: UP000008816). Trypsin/P was specified as the protease, allowing up to two missed cleavages. The minimum peptide length was set to seven amino acids. Carbamidomethylation (C) was set as a fixed modification. Methionine oxidation, protein N-terminal acetylation, and 2-hydroxyisobutyrylation (Khib) were set as variable modifications. The mass tolerance was set to ± 10 ppm for precursor ions and ± 0.02 Da for fragment ions. The false discovery rate (FDR) for peptide-spectrum matches was estimated at \u0026lt; 1% using a target-decoy approach. To ensure high-confidence identification of Khib-modified sites, we applied additional filtering criteria: only peptides with a Spectronaut cross-run normalized score \u0026gt; 40 and a modification site localization probability \u0026gt; 0.99 were retained. The abundance of quantified Khib peptides was normalized to the total intensity of their corresponding proteins derived from the global proteomic profile.\u003c/p\u003e\u003ch2\u003eBioinformatics Analysis\u003c/h2\u003e\u003cp\u003eKEGG and GO enrichment analyses were performed using the online service tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omicsolution.org/wu-kong-beta-linux/main/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Cytoscape software (version 1.5.1). Using the online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hiplot.com.cn/home/index.html\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for KEGG visualization analysis. Protein sequence alignment was conducted using the Clustal W server. Amino acid sequence motifs (seven amino acids upstream and downstream of the 2-hydroxyisobutyrylated lysine) were analyzed using pLogo\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The protein structures were visualized with PyMOL (v. 3.1) \u003csup\u003e44\u003c/sup\u003e. The structures of the AcuA and CodY mutants were simulated using Alphafold3\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eMouse pneumonia infection model\u003c/h2\u003e\u003cp\u003e6-weeks-old BALB/c female mice were obtained from the Experimental Animal Department of Guangzhou Southern Medical University (Guangzhou, China) and housed for one week before experiments. All experiments adhered to institutional guidelines and were approved by the Animal Experiment Ethics Committee of Jinan University. For the pneumonia infection model, mice were anesthetized with a mixture of 2,2,2-tribromoethanol and 2-methyl-2-butanol. Cultured \u003cem\u003eS. aureus\u003c/em\u003e (OD\u003csub\u003e600\u003c/sub\u003e = 0.8) strains were dissolved in 20 µL PBS solution and administered intranasally; mice receiving PBS alone served as negative controls. Twelve hours post-infection, treatment was initiated with intraperitoneal injections of ciprofloxacin (30 mg/kg) administered twice daily. Forty-eight hours later, mice were euthanized by cervical dislocation, and lung tissue homogenates were serially diluted with PBS and spread onto TSB plates to assess the bacterial load in the lungs.\u003c/p\u003e\u003ch2\u003eElectrophoresis migration shift assays (EMSA)\u003c/h2\u003e\u003cp\u003eThe target regulatory regions of \u003cem\u003ecap/ica\u003c/em\u003e operons were amplified by PCR. Then purified CodY variants were incubated with 30 ng DNA fragments in 20 µL of binding buffer containing 20 mM Tris-Cl [pH 8.0], 50 mM KCl, 2 mM MgCl₂, 5% [v/v] glycerol, 10 mM DTP, 10 mM each of valine, leucine, and isoleucine at room temperature for 30 min. Protein-DNA complexes were resolved on 8% native polyacrylamide gel in 0.5×TBE buffer at 100 V for 1 h in ice bath and transferred to a nylon membrane. Biotin-labeled DNA was detected using chemiluminescent substrate.\u003c/p\u003e\u003ch2\u003eFormaldehyde cross-linking\u003c/h2\u003e\u003cp\u003eIncubate wild-type or mutant CodY protein (1 µg) in 1× cross-linking buffer (100 mM KCl, 15 mM Tris-HCl, pH 7.5) at room temperature for 15 min, in a total volume of 20 µL. Then add formaldehyde to the reaction mixture to a final concentration of 1% (v/v), and incubate at room temperature for 20 min. Quench the formaldehyde-treated samples with 125 mM glycine and incubate at room temperature for 15 min. Then heat each sample for 10 min at 65°C in a reducing buffer containing 50 mM DTT, followed by western blot analysis using SDS-PAGE and specific antibodies.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll \u003cem\u003ein vitro\u003c/em\u003e experiments were performed with at least two independent replicates to ensure reproducibility. Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were carried out using appropriate tests (e.g., Student’s t-test or one-way ANOVA), with significance levels defined as follows: P \u0026lt; 0.05 (*), P \u0026lt; 0.01 (**), P \u0026lt; 0.001 (***), P \u0026lt; 0.0001 (****); “ns” denotes not significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw proteomic data and search results have been deposited to the ProteomeXchange Consortium via the PRIDE\u003csup\u003e45\u003c/sup\u003e partner repository on November 14, 2025, with the dataset identifier PXD070748, and can be accessed with the reviewer account at https://www.ebi.ac.uk/pride using the username: [email protected] and the password: DGf5EliZ919c.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols involving animals were reviewed, evaluated, and formally approved by the Ethics Committee for Animal Experiments of Jinan University (IACUC Approval No. 20260112-02). The entire study was strictly performed in accordance with the institutional guidelines for the care and use of laboratory animals, and all efforts were made to minimize the suffering of experimental animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Xue Ting (Anhui Agricultural University) for providing the knockout strains. This work was supported by the National Natural Science Foundation of China (22377035 and 21977037 to X.S), Guangdong National Science Foundation (2022A1515010674 and 2023A1515011750 to X.S.). International Atomic Energy Agency Coordinated Research Project (25076 to X.S.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are ranked in descending order of their contribution\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMOE Key Laboratory of Tumor Molecular Biology and State Key Laboratory of Bioactive Molecules and Druggability Assessment, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYun Liu, Zhen Wang, Jiamin Qiu, Haiming Wu, Jiayi Wu, Tairan Zhong, Yundan Zheng, Nan Li, Yunpeng Yang, Zhenghua Sun, Qing-Yu He, Xuesong Sun\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eThe experiment was designed by Y.L and X.S., and performed by Y.L., Z.W., J.Q., H.W., J.W., T.Z., and Y.Z.; Clinical multidrug-resistant strains were collected by N.L. and Y.Y.; LC-MS analysis was conducted by Z.S.; The draft manuscript was written by Y.L. and critically revised by X.S., and Q.H. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCorrespondence to Qing-Yu He or Xuesong Sun.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHo, C. 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A. \u003cem\u003eet al.\u003c/em\u003e 2016 update of the PRIDE database and its related tools. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e 44, D447\u0026ndash;D456 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"2-hydroxyisobutyrylation, Staphylococcus aureus, Antibiotic resistance, Fluoroquinolones, CodY","lastPublishedDoi":"10.21203/rs.3.rs-8837885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8837885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Lysine 2-hydroxyisobutyrylation (Khib) controls important cellular physiological processes, but its functions in antibiotic resistance remain poorly understood. Here, we report that Khib positively regulates fluoroquinolone resistance in (). Quantitative proteomics revealed remodeling of Khib across multiple metabolic pathways in ciprofloxacin-resistant strains, and manipulation of Khib altered susceptibility to fluoroquinolones. We identified AcuA as a novel 2-hydroxyisobutyryltransferase in . AcuA deletion impaired biofilm formation, and lowered fluoroquinolone resistance. Mechanistically, AcuA enhances resistance to fluoroquinolones by 2-hydroxyisobutyrylating CodY, which alters the structure and electrostatic potential of DNA-binding domain, thereby weakening its binding to the promoter. Critically, mouse infection models also confirmed that strains with elevated CodY Khib levels exhibited even stronger resistance. Importantly, this signaling pathway was also validated in clinically multidrug-resistant (MDR) isolates. Thus, our study dissects a novel mechanism by which covalent modifications regulate antibiotic resistance in important human pathogens.","manuscriptTitle":"A novel 2-hydroxyisobutyrylation signaling pathway drives fluoroquinolone resistance in Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 09:35:40","doi":"10.21203/rs.3.rs-8837885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-14T14:58:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282623193762200965791313367182467655514","date":"2026-02-23T08:48:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-21T09:42:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T09:23:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T09:09:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Biofilms and Microbiomes","date":"2026-02-10T07:21:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1afab61-4f1b-46fd-b19a-8b7927da9834","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67278066,"name":"Biological sciences/Biochemistry"},{"id":67278067,"name":"Health sciences/Diseases"},{"id":67278068,"name":"Biological sciences/Drug discovery"},{"id":67278069,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-30T09:35:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 09:35:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8837885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8837885","identity":"rs-8837885","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}