Host-derived arginine promotes Klebsiella pneumoniae colonization and dissemination through ArtP–ArgR–capsule signaling

Host-derived arginine promotes Klebsiella pneumoniae colonization and dissemination through ArtP–ArgR–capsule signaling | 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 Host-derived arginine promotes Klebsiella pneumoniae colonization and dissemination through ArtP–ArgR–capsule signaling Fangyou Yu, Ying Zhou, Haojin Gao, Peiyao Zhou, Chunyang Wu, chao cai, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8289577/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Intestinal colonization is a critical precursor to invasive Klebsiella pneumoniae infection, yet the host-derived metabolic cues that license this transition remain unclear. Here, we identify arginine as a consistently enriched amino acid across inflammation-, diet-, and antibiotic-induced gut perturbations. Arginine markedly enhances capsular polysaccharide production and virulence in both hypervirulent ST23-KL1 and carbapenem-resistant ST11-KL64 lineages under conditions reflective of the gut microenvironment. Mechanistically, arginine must be transported by the ArtP ATP-binding transporter to activate the regulator ArgR, which directly binds capsular polysaccharide operon promoters and indirectly upregulates rmpA , forming a conserved ArtP–ArgR–capsule signaling axis. In mouse models, elevated intestinal arginine increases gut colonization, accelerates mucosal invasion, and promotes systemic dissemination, whereas arginase-mediated arginine depletion or loss of ArtP/ArgR abrogates these effects. Together, our findings reveal intestinal arginine as a key host-derived signal that drives K. pneumoniae pathogenic progression and identify arginine metabolism as a tractable therapeutic target. Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology/Clinical microbiology Klebsiella pneumoniae intestinal colonization arginine metabolism Capsular polysaccharide ArgR regulatory pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Klebsiella pneumoniae (Kp) is a major global health threat recognized by the World Health Organization (WHO) as a top-priority pathogen and a member of the ESKAPE group due to its high antibiotic resistance and clinical impact 1 , 2 . It primarily exists in two forms: multidrug-resistant (MDR) and hypervirulent (hv) strains 3 . MDR-Kp accounts for 10–20% of deaths caused by multidrug-resistant bacterial infections over the past 30 years, ranking among the top three most lethal pathogens 4 – 6 . HvKp, typically causing severe systemic infections such as liver abscesses, meningitis, and bacteremia, is associated with mortality rates of 30%–85% 7 . Critically, both MDR, such as carbapenem resistance (CR), and hypervirulence traits are increasingly being found within the same strains, giving rise to hypervirulent carbapenem-resistant Kp (hv-CRKp) 8 – 11 . Among these, the ST11-KL64 lineage has emerged as a predominant epidemic clone in clinical settings and is closely associated with bloodstream infections, and poor patient outcomes 12 – 14 . The rapid global spread of hvKp and CRKp, particularly the epidemic ST11-KL64 lineage, underscores the urgent need to understand factors that enable K. pneumoniae to colonize, disseminate, and cause life-threatening systemic infections. Although K. pneumoniae commonly causes pulmonary, hepatic, and bloodstream infections, colonization typically begins in the gastrointestinal (GI) tract, which serves as the principal reservoir and portal of entry for invasive disease 3 , 15 . Only a small fraction of ingested organisms succeed in establishing gut colonization, and expansion within this niche is often required to initiate systemic infection 3 . Previous studies indicates that intestinal inflammation and antibiotic-induced dysbiosis compromise barrier function and colonization resistance 3 , 16 – 18 . Under such conditions, the risk of stable intestinal colonization increases substantially, and the weakened mucosal barrier predisposes the host to bacterial translocation and invasive infection. However, the key host- or microbe-derived factors that drive this heightened invasiveness within the altered intestinal environment remain poorly defined. Recent evidence highlights that metabolites within the gastrointestinal tract may act as important signals regulating function and pathogenicity of intestinal commensals and pathobionts, including Enterobacteriaceae such as K. pneumoniae 19 – 22 . During intestinal inflammation, the host undergoes metabolic reprogramming characterized by a significant rise in free amino acid levels within the gut lumen 19 , 20 . Consistently, studies of antibiotic treatment and dietary perturbations have demonstrated that several free amino acids become significantly enriched 16 , 17 . These shifts in amino acid availability may serve as nutrient signals or regulatory cues that modulate virulence gene expression in K. pneumoniae , promoting its shift from a commensal colonizer to an invasive pathogen. However, whether alterations in intestinal amino acid composition influence K. pneumoniae adaptation within the gut and its capacity to traverse the intestinal barrier to cause systemic infection remains unknown. In this study, we investigated how intestinal amino acid availability shapes the pathogenic potential of K. pneumoniae . By integrating metabolomic evidence from dietary perturbation, antibiotic treatment, and intestinal inflammation models 16 , 17 , 20 , we identified multiple amino acids consistently enriched in the gut. A major virulence determinant of K. pneumoniae is the production of a thick capsular polysaccharide (CPS), which shields the bacteria from phagocytosis and complement-mediated killing 7 . We therefore examined how these amino acids affect CPS synthesis and virulence. Arginine emerged as a particularly relevant candidate, not only does it accumulate to high levels across diverse perturbations, but it significantly enhanced CPS production in K. pneumoniae . Recent work has shown that arginine increases the mucoid phenotype of hypervirulent K. pneumoniae when provided in a glycerol-based minimal medium, without increasing total CPS abundance, acting primarily through ArgR-dependent activation of rmpADC 23 . These observations suggest that arginine can modulate CPS chain length and viscosity, but do not address whether arginine can enhance CPS biosynthesis itself 23 . Moreover, it remains unclear whether arginine exerts similar regulatory effects under metabolic conditions relevant to the intestinal environment, where glucose, not glycerol, is abundant 24 , or whether arginine influences the capacity of K. pneumoniae to cross the intestinal barrier and seed systemic infection. To answer these key questions, we investigated how arginine affects cps gene expression under glucose-based conditions that mimic the gut milieu. We further examined the molecular requirements for arginine sensing, focusing on the ATP-dependent transporter ArtP and the transcriptional regulator ArgR, both of which participate in arginine metabolism and virulence regulation. Finally, using a murine intestinal colonization model, we assessed whether arginine availability in the gut environment alters K. pneumoniae colonization dynamics, mucosal barrier interactions, and progression toward systemic infection. Together, these approaches allowed us to define how intestinal amino acid perturbations may serve as host-derived signals that modulate K. pneumoniae pathogenic potential. Results Host intestinal amino acid shifts shape K. pneumoniae virulence determinants Intestinal metabolic perturbations caused by host inflammation, dietary fiber deficiency 16 , or antibiotic treatment 17 have all been reported to reshape the gut metabolome in ways that promote K. pneumoniae disseminattion 25 – 27 . To explore the metabolic features shared across these translocation-prone states, we re-analyzed available metabolomic datasets and confirmed that: 1) Inflammation caused by treatment of mice with anti-CD3 antibody leads to elevated levels of free amino acids in the gut lumen 20 (Fig. 1 a); 2) a fiber-free enteral diet drives marked K. pneumoniae expansion accompanied by amino acid accumulation 16 (Fig. 1 b); 3) and antibiotic-induced microbiota depletion enriches nutrients, particularly amino acids, thereby enhancing the growth of carbapenem-resistant Enterobacteriaceae 17 (Fig. 1 c). To systematically evaluate the contribution of these amino acid shifts, we selected ten consistently enriched amino acids in the above studies for experimental assessment of their effects on K. pneumoniae pathogenicity. Given that both the ability to thrive in a given microenvironment and the production of capsular polysaccharide are major determinants of K. pneumoniae virulence, we selected bacterial growth and CPS synthesis as in vitro readouts for amino acid effects, using two representative strains—NTUH-K2044 (ST23-KL1, hvKP) and FK3009 (ST11-KL64, CRKp). All assays were performed in M9 minimal medium supplemented with glucose as the sole carbon source, enabling us to evaluate growth dynamics and CPS production under metabolic conditions relevant to the intestinal environment(rich in glucose) 24 . Most amino acids exerted little effect on bacterial proliferation (Fig. 1 d,e,f and Figure S1 ); alanine modestly inhibited FK3009 growth, tryptophan suppressed NTUH-K2044, while phenylalanine enhanced FK3009. In contrast, glutamine promoted growth in both strains (Figure S1 ). Notably, only arginine, glutamate, and leucine consistently increased CPS production and viscosity in both strains (Fig. 1 g,h). Although glutamate and leucine also enhanced virulence in vitro, their intestinal enrichment under fiber deprivation and antibiotic treatment was far less pronounced than the robust elevation observed for arginine (Fig. 1 b,c). Thus, arginine appears as a particularly relevant amino acid potentially capable of promoting K. pneumoniae pathogenicity in vivo . Arginine enhances capsule gene expression through ArtP-mediated intracellular transport and metabolic sensing Our findings indicated that exogenous arginine markedly enhanced CPS production in K. pneumoniae . This differs from earlier observations showing that, under glycerol-based conditions, arginine increases mucoviscosity without altering total CPS levels. Our results suggest that under glucose-based conditions that better reflect the intestinal environment, arginine can indeed promote CPS biosynthesis, revealing an additional regulatory feature that had not been previously appreciated.. We next sought to investigate the underlying molecular mechanisms. To confirm whether arginine affects the expression of the cps operon, we performed RNA-sequencing on NTUH-K2044 and FK3009 cultured in glucose-based M9 minimal medium with or without L-arginine supplementation. We observed that the astCADBE operon encoding the arginine succinyl transferase pathway for arginine catabolism 22 was significantly upregulated, indicating that exogenous arginine is actively sensed (Fig. 2 a,d). Importantly, several genes within the capsular polysaccharide synthesis cluster were also significantly upregulated in response to arginine exposure in both Kp strains (Fig. 2 b-f). These findings indicated that arginine not only serves as a metabolic substrate but also acts as a signaling cue that reprograms virulence gene expression, which is uncoupled from impacts on replication rates (Fig. 1 d). We next asked whether arginine functions as a signaling molecule or whether its intracellular metabolism is required to activate virulence programs. To answer this question, we attempted to block the transport of arginine into K. pneumoniae . In K.pneumoniae , L-arginine is initially bound by three distinct periplasmic binding proteins and subsequently transported into the cytoplasm via two separate transmembrane complexes (Fig. 2 g) 22 . ArtP is essential for the function of both arginine uptake systems (Fig. 2 g). Using an artP mutant of NTUH-K2044 we found that that when arginine uptake is impaired, exogenous arginine fails to promote CPS production (Fig. 2 h,i). These findings suggest that intracellular uptake of arginine is essential for its role in promoting virulence, implying that its regulatory function is tightly coupled to metabolic processing rather than mere extracellular sensing. The arginine sensor ArgR is a key transcriptional regulator of arginine metabolism in K.pneumoniae . It primarily functions as a repressor of genes involved in arginine biosynthesis in response to elevated intracellular arginine levels, and acts as a coactivator of the astCADBE operon, which mediates arginine catabolism. Previous studies demonstrated that deletion of argR significantly alters viscosity in K. pneumoniae 23 , 28 , raising the possibility that exogenous arginine may influence capsule expression by activating ArgR-dependent regulatory pathways. To test this hypothesis, we performed RT-qPCR and examined the expression levels of argR in the two K. pneumoniae strains following arginine supplementation. In addition, we also assessed the transcription of a key regulatory factor involved in CPS production ( rmpA ), as well as that of a representative transcript from the cps locus ( wzi , the first gene of the polysaccharide polymerization transcript), and a core gene ( wcaJ , encodes the initiating glycosyltransferase) within the cps operon 7 . In these settings, arginine not only upregulated the expression of key genes involved in capsular polysaccharide biosynthesis and known regulatory factor, but also increased the transcriptional level of argR (Fig. 2 j,k). Taken together, our results suggest a model in which arginine promotes capsular polysaccharide production in K. pneumoniae by enhancing the transcription of capsule-related genes. This effect requires arginine uptake and metabolic processing, with ArgR likely serving as a key regulatory mediator. ArgR is essential for arginine-induced capsule expression and pathogenicity in hypervirulent and multidrug-resistant K. pneumoniae strains To further define the role of ArgR in K. pneumoniae capsule formation and pathogenicity, we constructed argR deletion and complemented mutants in both NTUH-K2044 (KL-1) and FK3009 (ST11-KL64). Deletion of argR decreased the production of capsular polysaccharide in both strains, a phenotype that could be rescued upon complementation (Fig. 3 a-d). To comprehensively assess the impact of argR on K. pneumoniae virulence, we employed in vitro cell infection assays (RAW 264.7 Macrophages and primary Kupffer Cells) (Figue 3e,f) and in vivo models, namely Galleria mellonella and murine bloodstream infection (Figure S2 , Fig. 3 h-k). These models allowed us to evaluate the relative contribution of argR to both immune evasion and systemic pathogenicity. Capsular polysaccharide is a critical virulence factor that protects K. pneumoniae from phagocytic clearance. Phagocytosis assays revealed that deletion of the argR gene significantly increased bacterial uptake by both RAW 264.7 macrophages (Fig. 3 e) and primary murine Kupffer cells (Fig. 3 f), indicating that argR is required for K. pneumoniae to evade phagocytosis. Consistently, survival rates of Galleria mellonella (Figure S2 ) and mice (Fig. 3 h-k) systemically infected with the argR mutant were markedly higher compared to those infected with the wild-type strain. Notably, bacterial burdens in key organs such as the liver and spleen were also markedly reduced in the argR mutant (Fig. 3 i,k). These findings demonstrate that argR is essential for K. pneumoniae to evade host immune clearance and maintain full virulence in vivo , likely through its regulation of capsular polysaccharide expression. To determine whether arginine-mediated regulation of capsular polysaccharide depends on the transcriptional regulator ArgR, we assessed capsule production in wild-type, argR knockout, and complemented strains in the presence or absence of exogenous arginine. Arginine supplementation significantly increased capsule production in the wild-type strain, but this effect was significantly abolished in the argR mutant. Notably, restoration of argR expression rescued the arginine-induced capsule enhancement (Fig. 4 a,b). Furthermore, a murine bloodstream infection model was employed to assess whether the virulence-promoting effect of arginine also requires argR . Mice were challenged with either NTUH-K2044 (ST23, KL1, hvKP) or its argR deletion derivative. Supplementation with arginine increased the lethality of the wild-type strain, but had little to no effect on the pathogenicity of the argR mutant (Fig. 4 c). In addition, bacterial load quantification in major organs revealed a 1–2 log increase in the liver and spleen of wild-type–infected mice following arginine supplementation, whereas no significant differences were detected in the argR mutant (Fig. 4 d). These findings indicate that the arginine-induced enhancement of capsular polysaccharide production and virulence in K. pneumoniae is largely dependent on the transcriptional regulator ArgR. Importantly, these effects were observed in both the hypervirulent NTUH-K2044 and the multidrug-resistant FK3009 strains, supporting ArgR-dependent regulation as a generalizable mechanism in K. pneumoniae . Arginine enhances ArgR Binding to cps and rmpA promoters to regulate capsule gene expression Having established that arginine regulates capsular polysaccharide production through activation of ArgR, we next sought to elucidate how ArgR regulate the CPS gene expression. Given that arginine enhances capsule gene expression without affecting bacterial growth, and that ArgR functions as a transcriptional regulator, we hypothesized that arginine promotes CPS production by facilitating ArgR-dependent transcriptional activation. RT-qPCR analysis revealed that deletion of ArgR significantly reduced the transcription of both the capsule regulator rmpA and key genes within the cps cluster in both K. pneumoniae strains. Restoration of argR expression rescued the expression of these genes (Fig. 5 a,b). Furthermore, promoter activity assays confirmed that ArgR can directly enhance the promoter activities of rmpA and critical cps genes, indicating its role in transcriptional regulation of capsule synthesis (Fig. 5 c,d). However, it remained unclear whether ArgR regulates the cps locus directly or indirectly through RmpA. To address this question, we tested for ArgR direct binding to the promoter region of the cps operon independent of RmpA. A previous study demonstrated that ArgR regulates the rmp promoter by binding to a highly conserved ARG box, thereby modulating mucoviscosity in hypervirulent K. pneumoniae 23 . Consistent with these results, our in vitro EMSA assays confirmed ArgR binding to the rmpA promoter region, even at low protein concentrations, indicating a strong and specific interaction (Fig. 5 e). Notably, beyond rmpA , ArgR also bound to the promoter regions of galF and wzi , key transcripts within the cps locus (Fig. 5 g-j). These findings suggest that ArgR regulated capsule synthesis via a dual mechanism: indirectly, through RmpA activation and directly, by targeting cps locus promoters. Previous studies suggested that arginine activates ArgR not only by upregulating its expression, but also by directly binding to ArgR, inducing a conformational change that enhances its DNA-binding affinity and transcriptional regulatory activity. To test this hypothesis, we assessed binding capacity of ArgR to the promoter regions of its target genes in the presence of arginine. First, we determined a sub-saturating concentration of ArgR protein that did not bind to target promoters in standard EMSA conditions. Then, we performed EMSA using such sub-saturating concentrations of ArgR. In the absence of arginine, ArgR showed minimal binding to target promoters. Upon addition of increasing concentrations of L-arginine, DNA-binding was progressively enhanced. Specificity was confirmed by the absence of ArgR binding to the promoter of the negative control gene rpoB , and by competitive EMSA using excess unlabeled probes, which markedly reduced ArgR binding to biotin-labeled probes (Fig. 5 f, k-n). These results demonstrate that arginine directly enhances ArgR binding to target promoters, likely by inducing conformational changes in the transcription factors. Taken together, our results indicated arginine promotes capsular polysaccharide production in K. pneumoniae through activation of the transcriptional regulator ArgR. Upon sensing intracellular arginine, ArgR enhances the transcription of capsule-related genes via two mechanisms: direct binding to cps locus promoters (such as galF and wzi ) and indirect activation through upregulation of the capsule regulator rmpA . This dual regulatory strategy enables K. pneumoniae to coordinate arginine sensing with virulence gene expression. Importantly, this ArgR-dependent mechanism is conserved in both hypervirulent and multidrug-resistant strains, highlighting a general strategy by which K. pneumoniae links metabolic cues to pathogenicity. Local arginine enrichment promotes K. pneumoniae intestinal colonization The above data indicate that exogenous arginine enhances capsular polysaccharide synthesis in both representative hv and CR K. pneumoniae in vitro . We next asked whether arginine enrichment in the intestinal environment likewise affects K. pneumoniae pathogenicity in vivo . Because stable gut colonization represents the critical first step enabling K. pneumoniae to traverse the intestinal barrier and seed systemic infection, we established an intestinal colonization model to evaluate the impact of arginine on this process. Clinically, antibiotic exposure is a major driver of K. pneumoniae overgrowth and persistent colonization; therefore, we focused on an antibiotic-treated mouse model. Previous studies have shown that antibiotic treatment leads to intestinal accumulation of arginine (Fig. 1 c). To determine whether arginine similarly increases in our model and to establish the baseline amino acid landscape, we measured amino acid levels in the intestinal contents of our antibiotic-treated mice using targeted metabolomic profiling. Mice were first administered a broad-spectrum antibiotic cocktail (ampicillin, metronidazole, neomycin, and vancomycin; AMNV) to deplete the intestinal microbiota, followed by oral inoculation with K. pneumoniae and maintenance on vancomycin and metronidazole to sustain dysbiosis (Fig. 6 a). To characterize the amino acid environment generated by this model, we treated mice with the AMNV regimen and then provided PBS for 24 hours before euthanasia. Cecal contents were subsequently collected for targeted metabolomic analysis. Strikingly, arginine exhibited a pronounced accumulation in the antibiotic-treated gut and ranked as the fourth most abundant amino acid detected (Fig. 6 a). These results confirm that antibiotic-induced dysbiosis creates an arginine-enriched intestinal environment, providing a physiologically relevant context in which to assess its effects on K. pneumoniae colonization. Given our in vitro findings that arginine must be imported through the ArtP transporter to activate K. pneumoniae virulence and its downstream metabolic responses, and that ArgR functions as the key regulator mediating this arginine-driven virulence, we selected these two mutants to determine whether arginine metabolism influences K. pneumoniae intestinal colonization. Using an intestinal competition model in which wild-type and mutant strains colonized the same host, we found that deletion of artP , which blocks arginine uptake, abolished arginine-induced CPS enhancement (Fig. 6 b) and markedly impaired intestinal colonization (Fig. 6 c). Likewise, deletion of argR not only attenuated capsule formation and systemic infection but also reduced intestinal colonization by ~ 2 logs compared with the wild-type strain (Fig. 6 c). These results demonstrate that arginine metabolism in K. pneumoniae significantly influences its ability to colonize the gut. To determine whether elevated intestinal arginine directly influences K. pneumoniae colonization in vivo, we used the same antibiotic-treated intestinal colonization model, in which microbiota depletion was achieved with an AMNV cocktail before oral inoculation with NTUH-K2044 or FK3009. Mice were then provided either PBS or arginine-supplemented drinking water (Fig. 6 d,e). Notably, in mice colonized with the hypervirulent NTUH-K2044 strain, all animals receiving arginine supplementation succumbed within 3 days (Fig. 6 f). Fecal CFU counts collected on day 1 showed significantly higher intestinal colonization in the arginine-supplemented group than in controls (Fig. 6 h). Consistent with this, bacterial burdens in the liver and spleen were markedly elevated—by 1–2 logs—indicating enhanced systemic dissemination (Fig. 6 i). In contrast, mice colonized with the less virulent multidrug-resistant FK3009 strain (ST11-KL64, CRKp) exhibited no mortality during the two-week colonization period (Fig. 6 g). Nonetheless, arginine supplementation significantly increased intestinal colonization (Fig. 6 j), and at day 14, higher bacterial loads were detected in both the intestine and systemic organs, including the liver and spleen (Fig. 6 k). Together, these data demonstrate that increasing intestinal arginine availability enhances gut colonization and promotes systemic spread across both hypervirulent and CRKp strains. Elevated host arginine promotes systemic infection by K. pneumoniae Given that intestinal arginine accumulation enhanced gut colonization, facilitated systemic infection, and increased mortality over time, we next sought to determine the impact of arginine on systemic disease and on the colonization/invasion switch. First, we employed a bloodstream infection model, as this approach provides direct and unconfounded assessment of whether arginine supplementation increases K. pneumoniae systemic virulence (Fig. 7 a). We compared the systemic effects of arginine with those of leucine and glutamate, two additional amino acids that increased CPS production in vitro . In this bacteremia model, supplementation with arginine, glutamate, or leucine markedly reduced host survival and promoted bacterial dissemination to the liver, spleen, lungs, and kidneys (Figure S3). Notably, arginine ccelerated mortality in mice infected with either NTUH-K2044 (ST23-KL1, hvKp) or FK3009 (ST11-KL64, CRKp), demonstrating that arginine broadly potentiates K. pneumoniae virulence during systemic infection (Fig. 7 b). Having established that arginine directly enhances systemic virulence in the bloodstream, we next asked whether arginine also accelerates the transition from gut colonization to systemic infection. To determine whether arginine promotes mucosal invasion, we conducted short-term colonization experiments. AMNV-treated mice received oral K. pneumoniae inoculation and were given either control drinking water or arginine-supplemented water, then sacrificed 12 hours later (Fig. 7 c). Small intestinal tissue, cecal tissue, and systemic organs (liver and spleen) were collected to quantify the rapid effects of arginine on mucosal crossing versus systemic expansion. To further clarify whether this effect requires arginine uptake and signaling, we included Δ artP and Δ argR mutants as controls. In agreement with our previous data, intestinal arginine accumulation markedly increased the ability of the NTUH-K2044 wild-type strain to invade both small intestinal and cecal tissues, with bacterial loads rising by approximately two orders of magnitude compared with PBS controls (Fig. 7de). Consistent with this enhanced mucosal invasion, we observed elevated bacterial burdens in the liver and spleen, although the increase in systemic organs was more modest (~ 1 log) (Fig. 7fg). These findings indicate that, in hosts whose intestine has been colonized by Klebsiella penumoniae , arginine primarily promotes systemic infection by facilitating the breach of the intestinal mucosal barrier rather than by accelerating systemic expansion. Of note, Δ artP and Δ argR mutants failed to respond to exogenous arginine, showing no significant increase in tissue invasion or systemic dissemination. This confirms that the ability of arginine to promote K. pneumoniae systemic infection requires both ArtP-dependent arginine uptake and ArgR-mediated transcriptional regulation. Targeting intestinal arginine with arginase as a potential strategy to limit K. pneumoniae infection Building on the observation that arginine enhances K. pneumoniae intestinal colonization and virulence, we hypothesized that reducing luminal arginine might attenuate these effects. To test this possibility, we collected cecal contents from three antibiotic-treated mice and resuspended them in PBS to mimic the intestinal environment ex vivo (Fig. 8 a). NTUH-K2044 wild-type or the artP deletion mutant were inoculated at 10 6 CFU into cecal content left either untreated (PBS) or spiked-in with arginase, an enzyme that converts arginine into ornithine and urea, thus degrading luminal arginine (Fig. 8 a). Strikingly, arginase treatment significantly reduced the expansion of NTUH-K2044 in this simulated gut environment, whereas the artP knockout strain showed no detectable difference between control and arginase-treated conditions (Fig. 8 b,c). These results indicate that enzymatic depletion of luminal arginine directly impairs K. pneumoniae growth within the gut environment and that this effect strictly depends on ArtP-mediated arginine uptake. Although arginine does not significantly alter bacterial growth in vitro , the intestinal environment imposes additional selective pressures that differ from nutrient-rich laboratory conditions. Our findings show that arginine primarily enhances CPS production rather than functioning as a growth substrate. Because elevated capsule expression enables K. pneumoniae to resist complement-mediated killing—and active complement is known to be present in the gut—arginine likely promotes bacterial expansion by improving survival against host defenses rather than by increasing intrinsic replication. This model explains why removing arginine from the gut environment reduces bacterial expansion despite its minimal impact on growth in vitro. Moreover, The lack of response in the artP mutant highlights ArtP as a potential therapeutic target for disabling arginine-dependent virulence pathways. Together, these findings demonstrate that lowering intestinal arginine levels, such as through arginase treatment, may diminish K. pneumoniae intestinal colonization and reduce the risk of systemic infection, while simultaneously identifying ArtP as a promising metabolic–virulence target for therapeutic intervention. Discussion This study uncovers a previously unrecognized link between host amino acid metabolism and K.pneumoniae virulence. Increasing evidence highlights gut colonization as a critical reservoir for healthcare-associated pathogens, including K. pneumoniae 3 , 15 . However, the specific host-derived signals that drive virulence acquisition and enable bacterial translocation across the intestinal barrier remain poorly defined. Here, we show that perturbations of the intestinal environment, such as those occurring during inflammation 20 , antibiotic exposure 17 , or dietary imbalance 16 , lead to altered amino acid profiles, and that these metabolic changes, particularly arginine accumulation, directly enhance K. pneumoniae colonization and systemic pathogenicity (Fig. 9 ). Among the amino acids detected in the gut, arginine was particularly notable. Moreover, in diverse contexts including inflammation, diet-induced dysbiosis, and antibiotic treatment, arginine accumulates significantly in the intestinal lumen 16 , 17 , 20 . This consistent enrichment underscores arginine as a physiologically relevant signal that strongly influences K. pneumoniae pathogenesis. Previous studies have reported that exogenous arginine can induce the hypermucoviscous phenotype in hypervirulent K. pneumoniae under in vitro conditions 23 . Our findings build upon and extend these observations by showing that arginine not only promotes capsule production but also enhances intestinal colonization and systemic virulence in vivo , and that this effect is conserved across both hypervirulent and multidrug-resistant strains. Thus, our study provides a broader and more generalized mechanistic framework linking arginine availability to K. pneumoniae pathogenic potential. The arginine transporter ArtP proved indispensable for this phenotype. Deletion of artP abolished arginine-induced capsule enhancement and markedly reduced intestinal colonization, highlighting the requirement for intracellular uptake and metabolism. These findings indicate that arginine does not simply act as an extracellular cue but must be actively imported to influence bacterial physiology and persistence within the host gut. The transcriptional regulator ArgR emerged as another critical determinant. Loss of argR abolished the virulence-promoting effect of arginine and led to a ~ 2-log reduction in intestinal colonization. Consistent with previous reports, ArgR activity is enhanced by arginine abundance in the intestinal milieu, which has been shown to induce type VI secretion system (T6SS) expression, thereby promoting asymptomatic colonization 29 . Importantly, our work extends this knowledge by demonstrating that ArgR directly binds to promoters within the cps locus and drives capsule gene expression, with exogenous arginine strengthening this interaction. Thus, ArgR integrates nutrient sensing with both capsule regulation and secretion system activity, placing it at the center of a multifaceted regulatory network controlling colonization and systemic infection. The capsule polysaccharide itself represents an important determinant of intestinal persistence 30 . Prior studies have shown that capsule-deficient K. pneumoniae strains exhibit impaired colonization and reduced competitiveness within the gut niche 30 . Together with our findings, this supports a model in which arginine availability, ArtP-mediated uptake, and ArgR-dependent transcriptional regulation converge on capsule production and secretion system activation to enable successful intestinal colonization and dissemination. We suggest that changes in intestinal amino acid levels play an important role in promoting K. pneumoniae colonization and translocation into the bloodstream. This concept is supported by recent observations in other enteric pathogens 22 , 31 , 32 . For example, commensal yeast has been shown to increase intestinal arginine levels, which in turn promote Salmonella Typhimurium virulence by enhancing fitness and invasion 32 . Similarly, increased concentrations of ornithine and glutamate in the gut markedly enhance Acinetobacter baumannii colonization 31 . These studies, together with our findings, support a broader principle in which dietary or microbiota-derived amino acids act as conserved cues that are exploited by multiple gut pathobionts to trigger virulence programs and overcome colonization resistance. These results also carry important clinical significance. Intestinal metabolic disturbances are common in critically ill patients, premature infants, and individuals receiving broad-spectrum antibiotics 3 . In such settings, accumulation of free amino acids may inadvertently promote colonization, barrier translocation, and bloodstream infection by opportunistic pathogens such as K. pneumoniae . Recognizing this risk highlights the need to consider host metabolic context when designing nutritional or therapeutic strategies. Future integration of clinical nutrition studies with microbiome and metabolome profiling will be crucial to evaluate whether amino acid supplementation in vulnerable hosts contributes to the risk of invasive K. pneumoniae infections. Beyond defining the molecular basis through which intestinal arginine promotes K. pneumoniae virulence, our ex vivo analyses provide further functional support for the translational relevance of this metabolic–virulence axis. By enzymatically depleting arginine in antibiotic-treated cecal contents using arginase, we observed a marked reduction in the expansion of the wild-type NTUH-K2044 strain, whereas the artP mutant (unable to import arginine) remained unaffected. These findings reinforce that luminal arginine is not merely a nutrient but an active regulatory cue that enhances bacterial fitness within the gut environment. Importantly, they also suggest that targeted removal of intestinal arginine can attenuate K. pneumoniae proliferation in the presence of complex metabolites. This raises the possibility that enzymatic modulation of luminal arginine levels, or therapeutic strategies aimed at restricting arginine availability, could reduce gut colonization and limit progression to systemic infection. Moreover, the lack of response in the artP mutant highlights arginine transport as a druggable vulnerability within the ArtP–ArgR–CPS pathway, offering a mechanistically informed avenue for therapeutic intervention. In summary, our study identifies a conserved, host-driven mechanism in which intestinal amino acid alterations, particularly arginine enrichment, enhance K. pneumoniae virulence through ArtP- and ArgR-dependent pathways. These effects were consistently observed in both hypervirulent and carbapenem-resistant strains, underscoring their generalizability across clinically relevant lineages. By linking metabolic remodeling to capsule biosynthesis and intestinal persistence, our findings highlight host-derived arginine as a key driver of K. pneumoniae pathogenicity and a potential target for therapeutic intervention. Materials and Methods Bacterial strains NTUH-K2044, a well-characterized ST23-KL1 strain, was originally isolated from a patient with a liver abscess and serves as a classical hvKp model 33 . Another strain, FK3009 (Accension number: SAMN43180534) was isolated from a 72-year-old patient with septicemia. This strain represents a typical ST11-KL64 epidemic clone of CRKp that has acquired a virulence plasmid conferring a hypervirulent phenotype.All strain information is provided in the Table S1 . Metabolomics analysis Data on metabolic alterations in the intestinal environment under acute inflammatory 20 (Dataset-1), dietary perturbation 16 (Dataset-2), and antibiotic treatment 17 (Dataset-3) conditions were obtained from previously published studies and publicly available datasets. Detailed experimental procedures for each model have been described previously, and all metabolomic data were integrated into a unified dataset for comparative analysis in this study. Growth curve in vitro To evaluate whether amino acids elevated in the intestinal environment during inflammation affect K. pneumoniae growth, in vitro growth curve assays were performed in the presence of varying concentrations of individual amino acids. K.pneumoniae strains were cultured overnight in Luria–Bertani (LB) medium at 37°C with aeration. Overnight cultures were adjusted to an optical density at 600 nm (OD₆₀₀) corresponding to approximately 1 × 10⁸ CFU/mL, followed by a 1:100 dilution into M9 minimal medium supplemented with individual amino acids as indicated. For arginine-supplemented cultures, bacterial growth was monitored manually by measuring OD₆₀₀ at regular intervals over a 24 h period. For all other amino acids, OD₆₀₀ measurements were recorded every 15 min for 24 h using a TECAN Infinite 200 PRO plate reader (TECAN) under aerobic conditions. Mucoviscosity Assay and Capsule Quantification Because capsular polysaccharide is the major virulence determinant of K. pneumoniae , CPS production was evaluated in vitro using a semiquantitative mucoviscosity assay and uronic acid quantification. For the mucoviscosity assay, overnight cultures grown in LB broth were diluted 1:100 into fresh medium and incubated at 37°C with shaking. After 6 h of growth, cultures were centrifuged at 1,000 × g for 5 min, and the optical density of the supernatant at 600 nm (OD₆₀₀) was measured to assess mucoviscosity, as described previously (Ratio: OD600 final /OD600 innitial ) 34 . For uronic acid quantification, K. pneumoniae strains were cultured for 6 h, and 500 µL of culture was mixed with 100 µL of 1% Zwittergent 3–12 detergent. Samples were heated for 20 min at 50°C and centrifuged at 13,000 × g for 5 min. Supernatants (300 µL) were mixed with 1.2 mL of absolute ethanol, incubated on ice for 20 min, and centrifuged at 13,000 × g for 5 min. Pellets were dried and resuspended in 200 µL of sterile water, followed by the addition of 1.2 mL of 12.5 mM sodium tetraborate in concentrated sulfuric acid. After heating at 100°C for 5 min and cooling on ice for 10 min, 20 µL of 0.15% 3-hydroxydiphenyl reagent was added. Samples were incubated for 5 min at room temperature, and absorbance was measured at 520 nm to quantify uronic acid content, as described previously 34 . RNA-seq and RT-qPCR To investigate the mechanisms by which arginine promotes capsular polysaccharide production, total RNA was extracted from K. pneumoniae strains incubated with or without arginine, and transcriptome sequencing was performed. All raw sequencing data have been deposited in the NCBI database (SAMN51231398-SAMN51231401), and RNA-seq library preparation, sequencing, and bioinformatic analyses were conducted by Shanghai Meiji Biotechnology Co., Ltd.. To validate the RNA-seq results, RT-qPCR was performed to quantify the mRNA expression levels of key genes involved in arginine metabolism and CPS biosynthesis. In addition, RT-qPCR was used to examine the role of the transcriptional regulator ArgR in controlling the expression of major CPS transcripts. Primer sequences are listed in Table S2 , and detailed methods were performed as described in our previous publication. Construction of argR and artP mutants and pACYC- argR complementation plasmid Deletion mutants of argR and artP were generated using the λ-Red homologous recombination system as previously described. Primer sequences used for mutant construction are listed in Table S2 , and all bacterial strains and plasmids employed in this study are summarized in Table S1 . For genetic complementation, the pACYC- argR plasmid was assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs) according to the manufacturer’s instructions. The vector backbone and argR insert were PCR-amplified using Q5 High-Fidelity 2× PCR Master Mix (NEB), purified, and assembled into the final plasmid construct. The resulting plasmid was transformed into the appropriate electrocompetent K. pneumoniae strains as described previously. Transmission Electron Microscopy To directly visualize whether argR deletion affects capsular polysaccharide expression, transmission electron microscopy (TEM) was employed to compare capsule thickness among the wild-type, argR knockout, and complemented K. pneumoniae strains. K. pneumoniae cells in the mid-logarithmic growth phase were harvested and fixed overnight at 4°C in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4). Fixed samples were washed twice with 0.1 M phosphate buffer, post-fixed in 1% osmium tetroxide for 1 h at room temperature, and rinsed sequentially in 0.1 M phosphate buffer and distilled water. Samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 95% for 15 min each, followed by 100% ethanol twice for 10 min each). Dehydrated specimens were infiltrated and embedded in Spurr’s resin and polymerized at 60°C for 48 h. Ultrathin sections were prepared and examined using a Tecnai G2 Spirit Twin transmission electron microscope, and images were digitally recorded. Macrophage and Kupffer Cell Phagocytosis Assays The ability of K. pneumoniae strains to resist phagocytosis was evaluated using both murine macrophages and primary liver Kupffer cells. For infection assays, macrophages or Kupffer cells were seeded into 24-well plates at a density of 1 × 10⁵ cells/well and infected with K. pneumoniae strains at a multiplicity of infection (MOI) of 50. Plates were centrifuged at 200 × g for 5 min to facilitate bacterial contact and incubated at 37°C with 5% CO₂ for 1.5 h to allow phagocytosis. After infection, cells were washed three times with phosphate-buffered saline (PBS) and incubated for an additional 1.5 h in medium containing meropenem (100 µg/mL) to eliminate extracellular bacteria. Following three further washes with PBS, cells were lysed with 0.2% Triton X-100 for 20 min. Serial dilutions of the lysates were plated on LB agar to enumerate intracellular bacteria. All assays were performed in triplicate using three independent biological replicates per strain. β-galactosidase assays To investigate the regulation of ArgR on the promoter activity of genes within the cps operon, promoter regions of rmpA , galF , and wzi were amplified from K. pneumoniae strains NTUH-K2044 and FK3009 and cloned upstream of the lacZ reporter gene. Promoter activity was quantified by measuring β-galactosidase activity using a modified Miller assay in bacterial cultures harvested at mid-logarithmic growth phase. Enzyme inhibitors were added during the final step, and cell lysates were prepared using an ultrasonic disruptor to ensure complete release of β-galactosidase. All assays were performed in duplicate 35 . Protein Purification and Electrophoretic Mobility Shift Assay (EMSA) The ArgR expression plasmid was commercially constructed by GenScript (Nanjing, China). Briefly, the argR gene was synthesized and inserted into the pET-30a(+) vector using NdeI and HindIII restriction sites, generating a kanamycin-resistant construct. The recombinant plasmid was verified by restriction digestion and confirmed by sequencing. For recombinant protein production, the expression plasmid was transformed into E. coli BL21(DE3). Protein expression and purification were performed by GenScript using a standard Ni-IDA affinity chromatography system. In brief, expression was induced with IPTG at low temperature, bacterial cells were lysed by sonication, and the clarified lysate was applied to a Ni-IDA column. Bound protein was eluted with imidazole and dialyzed into storage buffer (10 mM HEPES, pH 7.8, 150 mM NaCl, 5% glycerol, 1 mM DTT). The purified ArgR protein was filtered through a 0.22 µm membrane, aliquoted, and stored at − 80°C. Protein purity was confirmed by SDS-PAGE. For EMSA, DNA probes corresponding to the promoter regions of target genes were amplified by PCR with primers listed in Table S2 , purified, and end-labeled with [γ-³²P]ATP using T4 polynucleotide kinase (New England Biolabs). Binding reactions were performed by incubating purified ArgR protein with labeled probes in binding buffer (10 mM Tris-HCl, pH 7.5; 50 mM KCl; 1 mM DTT; 5 mM MgCl₂; 0.05% Nonidet® P-40; 2.5% glycerol; 50 mM acetyl phosphate) at 37°C for 30 min. Samples were separated on 4% native polyacrylamide gels in 0.5× Tris-borate-EDTA (TBE) buffer at 90 V for 40 min at 4°C. Following electrophoresis, DNA–protein complexes were transferred to a nylon membrane in 0.5× TBE buffer at 4°C using a constant current of 380 mA for 1 h. Membranes were UV-crosslinked, blocked, and incubated with stabilized streptavidin–HRP conjugate (1:200 in blocking buffer). After washing, signals were detected by chemiluminescence using a digital imaging system. Murine Intestinal Colonization Model To assess gut colonization and translocation, mice were pretreated with a combination of ampicillin, metronidazole, neomycin, and vancomycin (AMNV; each at 0.5 g/L, put in the drinking water) for three days prior to K. pneumoniae inoculation, and then flush out with the combination of vancomycin and metronidazole (each at 0.5 g/L) for two days to deplete endogenous microbiota. A 200 µL of the bacterial suspension (10 4 CFU for NTUH-K2044;10 5 CFU for FK3009) was administered to mice via oral gavage. Following oral inoculation, mice received drinking water containing vancomycin (0.5 g/L) and metronidazole (0.5 g/L) either alone or in combination with the L-arginine throughout the study period. Vancomycin and metronidazole were included to suppress competing microbiota and maintain K. pneumoniae colonization. Fecal samples were collected on days 1, 3, 7, and 14 post-infections to monitor intestinal colonization. At corresponding time point, mice were euthanized, and bacterial burdens in key organs, including the liver and spleen, were quantified by plating serial dilutions of tissue homogenates on LB agar to assess systemic dissemination from the intestinal tract. Metabolomic analysis of cecal contents in the AMNV antibiotics treated mice To characterize amino acid alterations induced by the antibiotic-treated colonization model used in this study, eight SPF female BALB/c mice (6–8 weeks old) were subjected to microbiota depletion following the previously described AMNV protocol. Mice were then provided sterile PBS in drinking water for an additional 24 hours to remove residual antibiotics. All animals were euthanized thereafter, and cecal contents were immediately collected under sterile conditions, flash-frozen in liquid nitrogen, and stored at − 80°C. Targeted amino acid metabolomic profiling was performed to determine the baseline luminal amino acid distribution associated with the antibiotic-treated model employed throughout this study. Targeted amino acid metabolomic profiling of mouse cecal contents was conducted using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS; Agilent 1290 Infinity II system and QTRAP 6500 + mass spectrometer, SCIEX). Approximately 20 mg of frozen cecal content was extracted with prechilled methanol/water (4:1, v/v) containing internal standards, followed by homogenization, sonication, derivatization with FDAA, and centrifugation. Metabolites were separated on an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 µm) using a 5 mM ammonium acetate–acetonitrile gradient. Data were acquired in multiple reaction monitoring (MRM) mode, and analyte quantification was achieved using isotope-labeled internal standards and external calibration curves (R² >0.99). The limits of detection and quantification were established according to FDA bioanalytical method validation guidelines, ensuring analytical precision and reproducibility (RSD < 20%). All the original metabolomic data were listed in data-set-4. Intestinal Colonization Competition Assay in Mice To evaluate the role of arginine metabolism in intestinal colonization by K. pneumoniae , we performed colonization competition assays using the hypervirulent strain NTUH-K2044, along with isogenic deletion mutants lacking artP (a critical transporter) or argR (a key transcriptional regulator). The intestinal colonization model was established as described previously. Briefly, mice were inoculated by gavage with a 1:1 mixture of the wild-type strain and the corresponding mutant. Fecal samples were collected on days 1, 3, and 7 post-inoculation, and the competitive index was calculated by comparing mutant to wild-type bacterial counts to assess dynamic changes over time. Galleria mellonella Infection Model Galleria mellonella infection assays also applied to assess the bacterial virulence. Briefly, ten larvae weighing approximately 300 mg were randomly assigned to each experimental group. For infection, 10 µL of bacterial suspension (10⁶ CFU/mL in PBS) was injected into the last left proleg using a Hamilton syringe. Control larvae received 10 µL of sterile PBS. Following infection, larvae were incubated at 37°C in the dark and monitored for 96 h. Larvae were considered dead if they failed to respond to repeated physical stimulation. Survival was recorded at regular intervals, and data were analyzed to assess differences in virulence among strains. Murine Bloodstream Infection Model To evaluate K. pneumoniae virulence under different experimental conditions, murine infection models were established to assess host survival and bacterial dissemination to key organs. For systemic infection, K. pneumoniae strains were cultured overnight in LB broth at 37°C, washed, and resuspended in sterile PBS. Female BALB/c mice (6–8 weeks old; specific pathogen-free) were injected via the tail vein with 100 µL of bacterial suspension (10 4 CFU for NTUH-K2044;10 6 CFU for FK3009). For experiments involving exogenous amino acids, bacteria were pre-incubated with the indicated amino acid in PBS for 3 hours prior to injection and keep drinking water supplemented with corresponding amino acids. Survival was monitored over 7 days, and bacterial loads in the liver and spleen were quantified at designated time points by plating serial dilutions of tissue homogenates on LB agar. Short-term (12-hour) intestinal colonization model to assess early mucosal invasion and systemic dissemination To quantify the early impact of intestinal arginine enrichment on mucosal invasion and systemic spread, a short-term colonization model was established. Mice were first treated with the AMNV antibiotic protocol to deplete commensal microbiota, followed by vancomycin/metronidazole washout as described above. Animals were then orally inoculated with 1 × 10⁵ CFU of NTUH-K2044 (wild-type, ΔartP, or ΔargR strains). Immediately after gavage, mice were provided either sterile drinking water or water supplemented with 10mM L-arginine. At 12 h post-inoculation, mice were euthanized, and tissues were aseptically harvested, including small intestinal segments (ileum), cecal tissue, liver, and spleen. Intestinal tissues were thoroughly washed to remove luminal bacteria, homogenized, and plated to determine tissue-associated bacterial burdens. Liver and spleen homogenates were similarly plated to quantify early systemic dissemination. This model allowed discrimination between arginine-driven enhancement of mucosal penetration and downstream systemic expansion, and enabled assessment of whether these effects require ArtP-mediated arginine uptake and ArgR-dependent transcriptional regulation. Ex vivo arginase treatment of cecal contents to assess arginine-dependent K. pneumoniae expansion To evaluate whether enzymatic depletion of luminal arginine limits K. pneumoniae expansion in the gut environment, an ex vivo cecal content assay was established. Cecal contents were collected from three antibiotic-treated mice (AMNV protocol as described above), pooled, and resuspended in sterile PBS at a ratio of 100 mg/mL to mimic the intestinal milieu. The suspension was homogenized, briefly centrifuged to remove large debris, and aliquoted into equal volumes. Recombinant L-arginase (MCE) was added to the treatment group at a final total amount of 1 U per tube, while control tubes received an equal volume of PBS. After a 30-min pre-incubation at 37°C to allow enzymatic depletion of free arginine, each tube was inoculated with 1 × 10⁶ CFU of NTUH-K2044 wild-type or the Δ artP mutant. Samples were incubated at 37°C for 6h, 12h, and 24h under anaerobic environment, after which bacterial counts were determined by serial dilution and plating on LB agar. Statistical analysis All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA) and R (version 4.2.2). Data are presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) as indicated in figure legends. For comparisons between two groups, a two-tailed unpaired Student’s t -test was used for normally distributed data, and a Mann–Whitney U test was applied for nonparametric data. Comparisons among multiple groups were analyzed using a one-way or two-way analysis of variance (ANOVA) followed by Tukey’s or Sidak’s multiple-comparison test. Competitive index assays were evaluated using a one-sample t-test or Wilcoxon signed-rank test to determine whether the mean log₁₀(CI) differed from zero. RNA-seq differential expression analysis was performed using DESeq2, with significance defined as adjusted p < 0.05 and |log₂(fold change)| ≥ 1. All experiments were performed in at least three independent biological replicates, and statistical significance was defined as p < 0.05. Exact p -values and statistical tests used for each dataset are reported in the corresponding figure legends. Declarations Contributors FY, LC, and SB contributed to conceptualization, supervision, and designed the study. YZ contributed to funding acquisition. YZ, and HG performed data analysis, including methodology, software, and interpretation. YZ, HG, SB, LC, and FY verified the underlying data. YZ, FY, LC, and SB wrote the original draft. PZ, CW, CC, BW, HZ, JF, and JZ contributed to the interpretation of data. All the authors contributed to the manuscript drafting. All authors read and approved the final version of the manuscript. Data availability statement All raw RNA sequencing data have been deposited in the NCBI database (SAMN51231398-SAMN51231401). https://www.ncbi.nlm.nih.gov/bioproject/1328593 Acknowledgements We thank the authority of NTUH-K2044 by Professor Jin-Town Wang from Department of Internal Medicine, National Taiwan University Hospital. Ethics approval and consent to participate The informed consent was obtained from all subjects and/or their legal guardian. The research protocol was approved by the Ethics Committee of Shanghai Pulmonary Hospital(K24-096Y). Competing interests The authors report that there are no competing interests to declare. Funding This study was supported by the [National Natural Science Foundation of China] under Grant number [82472287, 82202564], and [Shanghai “Chen Guang” project] under Grant number [23CGA22]. 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Supplementary Files suggestedreviewers.docx suggested reviewers Metabolomicsdataset141122.xlsx metabolomic dataset SupplementaryMaterials.docx Supplementary materials Cite Share Download PDF Status: Under Review Version 1 posted 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. 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05:58:50","extension":"html","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167030,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/4a2295170affcb7b6d031844.html"},{"id":98440952,"identity":"7f6207c6-1366-4d5d-968b-655720e12055","added_by":"auto","created_at":"2025-12-17 17:04:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":740614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntestinal amino acid perturbations promote capsular polysaccharide production in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK.pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.(abc) \u003c/strong\u003eIntestinal metabolomic shifts under different perturbations. (\u003cstrong\u003ea)\u003c/strong\u003e, Targeted metabolomic profiling of anti-CD3–induced acute intestinal inflammation (Dataset-1) shows significant accumulation of multiple free amino acids in the gut lumen, including a robust increase in arginine. (\u003cstrong\u003eb)\u003c/strong\u003e, Metabolomic analysis of mice fed a fiber-free enteral diet (Dataset-2) demonstrates marked expansion of \u003cem\u003eK. pneumoniae\u003c/em\u003e accompanied by elevated luminal amino acids, with arginine among the most enriched metabolites. (\u003cstrong\u003ec)\u003c/strong\u003e, Antibiotic-induced microbiota depletion (Dataset-3) results in nutrient enrichment dominated by free amino acids, including substantial arginine accumulation, which favors the expansion of carbapenem-resistant \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. (\u003cstrong\u003ed–f)\u003c/strong\u003e, Growth curves of NTUH-K2044 (ST23-KL1) and FK3009 (ST11-KL64) cultured in glucose-based M9 minimal medium supplemented with the indicated amino acids show minimal effects on proliferation. (\u003cstrong\u003eg–h)\u003c/strong\u003e, Capsules polysaccharide (CPS) viscosity assays reveal that only arginine, glutamate, and leucine increase mucoviscosity and CPS production across both strains. Collectively, these data identify arginine as a consistently enriched intestinal metabolite across translocation-permissive states and a potent enhancer of \u003cem\u003eK. pneumoniae\u003c/em\u003eCPS-associated virulence traits \u003cem\u003ein vitro\u003c/em\u003e.Data represent mean ± SD of three biological replicates. Statistical significance was determined using one-way ANOVA with multiple comparisons (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"OnlineFigure1XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/8dee18344f47efa573187f45.png"},{"id":98371924,"identity":"c5c9bea9-f93b-4df5-bd1c-7a3b22a23660","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1539632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArginine uptake is required for induction of capsular polysaccharide biosynthesis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK.pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eTranscriptomic profiling of NTUH-K2044 and FK3009 in response to exogenous arginine.\u003cstrong\u003e (a,d) \u003c/strong\u003eKEGG enrichment analysis of upregulated genes highlights arginine and proline metabolism as significantly enriched pathways. \u003cstrong\u003e(b,e) \u003c/strong\u003eVFDB classification shows that arginine exposure increases expression of virulence-associated genes, particularly those related to immune modulation. \u003cstrong\u003e(c,f) \u003c/strong\u003eCircos plots illustrate representative arginine-induced genes (\u003cem\u003egalF, ugd,\u003c/em\u003e and \u003cem\u003ewcaJ\u003c/em\u003e) within the \u003cem\u003ecps \u003c/em\u003ecluster, indicating activation of capsule biosynthesis. Requirement of arginine transporter ArtP for capsule induction.\u003cstrong\u003e (g)\u003c/strong\u003e Schematic model of arginine transport via the ArtPQM system and its metabolic utilization. Quantification of CPS production using uronic acid assay\u003cstrong\u003e(h)\u003c/strong\u003e and mucoviscosity test\u003cstrong\u003e(i) \u003c/strong\u003eshows that exogenous arginine enhances capsule formation in NTUH-K2044, whereas deletion of \u003cem\u003eartP\u003c/em\u003e abolishes this effect. Data are presented as mean ± SD from three biological replicates (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, one-way ANOVA). Arginine enhances expression of capsule-related genes. RT-qPCR analysis demonstrates that arginine supplementation significantly upregulates transcription of capsule biosynthesis genes (\u003cem\u003ewcaJ, wzi\u003c/em\u003e) and the capsule regulator \u003cem\u003ermpA\u003c/em\u003ein both NTUH-K2044 \u003cstrong\u003e(j)\u003c/strong\u003e and FK3009 \u003cstrong\u003e(k)\u003c/strong\u003e. These findings confirm that arginine-mediated activation of capsule formation involves both structural and regulatory components and is conserved across hypervirulent and carbapenem-resistant strains. Data represent mean ± SD of three independent experiments (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"OnlineFigure2XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/6dc9262a157d0e294fcef940.png"},{"id":98371937,"identity":"9f011682-c23d-4ba7-883f-42cbfcef62da","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1971183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArgR is required for capsule production, resistance to phagocytosis, and full virulence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eQuantification of capsular polysaccharide production in wild-type, Δ\u003cem\u003eargR\u003c/em\u003e, and complemented strains. \u003cstrong\u003e(a,b)\u003c/strong\u003e NTUH-K2044 and FK3009 strains were assessed by uronic acid content and mucoviscosity ratio. Deletion of \u003cem\u003eargR\u003c/em\u003emarkedly reduced CPS levels, which were restored upon complementation. Data represent mean ± SD from three independent experiments (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, one-way ANOVA). Transmission electron microscopy (TEM) images of NTUH-K2044 \u003cstrong\u003e(c)\u003c/strong\u003e and FK3009 \u003cstrong\u003e(d) \u003c/strong\u003ewild-type, Δ\u003cem\u003eargR\u003c/em\u003e, and complemented strains. Red arrows indicate capsule thickness; capsule was significantly reduced in Δ\u003cem\u003eargR\u003c/em\u003e mutants and restored in complemented strains. Phagocytosis assays. \u003cstrong\u003e(e)\u003c/strong\u003eRAW 264.7 macrophages and \u003cstrong\u003e(f)\u003c/strong\u003eprimary murine Kupffer cells infected with wild-type or Δ\u003cem\u003eargR\u003c/em\u003e strains. Deletion of \u003cem\u003eargR\u003c/em\u003e significantly increased bacterial uptake, indicating impaired resistance to phagocytosis. Data are presented as mean ± SD from three biological replicates (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test). Murine bloodstream infection model. Survival curves of mice infected with NTUH-K2044 \u003cstrong\u003e(h,i) \u003c/strong\u003eor FK3009 \u003cstrong\u003e(j,k)\u003c/strong\u003e. Deletion of \u003cem\u003eargR\u003c/em\u003e attenuated lethality compared to wild type. Bacterial burdens in organs collected at 48 h (NTUH-K2044, i) and 120 h (FK3009, k) post-infection. Δ\u003cem\u003eargR\u003c/em\u003estrains exhibited significantly reduced colonization of the liver, lung, kidney, and spleen. Data represent mean ± SD (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"OnlineFigure3XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/75d306dcc2eabab87d370de3.png"},{"id":98371927,"identity":"38b88e75-10ae-43e5-a9e2-f3a293cb97af","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArginine-induced capsule production and virulence depend on ArgR in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK.pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eCapsule quantification in wild-type, Δ\u003cem\u003eargR\u003c/em\u003e, and complemented (Δ\u003cem\u003eargR-C\u003c/em\u003e) strains with or without exogenous arginine. \u003cstrong\u003e(a)\u003c/strong\u003eNTUH-K2044 and \u003cstrong\u003e(b) \u003c/strong\u003eFK3009. Arginine supplementation significantly enhanced CPS production in wild-type and complemented strains but not in the Δ\u003cem\u003eargR\u003c/em\u003emutant. Data represent mean ± SD from three independent experiments (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, one-way ANOVA). Murine bloodstream infection model using NTUH-K2044 and its Δ\u003cem\u003eargR\u003c/em\u003e mutant.\u003cstrong\u003e (c)\u003c/strong\u003e Kaplan–Meier survival curves. Arginine supplementation increased lethality in wild-type but not in Δ\u003cem\u003eargR\u003c/em\u003e-infected mice. \u003cstrong\u003e(d) \u003c/strong\u003eBacterial burdens in major organs at 48 h post-infection. Arginine supplementation significantly increased dissemination of wild-type bacteria to the spleen, lung, liver, and kidney, whereas Δ\u003cem\u003eargR\u003c/em\u003e mutants showed no significant differences. Data represent mean ± SD (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"OnlineFigure4XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/ea5b9dd5393740fc38b99ac9.png"},{"id":98439599,"identity":"08ad4200-5c6a-43e6-b72f-36d855b665ff","added_by":"auto","created_at":"2025-12-17 17:02:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1760289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArgR directly regulates capsule gene expression through promoter binding in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e RT-qPCR analysis of capsule-associated genes (\u003cem\u003ermpA, argR, wcaJ, galF, wzi\u003c/em\u003e) in wild-type, Δ\u003cem\u003eargR\u003c/em\u003e, and complemented (Δ\u003cem\u003eargR-C\u003c/em\u003e) strains. \u003cstrong\u003e(a)\u003c/strong\u003e NTUH-K2044 and \u003cstrong\u003e(b)\u003c/strong\u003e FK3009. Deletion of \u003cem\u003eargR\u003c/em\u003e significantly reduced expression of these genes, which was restored in complemented strains. Data represent mean ± SD from three biological replicates (**\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test). Promoter activity assays. β-galactosidase activity from \u003cem\u003elacZ\u003c/em\u003etranscriptional fusions demonstrated that \u003cem\u003eargR\u003c/em\u003edeletion markedly decreased promoter activity of \u003cem\u003ermpA, wzi,\u003c/em\u003e and \u003cem\u003egalF\u003c/em\u003ein both NTUH-K2044 \u003cstrong\u003e(c)\u003c/strong\u003e and FK3009 \u003cstrong\u003e(d)\u003c/strong\u003e. Data represent mean ± SD from three independent experiments (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001). Electrophoretic mobility shift assays (EMSA). \u003cstrong\u003e(e) \u003c/strong\u003eArgR binds directly to the \u003cem\u003ermpA\u003c/em\u003epromoter in a dose-dependent manner.\u003cstrong\u003e (f) \u003c/strong\u003eCompetition assays using unlabeled probes confirmed specific ArgR binding. \u003cstrong\u003e(g,h) \u003c/strong\u003eArgR binding to the \u003cem\u003ewzi\u003c/em\u003e and \u003cem\u003egalF\u003c/em\u003epromoters in NTUH-K2044. \u003cstrong\u003e(i,j) \u003c/strong\u003eArgR binding to the \u003cem\u003egalF\u003c/em\u003e and \u003cem\u003ewzi\u003c/em\u003e promoters in FK3009. \u003cstrong\u003e(f, k-n) \u003c/strong\u003eAddition of arginine enhanced ArgR–DNA interactions at target promoters, further supporting arginine-dependent regulation.\u003c/p\u003e","description":"","filename":"OnlineFigure5XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/d6204bf8d70e49af244718b7.png"},{"id":98371930,"identity":"d86bd811-066b-4c48-a3d5-61a8da9c0e0d","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":720306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated intestinal arginine enhances gut colonization and systemic dissemination of hypervirulent and carbapenem-resistant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a\u003c/strong\u003e) Schematic of the AMNV-treated murine intestinal colonization model and targeted amino acid metabolomics workflow, confirming marked luminal arginine enrichment. Heatmap shows amino acid profiles in cecal contents following antibiotic treatment. Intestinal colonization competition assay. (\u003cstrong\u003eb,c)\u003c/strong\u003e Mice were co-inoculated with a 1:1 mixture of wild-type and either \u003cstrong\u003e(b)\u003c/strong\u003eΔ\u003cem\u003eartP\u003c/em\u003e or \u003cstrong\u003e(c)\u003c/strong\u003e Δ\u003cem\u003eargR\u003c/em\u003emutants. Competitive index was calculated as the mutant-to-wild-type ratio in fecal samples collected on days 1, 3, and 7 post-inoculations. Both Δ\u003cem\u003eartP\u003c/em\u003e and Δ\u003cem\u003eargR\u003c/em\u003emutants exhibited significantly reduced colonization capacity compared with wild type. Data represent mean ± SD (***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, one sample t and Wilcoxon test). Murine intestinal colonization models: \u003cstrong\u003e(d-e) \u003c/strong\u003eSchematic overview of experimental design: mice pretreated with an antibiotic cocktail were orally gavaged with either NTUH-K2044 or FK3009 and provided drinking water with or without L-arginine throughout the study. \u003cstrong\u003e(e-f)\u003c/strong\u003eSurvival analysis of colonized mice. All animals colonized with NTUH-K2044 and supplemented with arginine succumbed by day 3, while no deaths occurred in FK3009-colonized groups. \u003cstrong\u003e(g)\u003c/strong\u003e Fecal bacterial loads on day 1 post-infection showed significantly increased NTUH-K2044 colonization in the arginine group compared to controls. \u003cstrong\u003e(h)\u003c/strong\u003e On day 3, bacterial burdens in the liver and spleen were markedly higher in arginine-supplemented NTUH-K2044 mice, consistent with systemic dissemination. \u003cstrong\u003e(i) \u003c/strong\u003eIn FK3009-colonized mice, arginine supplementation modestly increased fecal colonization on days 3 and 7, with differences diminishing by day 14.\u003cstrong\u003e (j) \u003c/strong\u003eAt day 14, bacterial burdens in the liver and spleen were significantly higher in the arginine-supplemented FK3009 group compared to controls. Data are presented as mean ± SEM, and statistical significance was determined using unpaired Student’s \u003cem\u003et\u003c/em\u003e-test for colonization and organ bacterial loads (exact \u003cem\u003ep\u003c/em\u003e values shown).\u003c/p\u003e","description":"","filename":"OnlineFigure6XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/a2d21ef9a90697713c38ad5f.png"},{"id":98371947,"identity":"160263fd-9d2c-4553-917d-2299df0fce2e","added_by":"auto","created_at":"2025-12-17 05:58:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":876456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArginine enhances systemic infection and promotes early mucosal invasion in an ArtP–ArgR-dependent manner. (a) \u003c/strong\u003eSchematic of the bloodstream infection model. NTUH-K2044 or FK3009, with or without prior arginine exposure, was injected intravenously into mice, which subsequently received arginine- or PBS-supplemented drinking water. \u003cstrong\u003e(b) \u003c/strong\u003eSurvival curves of mice infected with NTUH-K2044 (left) or FK3009 (right). Arginine supplementation markedly accelerates mortality in NTUH-K2044 and FK3009 infected mice. \u003cstrong\u003e(c) \u003c/strong\u003eSchematic of the 12-hour short-term intestinal colonization model. AMNV-treated mice were orally inoculated with wild-type NTUH-K2044 or \u003cem\u003eΔartP\u003c/em\u003e/ \u003cem\u003eΔargR\u003c/em\u003e mutants and given arginine- or PBS-supplemented drinking water before sacrifice at 12 h to assess mucosal invasion and early dissemination. (\u003cstrong\u003ed–e)\u003c/strong\u003e, Tissue-associated bacterial burdens in cecum \u003cstrong\u003e(d)\u003c/strong\u003eand small intestine \u003cstrong\u003e(e)\u003c/strong\u003e. Arginine significantly increases mucosal invasion by wild-type NTUH-K2044 (10–100× fold increase), whereas Δ\u003cem\u003eartP\u003c/em\u003e and Δ\u003cem\u003eargR\u003c/em\u003e mutants fail to respond to arginine.(\u003cstrong\u003ef–g)\u003c/strong\u003e, Bacterial loads in spleen \u003cstrong\u003e(f)\u003c/strong\u003e and liver \u003cstrong\u003e(g)\u003c/strong\u003e at 12 h. Arginine supplementation enhances early systemic dissemination of NTUH-K2044 (~1 log increase), while no arginine-dependent increase is observed for Δ\u003cem\u003eartP\u003c/em\u003e or Δ\u003cem\u003eargR\u003c/em\u003e strains. Statistical significance was determined using unpaired Student’s \u003cem\u003et\u003c/em\u003e-test for tissue bacterial loads (exact \u003cem\u003ep\u003c/em\u003e values shown).\u003c/p\u003e","description":"","filename":"OnlineFigure7XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/c35b4605b9674f40e04f7fec.png"},{"id":98371941,"identity":"edc15b51-250c-4b5c-ad09-456fe610ab55","added_by":"auto","created_at":"2025-12-17 05:58:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":371634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArginase-mediated depletion of luminal arginine suppresses \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpansion\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e ex vivo\u003c/strong\u003e\u003c/em\u003e.\u003cstrong\u003e (a) \u003c/strong\u003eSchematic of the ex vivo cecal content assay. Cecal contents were collected from 3 antibiotic-treated mice (AMNV protocol), pooled, resuspended in PBS, and aliquoted for treatment with either PBS (control) or recombinant L-arginase (1 U; MCE) to enzymatically deplete free arginine. NTUH-K2044 or the Δ\u003cem\u003eartP\u003c/em\u003emutant (10⁶ CFU) was inoculated into each condition and incubated for the indicated time points. \u003cstrong\u003e(b) \u003c/strong\u003eArginase treatment markedly reduced the expansion of NTUH-K2044 in cecal content suspensions across 6 h, 12 h, and 24 h compared with PBS controls. \u003cstrong\u003e(c) \u003c/strong\u003eExpansion of the ΔartP mutant was unaffected by arginase treatment, demonstrating that the inhibitory effect requires ArtP-mediated arginine uptake.Data represent individual biological replicates and are shown as mean ± SEM. Statistical significance was assessed by two-tailed unpaired Student’s t-test at each time point. Exact \u003cem\u003ep\u003c/em\u003e-values are indicated above comparisons.\u003c/p\u003e","description":"","filename":"OnlineFigure8XXXXXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/4dc156eb29848144a56e927b.png"},{"id":98371940,"identity":"35b72104-6e26-4b99-b353-e6d9c3b32fd9","added_by":"auto","created_at":"2025-12-17 05:58:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1187400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model of intestinal arginine–ArtP-ArgR–CPS regulation driving \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK.pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e pathogenicity. \u003c/strong\u003eElevated intestinal arginine levels, induced by antibiotic exposure, dietary or infection-related factors, are imported into \u003cem\u003eK. pneumoniae\u003c/em\u003e through the ATP-dependent transporter ArtPQM. Intracellular arginine binds to and activates the transcriptional regulator ArgR, which exerts a dual regulatory function: ArgR directly binds to the promoters of \u003cem\u003ecps\u003c/em\u003e biosynthesis genes (\u003cem\u003egalF\u003c/em\u003e, \u003cem\u003ewzi\u003c/em\u003e) and indirectly enhances \u003cem\u003ermpA\u003c/em\u003e expression. These coordinated actions collectively boost capsular polysaccharide (CPS) production, increasing capsule thickness, immune evasion, and mucosal barrier penetration. Consequently, the enhanced CPS facilitates bacterial translocation across the intestinal epithelium and dissemination into the bloodstream. This intestinal arginine–ArtP-ArgR–CPS regulatory axis links gut metabolic alterations to systemic hypervirulence in both hypervirulent and carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e lineages.\u003c/p\u003e","description":"","filename":"OnlineFigure901.png","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/1e67fd987cb0c483365660ed.png"},{"id":98775014,"identity":"c55a1d9e-f299-4213-ba53-83ca3a935515","added_by":"auto","created_at":"2025-12-22 12:17:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4054304,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/9dac3e1b-7537-4bee-9f94-d02a516dfc1f.pdf"},{"id":98371923,"identity":"b757e53c-f33d-4746-943e-a9837cb77d26","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17916,"visible":true,"origin":"","legend":"suggested reviewers","description":"","filename":"suggestedreviewers.docx","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/e74792e8c39c70ea51aa97e1.docx"},{"id":98371925,"identity":"02837996-4deb-4163-b81e-437eca0c8ede","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":175775,"visible":true,"origin":"","legend":"metabolomic dataset","description":"","filename":"Metabolomicsdataset141122.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/2a4ac278265b41d94ad97c81.xlsx"},{"id":98371926,"identity":"b25dc148-cb9b-4610-9204-7dde17547d51","added_by":"auto","created_at":"2025-12-17 05:58:49","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2847957,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8289577/v1/c71796e12bda23c4444cde84.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Host-derived arginine promotes Klebsiella pneumoniae colonization and dissemination through ArtP–ArgR–capsule signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e pneumoniae (Kp) is a major global health threat recognized by the World Health Organization (WHO) as a top-priority pathogen and a member of the ESKAPE group due to its high antibiotic resistance and clinical impact\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It primarily exists in two forms: multidrug-resistant (MDR) and hypervirulent (hv) strains\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. MDR-Kp accounts for 10\u0026ndash;20% of deaths caused by multidrug-resistant bacterial infections over the past 30 years, ranking among the top three most lethal pathogens\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. HvKp, typically causing severe systemic infections such as liver abscesses, meningitis, and bacteremia, is associated with mortality rates of 30%\u0026ndash;85%\u003csup\u003e7\u003c/sup\u003e. Critically, both MDR, such as carbapenem resistance (CR), and hypervirulence traits are increasingly being found within the same strains, giving rise to hypervirulent carbapenem-resistant Kp (hv-CRKp)\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among these, the ST11-KL64 lineage has emerged as a predominant epidemic clone in clinical settings and is closely associated with bloodstream infections, and poor patient outcomes\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The rapid global spread of hvKp and CRKp, particularly the epidemic ST11-KL64 lineage, underscores the urgent need to understand factors that enable \u003cem\u003eK. pneumoniae\u003c/em\u003e to colonize, disseminate, and cause life-threatening systemic infections.\u003c/p\u003e\u003cp\u003eAlthough \u003cem\u003eK. pneumoniae\u003c/em\u003e commonly causes pulmonary, hepatic, and bloodstream infections, colonization typically begins in the gastrointestinal (GI) tract, which serves as the principal reservoir and portal of entry for invasive disease\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Only a small fraction of ingested organisms succeed in establishing gut colonization, and expansion within this niche is often required to initiate systemic infection\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Previous studies indicates that intestinal inflammation and antibiotic-induced dysbiosis compromise barrier function and colonization resistance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Under such conditions, the risk of stable intestinal colonization increases substantially, and the weakened mucosal barrier predisposes the host to bacterial translocation and invasive infection. However, the key host- or microbe-derived factors that drive this heightened invasiveness within the altered intestinal environment remain poorly defined.\u003c/p\u003e\u003cp\u003eRecent evidence highlights that metabolites within the gastrointestinal tract may act as important signals regulating function and pathogenicity of intestinal commensals and pathobionts, including \u003cem\u003eEnterobacteriaceae\u003c/em\u003e such as \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. During intestinal inflammation, the host undergoes metabolic reprogramming characterized by a significant rise in free amino acid levels within the gut lumen\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Consistently, studies of antibiotic treatment and dietary perturbations have demonstrated that several free amino acids become significantly enriched\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These shifts in amino acid availability may serve as nutrient signals or regulatory cues that modulate virulence gene expression in \u003cem\u003eK. pneumoniae\u003c/em\u003e, promoting its shift from a commensal colonizer to an invasive pathogen. However, whether alterations in intestinal amino acid composition influence \u003cem\u003eK. pneumoniae\u003c/em\u003e adaptation within the gut and its capacity to traverse the intestinal barrier to cause systemic infection remains unknown.\u003c/p\u003e\u003cp\u003eIn this study, we investigated how intestinal amino acid availability shapes the pathogenic potential of \u003cem\u003eK. pneumoniae\u003c/em\u003e. By integrating metabolomic evidence from dietary perturbation, antibiotic treatment, and intestinal inflammation models\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, we identified multiple amino acids consistently enriched in the gut. A major virulence determinant of \u003cem\u003eK. pneumoniae\u003c/em\u003e is the production of a thick capsular polysaccharide (CPS), which shields the bacteria from phagocytosis and complement-mediated killing\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. We therefore examined how these amino acids affect CPS synthesis and virulence. Arginine emerged as a particularly relevant candidate, not only does it accumulate to high levels across diverse perturbations, but it significantly enhanced CPS production in \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eRecent work has shown that arginine increases the mucoid phenotype of hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e when provided in a glycerol-based minimal medium, without increasing total CPS abundance, acting primarily through ArgR-dependent activation of \u003cem\u003ermpADC\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These observations suggest that arginine can modulate CPS chain length and viscosity, but do not address whether arginine can enhance CPS biosynthesis itself\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, it remains unclear whether arginine exerts similar regulatory effects under metabolic conditions relevant to the intestinal environment, where glucose, not glycerol, is abundant\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, or whether arginine influences the capacity of \u003cem\u003eK. pneumoniae\u003c/em\u003e to cross the intestinal barrier and seed systemic infection.\u003c/p\u003e\u003cp\u003eTo answer these key questions, we investigated how arginine affects \u003cem\u003ecps\u003c/em\u003e gene expression under glucose-based conditions that mimic the gut milieu. We further examined the molecular requirements for arginine sensing, focusing on the ATP-dependent transporter ArtP and the transcriptional regulator ArgR, both of which participate in arginine metabolism and virulence regulation. Finally, using a murine intestinal colonization model, we assessed whether arginine availability in the gut environment alters \u003cem\u003eK. pneumoniae\u003c/em\u003e colonization dynamics, mucosal barrier interactions, and progression toward systemic infection. Together, these approaches allowed us to define how intestinal amino acid perturbations may serve as host-derived signals that modulate \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenic potential.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eHost intestinal amino acid shifts shape\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e \u003cb\u003evirulence determinants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIntestinal metabolic perturbations caused by host inflammation, dietary fiber deficiency\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, or antibiotic treatment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e have all been reported to reshape the gut metabolome in ways that promote \u003cem\u003eK. pneumoniae\u003c/em\u003e disseminattion\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To explore the metabolic features shared across these translocation-prone states, we re-analyzed available metabolomic datasets and confirmed that: 1) Inflammation caused by treatment of mice with anti-CD3 antibody leads to elevated levels of free amino acids in the gut lumen\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea); 2) a fiber-free enteral diet drives marked \u003cem\u003eK. pneumoniae\u003c/em\u003e expansion accompanied by amino acid accumulation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb); 3) and antibiotic-induced microbiota depletion enriches nutrients, particularly amino acids, thereby enhancing the growth of carbapenem-resistant \u003cem\u003eEnterobacteriaceae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). To systematically evaluate the contribution of these amino acid shifts, we selected ten consistently enriched amino acids in the above studies for experimental assessment of their effects on \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that both the ability to thrive in a given microenvironment and the production of capsular polysaccharide are major determinants of \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence, we selected bacterial growth and CPS synthesis as in vitro readouts for amino acid effects, using two representative strains\u0026mdash;NTUH-K2044 (ST23-KL1, hvKP) and FK3009 (ST11-KL64, CRKp). All assays were performed in M9 minimal medium supplemented with glucose as the sole carbon source, enabling us to evaluate growth dynamics and CPS production under metabolic conditions relevant to the intestinal environment(rich in glucose)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Most amino acids exerted little effect on bacterial proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e,f and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e); alanine modestly inhibited FK3009 growth, tryptophan suppressed NTUH-K2044, while phenylalanine enhanced FK3009. In contrast, glutamine promoted growth in both strains (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, only arginine, glutamate, and leucine consistently increased CPS production and viscosity in both strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg,h).\u003c/p\u003e\u003cp\u003eAlthough glutamate and leucine also enhanced virulence in vitro, their intestinal enrichment under fiber deprivation and antibiotic treatment was far less pronounced than the robust elevation observed for arginine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). Thus, arginine appears as a particularly relevant amino acid potentially capable of promoting \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenicity \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eArginine enhances capsule gene expression through ArtP-mediated intracellular transport and metabolic sensing\u003c/h2\u003e\u003cp\u003eOur findings indicated that exogenous arginine markedly enhanced CPS production in \u003cem\u003eK. pneumoniae\u003c/em\u003e. This differs from earlier observations showing that, under glycerol-based conditions, arginine increases mucoviscosity without altering total CPS levels. Our results suggest that under glucose-based conditions that better reflect the intestinal environment, arginine can indeed promote CPS biosynthesis, revealing an additional regulatory feature that had not been previously appreciated.. We next sought to investigate the underlying molecular mechanisms. To confirm whether arginine affects the expression of the \u003cem\u003ecps\u003c/em\u003e operon, we performed RNA-sequencing on NTUH-K2044 and FK3009 cultured in glucose-based M9 minimal medium with or without L-arginine supplementation. We observed that the \u003cem\u003eastCADBE\u003c/em\u003e operon encoding the arginine succinyl transferase pathway for arginine catabolism\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e was significantly upregulated, indicating that exogenous arginine is actively sensed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,d). Importantly, several genes within the capsular polysaccharide synthesis cluster were also significantly upregulated in response to arginine exposure in both Kp strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-f). These findings indicated that arginine not only serves as a metabolic substrate but also acts as a signaling cue that reprograms virulence gene expression, which is uncoupled from impacts on replication rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next asked whether arginine functions as a signaling molecule or whether its intracellular metabolism is required to activate virulence programs. To answer this question, we attempted to block the transport of arginine into \u003cem\u003eK. pneumoniae\u003c/em\u003e. In \u003cem\u003eK.pneumoniae\u003c/em\u003e, L-arginine is initially bound by three distinct periplasmic binding proteins and subsequently transported into the cytoplasm via two separate transmembrane complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. ArtP is essential for the function of both arginine uptake systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Using an \u003cem\u003eartP\u003c/em\u003e mutant of NTUH-K2044 we found that that when arginine uptake is impaired, exogenous arginine fails to promote CPS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh,i). These findings suggest that intracellular uptake of arginine is essential for its role in promoting virulence, implying that its regulatory function is tightly coupled to metabolic processing rather than mere extracellular sensing.\u003c/p\u003e\u003cp\u003eThe arginine sensor ArgR is a key transcriptional regulator of arginine metabolism in \u003cem\u003eK.pneumoniae\u003c/em\u003e. It primarily functions as a repressor of genes involved in arginine biosynthesis in response to elevated intracellular arginine levels, and acts as a coactivator of the \u003cem\u003eastCADBE\u003c/em\u003e operon, which mediates arginine catabolism. Previous studies demonstrated that deletion of \u003cem\u003eargR\u003c/em\u003e significantly alters viscosity in \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, raising the possibility that exogenous arginine may influence capsule expression by activating ArgR-dependent regulatory pathways. To test this hypothesis, we performed RT-qPCR and examined the expression levels of \u003cem\u003eargR\u003c/em\u003e in the two \u003cem\u003eK. pneumoniae\u003c/em\u003e strains following arginine supplementation. In addition, we also assessed the transcription of a key regulatory factor involved in CPS production (\u003cem\u003ermpA\u003c/em\u003e), as well as that of a representative transcript from the \u003cem\u003ecps\u003c/em\u003e locus (\u003cem\u003ewzi\u003c/em\u003e, the first gene of the polysaccharide polymerization transcript), and a core gene (\u003cem\u003ewcaJ\u003c/em\u003e, encodes the initiating glycosyltransferase) within the \u003cem\u003ecps\u003c/em\u003e operon\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In these settings, arginine not only upregulated the expression of key genes involved in capsular polysaccharide biosynthesis and known regulatory factor, but also increased the transcriptional level of \u003cem\u003eargR\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej,k). Taken together, our results suggest a model in which arginine promotes capsular polysaccharide production in \u003cem\u003eK. pneumoniae\u003c/em\u003e by enhancing the transcription of capsule-related genes. This effect requires arginine uptake and metabolic processing, with ArgR likely serving as a key regulatory mediator.\u003c/p\u003e\u003cp\u003e\u003cb\u003eArgR is essential for arginine-induced capsule expression and pathogenicity in hypervirulent and multidrug-resistant\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further define the role of ArgR in \u003cem\u003eK. pneumoniae\u003c/em\u003e capsule formation and pathogenicity, we constructed \u003cem\u003eargR\u003c/em\u003e deletion and complemented mutants in both NTUH-K2044 (KL-1) and FK3009 (ST11-KL64). Deletion of \u003cem\u003eargR\u003c/em\u003e decreased the production of capsular polysaccharide in both strains, a phenotype that could be rescued upon complementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo comprehensively assess the impact of \u003cem\u003eargR\u003c/em\u003e on \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence, we employed \u003cem\u003ein vitro\u003c/em\u003e cell infection assays (RAW 264.7 Macrophages and primary Kupffer Cells) (Figue 3e,f) and \u003cem\u003ein vivo\u003c/em\u003e models, namely \u003cem\u003eGalleria mellonella\u003c/em\u003e and murine bloodstream infection (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-k). These models allowed us to evaluate the relative contribution of \u003cem\u003eargR\u003c/em\u003e to both immune evasion and systemic pathogenicity. Capsular polysaccharide is a critical virulence factor that protects \u003cem\u003eK. pneumoniae\u003c/em\u003e from phagocytic clearance. Phagocytosis assays revealed that deletion of the \u003cem\u003eargR\u003c/em\u003e gene significantly increased bacterial uptake by both RAW 264.7 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) and primary murine Kupffer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), indicating that \u003cem\u003eargR\u003c/em\u003e is required for \u003cem\u003eK. pneumoniae\u003c/em\u003e to evade phagocytosis. Consistently, survival rates of \u003cem\u003eGalleria mellonella\u003c/em\u003e (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) and mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-k) systemically infected with the \u003cem\u003eargR\u003c/em\u003e mutant were markedly higher compared to those infected with the wild-type strain. Notably, bacterial burdens in key organs such as the liver and spleen were also markedly reduced in the \u003cem\u003eargR\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei,k). These findings demonstrate that \u003cem\u003eargR\u003c/em\u003e is essential for \u003cem\u003eK. pneumoniae\u003c/em\u003e to evade host immune clearance and maintain full virulence \u003cem\u003ein vivo\u003c/em\u003e, likely through its regulation of capsular polysaccharide expression.\u003c/p\u003e\u003cp\u003eTo determine whether arginine-mediated regulation of capsular polysaccharide depends on the transcriptional regulator ArgR, we assessed capsule production in wild-type, \u003cem\u003eargR\u003c/em\u003e knockout, and complemented strains in the presence or absence of exogenous arginine. Arginine supplementation significantly increased capsule production in the wild-type strain, but this effect was significantly abolished in the \u003cem\u003eargR\u003c/em\u003e mutant. Notably, restoration of \u003cem\u003eargR\u003c/em\u003e expression rescued the arginine-induced capsule enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Furthermore, a murine bloodstream infection model was employed to assess whether the virulence-promoting effect of arginine also requires \u003cem\u003eargR\u003c/em\u003e. Mice were challenged with either NTUH-K2044 (ST23, KL1, hvKP) or its \u003cem\u003eargR\u003c/em\u003e deletion derivative. Supplementation with arginine increased the lethality of the wild-type strain, but had little to no effect on the pathogenicity of the \u003cem\u003eargR\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In addition, bacterial load quantification in major organs revealed a 1\u0026ndash;2 log increase in the liver and spleen of wild-type\u0026ndash;infected mice following arginine supplementation, whereas no significant differences were detected in the \u003cem\u003eargR\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings indicate that the arginine-induced enhancement of capsular polysaccharide production and virulence in \u003cem\u003eK. pneumoniae\u003c/em\u003e is largely dependent on the transcriptional regulator ArgR. Importantly, these effects were observed in both the hypervirulent NTUH-K2044 and the multidrug-resistant FK3009 strains, supporting ArgR-dependent regulation as a generalizable mechanism in \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eArginine enhances ArgR Binding to\u003c/b\u003e \u003cb\u003ecps\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ermpA\u003c/b\u003e \u003cb\u003epromoters to regulate capsule gene expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving established that arginine regulates capsular polysaccharide production through activation of ArgR, we next sought to elucidate how ArgR regulate the CPS gene expression. Given that arginine enhances capsule gene expression without affecting bacterial growth, and that ArgR functions as a transcriptional regulator, we hypothesized that arginine promotes CPS production by facilitating ArgR-dependent transcriptional activation.\u003c/p\u003e\u003cp\u003eRT-qPCR analysis revealed that deletion of ArgR significantly reduced the transcription of both the capsule regulator \u003cem\u003ermpA\u003c/em\u003e and key genes within the \u003cem\u003ecps\u003c/em\u003e cluster in both \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. Restoration of \u003cem\u003eargR\u003c/em\u003e expression rescued the expression of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). Furthermore, promoter activity assays confirmed that ArgR can directly enhance the promoter activities of \u003cem\u003ermpA\u003c/em\u003e and critical \u003cem\u003ecps\u003c/em\u003e genes, indicating its role in transcriptional regulation of capsule synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d). However, it remained unclear whether ArgR regulates the \u003cem\u003ecps\u003c/em\u003e locus directly or indirectly through RmpA. To address this question, we tested for ArgR direct binding to the promoter region of the \u003cem\u003ecps\u003c/em\u003e operon independent of RmpA. A previous study demonstrated that ArgR regulates the \u003cem\u003ermp\u003c/em\u003e promoter by binding to a highly conserved ARG box, thereby modulating mucoviscosity in hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Consistent with these results, our \u003cem\u003ein vitro\u003c/em\u003e EMSA assays confirmed ArgR binding to the \u003cem\u003ermpA\u003c/em\u003e promoter region, even at low protein concentrations, indicating a strong and specific interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Notably, beyond \u003cem\u003ermpA\u003c/em\u003e, ArgR also bound to the promoter regions of \u003cem\u003egalF\u003c/em\u003e and \u003cem\u003ewzi\u003c/em\u003e, key transcripts within the \u003cem\u003ecps\u003c/em\u003e locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-j). These findings suggest that ArgR regulated capsule synthesis via a dual mechanism: indirectly, through RmpA activation and directly, by targeting \u003cem\u003ecps\u003c/em\u003e locus promoters.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies suggested that arginine activates ArgR not only by upregulating its expression, but also by directly binding to ArgR, inducing a conformational change that enhances its DNA-binding affinity and transcriptional regulatory activity. To test this hypothesis, we assessed binding capacity of ArgR to the promoter regions of its target genes in the presence of arginine. First, we determined a sub-saturating concentration of ArgR protein that did not bind to target promoters in standard EMSA conditions. Then, we performed EMSA using such sub-saturating concentrations of ArgR. In the absence of arginine, ArgR showed minimal binding to target promoters. Upon addition of increasing concentrations of L-arginine, DNA-binding was progressively enhanced. Specificity was confirmed by the absence of ArgR binding to the promoter of the negative control gene \u003cem\u003erpoB\u003c/em\u003e, and by competitive EMSA using excess unlabeled probes, which markedly reduced ArgR binding to biotin-labeled probes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, k-n). These results demonstrate that arginine directly enhances ArgR binding to target promoters, likely by inducing conformational changes in the transcription factors.\u003c/p\u003e\u003cp\u003eTaken together, our results indicated arginine promotes capsular polysaccharide production in \u003cem\u003eK. pneumoniae\u003c/em\u003e through activation of the transcriptional regulator ArgR. Upon sensing intracellular arginine, ArgR enhances the transcription of capsule-related genes via two mechanisms: direct binding to \u003cem\u003ecps\u003c/em\u003e locus promoters (such as \u003cem\u003egalF\u003c/em\u003e and \u003cem\u003ewzi\u003c/em\u003e) and indirect activation through upregulation of the capsule regulator \u003cem\u003ermpA\u003c/em\u003e. This dual regulatory strategy enables \u003cem\u003eK. pneumoniae\u003c/em\u003e to coordinate arginine sensing with virulence gene expression. Importantly, this ArgR-dependent mechanism is conserved in both hypervirulent and multidrug-resistant strains, highlighting a general strategy by which \u003cem\u003eK. pneumoniae\u003c/em\u003e links metabolic cues to pathogenicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLocal arginine enrichment promotes\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e \u003cb\u003eintestinal colonization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe above data indicate that exogenous arginine enhances capsular polysaccharide synthesis in both representative hv and CR \u003cem\u003eK. pneumoniae in vitro\u003c/em\u003e. We next asked whether arginine enrichment in the intestinal environment likewise affects \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenicity \u003cem\u003ein vivo\u003c/em\u003e. Because stable gut colonization represents the critical first step enabling \u003cem\u003eK. pneumoniae\u003c/em\u003e to traverse the intestinal barrier and seed systemic infection, we established an intestinal colonization model to evaluate the impact of arginine on this process. Clinically, antibiotic exposure is a major driver of \u003cem\u003eK. pneumoniae\u003c/em\u003e overgrowth and persistent colonization; therefore, we focused on an antibiotic-treated mouse model.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that antibiotic treatment leads to intestinal accumulation of arginine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). To determine whether arginine similarly increases in our model and to establish the baseline amino acid landscape, we measured amino acid levels in the intestinal contents of our antibiotic-treated mice using targeted metabolomic profiling. Mice were first administered a broad-spectrum antibiotic cocktail (ampicillin, metronidazole, neomycin, and vancomycin; AMNV) to deplete the intestinal microbiota, followed by oral inoculation with \u003cem\u003eK. pneumoniae\u003c/em\u003e and maintenance on vancomycin and metronidazole to sustain dysbiosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). To characterize the amino acid environment generated by this model, we treated mice with the AMNV regimen and then provided PBS for 24 hours before euthanasia. Cecal contents were subsequently collected for targeted metabolomic analysis. Strikingly, arginine exhibited a pronounced accumulation in the antibiotic-treated gut and ranked as the fourth most abundant amino acid detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). These results confirm that antibiotic-induced dysbiosis creates an arginine-enriched intestinal environment, providing a physiologically relevant context in which to assess its effects on \u003cem\u003eK. pneumoniae\u003c/em\u003e colonization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven our \u003cem\u003ein vitro\u003c/em\u003e findings that arginine must be imported through the ArtP transporter to activate \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence and its downstream metabolic responses, and that ArgR functions as the key regulator mediating this arginine-driven virulence, we selected these two mutants to determine whether arginine metabolism influences \u003cem\u003eK. pneumoniae\u003c/em\u003e intestinal colonization. Using an intestinal competition model in which wild-type and mutant strains colonized the same host, we found that deletion of \u003cem\u003eartP\u003c/em\u003e, which blocks arginine uptake, abolished arginine-induced CPS enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and markedly impaired intestinal colonization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Likewise, deletion of \u003cem\u003eargR\u003c/em\u003e not only attenuated capsule formation and systemic infection but also reduced intestinal colonization by ~\u0026thinsp;2 logs compared with the wild-type strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). These results demonstrate that arginine metabolism in \u003cem\u003eK. pneumoniae\u003c/em\u003e significantly influences its ability to colonize the gut.\u003c/p\u003e\u003cp\u003eTo determine whether elevated intestinal arginine directly influences \u003cem\u003eK. pneumoniae\u003c/em\u003e colonization in vivo, we used the same antibiotic-treated intestinal colonization model, in which microbiota depletion was achieved with an AMNV cocktail before oral inoculation with NTUH-K2044 or FK3009. Mice were then provided either PBS or arginine-supplemented drinking water (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed,e).\u003c/p\u003e\u003cp\u003eNotably, in mice colonized with the hypervirulent NTUH-K2044 strain, all animals receiving arginine supplementation succumbed within 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Fecal CFU counts collected on day 1 showed significantly higher intestinal colonization in the arginine-supplemented group than in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). Consistent with this, bacterial burdens in the liver and spleen were markedly elevated\u0026mdash;by 1\u0026ndash;2 logs\u0026mdash;indicating enhanced systemic dissemination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). In contrast, mice colonized with the less virulent multidrug-resistant FK3009 strain (ST11-KL64, CRKp) exhibited no mortality during the two-week colonization period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Nonetheless, arginine supplementation significantly increased intestinal colonization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej), and at day 14, higher bacterial loads were detected in both the intestine and systemic organs, including the liver and spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek).\u003c/p\u003e\u003cp\u003eTogether, these data demonstrate that increasing intestinal arginine availability enhances gut colonization and promotes systemic spread across both hypervirulent and CRKp strains.\u003c/p\u003e\u003cp\u003e\u003cb\u003eElevated host arginine promotes systemic infection by\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that intestinal arginine accumulation enhanced gut colonization, facilitated systemic infection, and increased mortality over time, we next sought to determine the impact of arginine on systemic disease and on the colonization/invasion switch.\u003c/p\u003e\u003cp\u003eFirst, we employed a bloodstream infection model, as this approach provides direct and unconfounded assessment of whether arginine supplementation increases \u003cem\u003eK. pneumoniae\u003c/em\u003e systemic virulence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). We compared the systemic effects of arginine with those of leucine and glutamate, two additional amino acids that increased CPS production in \u003cem\u003evitro\u003c/em\u003e. In this bacteremia model, supplementation with arginine, glutamate, or leucine markedly reduced host survival and promoted bacterial dissemination to the liver, spleen, lungs, and kidneys (Figure S3). Notably, arginine ccelerated mortality in mice infected with either NTUH-K2044 (ST23-KL1, hvKp) or FK3009 (ST11-KL64, CRKp), demonstrating that arginine broadly potentiates \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence during systemic infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHaving established that arginine directly enhances systemic virulence in the bloodstream, we next asked whether arginine also accelerates the transition from gut colonization to systemic infection. To determine whether arginine promotes mucosal invasion, we conducted short-term colonization experiments. AMNV-treated mice received oral \u003cem\u003eK. pneumoniae\u003c/em\u003e inoculation and were given either control drinking water or arginine-supplemented water, then sacrificed 12 hours later (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Small intestinal tissue, cecal tissue, and systemic organs (liver and spleen) were collected to quantify the rapid effects of arginine on mucosal crossing versus systemic expansion. To further clarify whether this effect requires arginine uptake and signaling, we included Δ\u003cem\u003eartP\u003c/em\u003e and Δ\u003cem\u003eargR\u003c/em\u003e mutants as controls.\u003c/p\u003e\u003cp\u003eIn agreement with our previous data, intestinal arginine accumulation markedly increased the ability of the NTUH-K2044 wild-type strain to invade both small intestinal and cecal tissues, with bacterial loads rising by approximately two orders of magnitude compared with PBS controls (Fig.\u0026nbsp;7de). Consistent with this enhanced mucosal invasion, we observed elevated bacterial burdens in the liver and spleen, although the increase in systemic organs was more modest (~\u0026thinsp;1 log) (Fig.\u0026nbsp;7fg). These findings indicate that, in hosts whose intestine has been colonized by \u003cem\u003eKlebsiella penumoniae\u003c/em\u003e, arginine primarily promotes systemic infection by facilitating the breach of the intestinal mucosal barrier rather than by accelerating systemic expansion. Of note, Δ\u003cem\u003eartP\u003c/em\u003e and Δ\u003cem\u003eargR\u003c/em\u003e mutants failed to respond to exogenous arginine, showing no significant increase in tissue invasion or systemic dissemination. This confirms that the ability of arginine to promote \u003cem\u003eK. pneumoniae\u003c/em\u003e systemic infection requires both ArtP-dependent arginine uptake and ArgR-mediated transcriptional regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTargeting intestinal arginine with arginase as a potential strategy to limit\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBuilding on the observation that arginine enhances \u003cem\u003eK. pneumoniae\u003c/em\u003e intestinal colonization and virulence, we hypothesized that reducing luminal arginine might attenuate these effects. To test this possibility, we collected cecal contents from three antibiotic-treated mice and resuspended them in PBS to mimic the intestinal environment \u003cem\u003eex vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). NTUH-K2044 wild-type or the \u003cem\u003eartP\u003c/em\u003e deletion mutant were inoculated at 10\u003csup\u003e6\u003c/sup\u003e CFU into cecal content left either untreated (PBS) or spiked-in with arginase, an enzyme that converts arginine into ornithine and urea, thus degrading luminal arginine (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStrikingly, arginase treatment significantly reduced the expansion of NTUH-K2044 in this simulated gut environment, whereas the \u003cem\u003eartP\u003c/em\u003e knockout strain showed no detectable difference between control and arginase-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb,c). These results indicate that enzymatic depletion of luminal arginine directly impairs \u003cem\u003eK. pneumoniae\u003c/em\u003e growth within the gut environment and that this effect strictly depends on ArtP-mediated arginine uptake. Although arginine does not significantly alter bacterial growth \u003cem\u003ein vitro\u003c/em\u003e, the intestinal environment imposes additional selective pressures that differ from nutrient-rich laboratory conditions. Our findings show that arginine primarily enhances CPS production rather than functioning as a growth substrate. Because elevated capsule expression enables \u003cem\u003eK. pneumoniae\u003c/em\u003e to resist complement-mediated killing\u0026mdash;and active complement is known to be present in the gut\u0026mdash;arginine likely promotes bacterial expansion by improving survival against host defenses rather than by increasing intrinsic replication. This model explains why removing arginine from the gut environment reduces bacterial expansion despite its minimal impact on growth \u003cem\u003ein vitro.\u003c/em\u003e Moreover, The lack of response in the \u003cem\u003eartP\u003c/em\u003e mutant highlights ArtP as a potential therapeutic target for disabling arginine-dependent virulence pathways.\u003c/p\u003e\u003cp\u003eTogether, these findings demonstrate that lowering intestinal arginine levels, such as through arginase treatment, may diminish \u003cem\u003eK. pneumoniae\u003c/em\u003e intestinal colonization and reduce the risk of systemic infection, while simultaneously identifying ArtP as a promising metabolic\u0026ndash;virulence target for therapeutic intervention.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study uncovers a previously unrecognized link between host amino acid metabolism and \u003cem\u003eK.pneumoniae\u003c/em\u003e virulence. Increasing evidence highlights gut colonization as a critical reservoir for healthcare-associated pathogens, including \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the specific host-derived signals that drive virulence acquisition and enable bacterial translocation across the intestinal barrier remain poorly defined. Here, we show that perturbations of the intestinal environment, such as those occurring during inflammation\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, antibiotic exposure\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, or dietary imbalance\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, lead to altered amino acid profiles, and that these metabolic changes, particularly arginine accumulation, directly enhance \u003cem\u003eK. pneumoniae\u003c/em\u003e colonization and systemic pathogenicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the amino acids detected in the gut, arginine was particularly notable. Moreover, in diverse contexts including inflammation, diet-induced dysbiosis, and antibiotic treatment, arginine accumulates significantly in the intestinal lumen\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This consistent enrichment underscores arginine as a physiologically relevant signal that strongly influences \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenesis. Previous studies have reported that exogenous arginine can induce the hypermucoviscous phenotype in hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Our findings build upon and extend these observations by showing that arginine not only promotes capsule production but also enhances intestinal colonization and systemic virulence \u003cem\u003ein vivo\u003c/em\u003e, and that this effect is conserved across both hypervirulent and multidrug-resistant strains. Thus, our study provides a broader and more generalized mechanistic framework linking arginine availability to \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenic potential.\u003c/p\u003e\u003cp\u003eThe arginine transporter ArtP proved indispensable for this phenotype. Deletion of \u003cem\u003eartP\u003c/em\u003e abolished arginine-induced capsule enhancement and markedly reduced intestinal colonization, highlighting the requirement for intracellular uptake and metabolism. These findings indicate that arginine does not simply act as an extracellular cue but must be actively imported to influence bacterial physiology and persistence within the host gut.\u003c/p\u003e\u003cp\u003eThe transcriptional regulator ArgR emerged as another critical determinant. Loss of \u003cem\u003eargR\u003c/em\u003e abolished the virulence-promoting effect of arginine and led to a\u0026thinsp;~\u0026thinsp;2-log reduction in intestinal colonization. Consistent with previous reports, ArgR activity is enhanced by arginine abundance in the intestinal milieu, which has been shown to induce type VI secretion system (T6SS) expression, thereby promoting asymptomatic colonization\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Importantly, our work extends this knowledge by demonstrating that ArgR directly binds to promoters within the \u003cem\u003ecps\u003c/em\u003e locus and drives capsule gene expression, with exogenous arginine strengthening this interaction. Thus, ArgR integrates nutrient sensing with both capsule regulation and secretion system activity, placing it at the center of a multifaceted regulatory network controlling colonization and systemic infection.\u003c/p\u003e\u003cp\u003eThe capsule polysaccharide itself represents an important determinant of intestinal persistence\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Prior studies have shown that capsule-deficient \u003cem\u003eK. pneumoniae\u003c/em\u003e strains exhibit impaired colonization and reduced competitiveness within the gut niche\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Together with our findings, this supports a model in which arginine availability, ArtP-mediated uptake, and ArgR-dependent transcriptional regulation converge on capsule production and secretion system activation to enable successful intestinal colonization and dissemination.\u003c/p\u003e\u003cp\u003eWe suggest that changes in intestinal amino acid levels play an important role in promoting \u003cem\u003eK. pneumoniae\u003c/em\u003e colonization and translocation into the bloodstream. This concept is supported by recent observations in other enteric pathogens\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. For example, commensal yeast has been shown to increase intestinal arginine levels, which in turn promote \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e virulence by enhancing fitness and invasion\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Similarly, increased concentrations of ornithine and glutamate in the gut markedly enhance \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e colonization\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These studies, together with our findings, support a broader principle in which dietary or microbiota-derived amino acids act as conserved cues that are exploited by multiple gut pathobionts to trigger virulence programs and overcome colonization resistance.\u003c/p\u003e\u003cp\u003eThese results also carry important clinical significance. Intestinal metabolic disturbances are common in critically ill patients, premature infants, and individuals receiving broad-spectrum antibiotics\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In such settings, accumulation of free amino acids may inadvertently promote colonization, barrier translocation, and bloodstream infection by opportunistic pathogens such as \u003cem\u003eK. pneumoniae\u003c/em\u003e. Recognizing this risk highlights the need to consider host metabolic context when designing nutritional or therapeutic strategies. Future integration of clinical nutrition studies with microbiome and metabolome profiling will be crucial to evaluate whether amino acid supplementation in vulnerable hosts contributes to the risk of invasive \u003cem\u003eK. pneumoniae\u003c/em\u003e infections.\u003c/p\u003e\u003cp\u003eBeyond defining the molecular basis through which intestinal arginine promotes \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence, our ex vivo analyses provide further functional support for the translational relevance of this metabolic\u0026ndash;virulence axis. By enzymatically depleting arginine in antibiotic-treated cecal contents using arginase, we observed a marked reduction in the expansion of the wild-type NTUH-K2044 strain, whereas the \u003cem\u003eartP\u003c/em\u003e mutant (unable to import arginine) remained unaffected. These findings reinforce that luminal arginine is not merely a nutrient but an active regulatory cue that enhances bacterial fitness within the gut environment. Importantly, they also suggest that targeted removal of intestinal arginine can attenuate \u003cem\u003eK. pneumoniae\u003c/em\u003e proliferation in the presence of complex metabolites. This raises the possibility that enzymatic modulation of luminal arginine levels, or therapeutic strategies aimed at restricting arginine availability, could reduce gut colonization and limit progression to systemic infection. Moreover, the lack of response in the \u003cem\u003eartP\u003c/em\u003e mutant highlights arginine transport as a druggable vulnerability within the ArtP\u0026ndash;ArgR\u0026ndash;CPS pathway, offering a mechanistically informed avenue for therapeutic intervention.\u003c/p\u003e\u003cp\u003eIn summary, our study identifies a conserved, host-driven mechanism in which intestinal amino acid alterations, particularly arginine enrichment, enhance \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence through ArtP- and ArgR-dependent pathways. These effects were consistently observed in both hypervirulent and carbapenem-resistant strains, underscoring their generalizability across clinically relevant lineages. By linking metabolic remodeling to capsule biosynthesis and intestinal persistence, our findings highlight host-derived arginine as a key driver of \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenicity and a potential target for therapeutic intervention.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains\u003c/h2\u003e\u003cp\u003eNTUH-K2044, a well-characterized ST23-KL1 strain, was originally isolated from a patient with a liver abscess and serves as a classical hvKp model\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Another strain, FK3009 (Accension number: SAMN43180534) was isolated from a 72-year-old patient with septicemia. This strain represents a typical ST11-KL64 epidemic clone of CRKp that has acquired a virulence plasmid conferring a hypervirulent phenotype.All strain information is provided in the Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMetabolomics analysis\u003c/h3\u003e\n\u003cp\u003eData on metabolic alterations in the intestinal environment under acute inflammatory\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (Dataset-1), dietary perturbation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (Dataset-2), and antibiotic treatment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Dataset-3) conditions were obtained from previously published studies and publicly available datasets. Detailed experimental procedures for each model have been described previously, and all metabolomic data were integrated into a unified dataset for comparative analysis in this study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth curve\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate whether amino acids elevated in the intestinal environment during inflammation affect \u003cem\u003eK. pneumoniae\u003c/em\u003e growth, \u003cem\u003ein vitro\u003c/em\u003e growth curve assays were performed in the presence of varying concentrations of individual amino acids. \u003cem\u003eK.pneumoniae\u003c/em\u003e strains were cultured overnight in Luria\u0026ndash;Bertani (LB) medium at 37\u0026deg;C with aeration. Overnight cultures were adjusted to an optical density at 600 nm (OD₆₀₀) corresponding to approximately 1 \u0026times; 10⁸ CFU/mL, followed by a 1:100 dilution into M9 minimal medium supplemented with individual amino acids as indicated. For arginine-supplemented cultures, bacterial growth was monitored manually by measuring OD₆₀₀ at regular intervals over a 24 h period. For all other amino acids, OD₆₀₀ measurements were recorded every 15 min for 24 h using a TECAN Infinite 200 PRO plate reader (TECAN) under aerobic conditions.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMucoviscosity Assay and Capsule Quantification\u003c/h2\u003e\u003cp\u003eBecause capsular polysaccharide is the major virulence determinant of \u003cem\u003eK. pneumoniae\u003c/em\u003e, CPS production was evaluated \u003cem\u003ein vitro\u003c/em\u003e using a semiquantitative mucoviscosity assay and uronic acid quantification.\u003c/p\u003e\u003cp\u003eFor the mucoviscosity assay, overnight cultures grown in LB broth were diluted 1:100 into fresh medium and incubated at 37\u0026deg;C with shaking. After 6 h of growth, cultures were centrifuged at 1,000 \u0026times; g for 5 min, and the optical density of the supernatant at 600 nm (OD₆₀₀) was measured to assess mucoviscosity, as described previously (Ratio: OD600\u003csub\u003efinal\u003c/sub\u003e/OD600\u003csub\u003einnitial\u003c/sub\u003e)\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor uronic acid quantification, \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were cultured for 6 h, and 500 \u0026micro;L of culture was mixed with 100 \u0026micro;L of 1% Zwittergent 3\u0026ndash;12 detergent. Samples were heated for 20 min at 50\u0026deg;C and centrifuged at 13,000 \u0026times; g for 5 min. Supernatants (300 \u0026micro;L) were mixed with 1.2 mL of absolute ethanol, incubated on ice for 20 min, and centrifuged at 13,000 \u0026times; g for 5 min. Pellets were dried and resuspended in 200 \u0026micro;L of sterile water, followed by the addition of 1.2 mL of 12.5 mM sodium tetraborate in concentrated sulfuric acid. After heating at 100\u0026deg;C for 5 min and cooling on ice for 10 min, 20 \u0026micro;L of 0.15% 3-hydroxydiphenyl reagent was added. Samples were incubated for 5 min at room temperature, and absorbance was measured at 520 nm to quantify uronic acid content, as described previously \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA-seq and RT-qPCR\u003c/h3\u003e\n\u003cp\u003eTo investigate the mechanisms by which arginine promotes capsular polysaccharide production, total RNA was extracted from \u003cem\u003eK. pneumoniae\u003c/em\u003e strains incubated with or without arginine, and transcriptome sequencing was performed. All raw sequencing data have been deposited in the NCBI database (SAMN51231398-SAMN51231401), and RNA-seq library preparation, sequencing, and bioinformatic analyses were conducted by Shanghai Meiji Biotechnology Co., Ltd..\u003c/p\u003e\u003cp\u003eTo validate the RNA-seq results, RT-qPCR was performed to quantify the mRNA expression levels of key genes involved in arginine metabolism and CPS biosynthesis. In addition, RT-qPCR was used to examine the role of the transcriptional regulator ArgR in controlling the expression of major CPS transcripts. Primer sequences are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, and detailed methods were performed as described in our previous publication.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of\u003c/b\u003e \u003cb\u003eargR\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eartP\u003c/b\u003e \u003cb\u003emutants and pACYC-\u003c/b\u003e\u003cb\u003eargR\u003c/b\u003e \u003cb\u003ecomplementation plasmid\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDeletion mutants of \u003cem\u003eargR\u003c/em\u003e and \u003cem\u003eartP\u003c/em\u003e were generated using the λ-Red homologous recombination system as previously described. Primer sequences used for mutant construction are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, and all bacterial strains and plasmids employed in this study are summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFor genetic complementation, the pACYC-\u003cem\u003eargR\u003c/em\u003e plasmid was assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs) according to the manufacturer\u0026rsquo;s instructions. The vector backbone and \u003cem\u003eargR\u003c/em\u003e insert were PCR-amplified using Q5 High-Fidelity 2\u0026times; PCR Master Mix (NEB), purified, and assembled into the final plasmid construct. The resulting plasmid was transformed into the appropriate electrocompetent \u003cem\u003eK. pneumoniae\u003c/em\u003e strains as described previously.\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy\u003c/h3\u003e\n\u003cp\u003eTo directly visualize whether \u003cem\u003eargR\u003c/em\u003e deletion affects capsular polysaccharide expression, transmission electron microscopy (TEM) was employed to compare capsule thickness among the wild-type, \u003cem\u003eargR\u003c/em\u003e knockout, and complemented \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. \u003cem\u003eK. pneumoniae\u003c/em\u003e cells in the mid-logarithmic growth phase were harvested and fixed overnight at 4\u0026deg;C in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4). Fixed samples were washed twice with 0.1 M phosphate buffer, post-fixed in 1% osmium tetroxide for 1 h at room temperature, and rinsed sequentially in 0.1 M phosphate buffer and distilled water.\u003c/p\u003e\u003cp\u003eSamples were dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 95% for 15 min each, followed by 100% ethanol twice for 10 min each). Dehydrated specimens were infiltrated and embedded in Spurr\u0026rsquo;s resin and polymerized at 60\u0026deg;C for 48 h. Ultrathin sections were prepared and examined using a Tecnai G2 Spirit Twin transmission electron microscope, and images were digitally recorded.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMacrophage and Kupffer Cell Phagocytosis Assays\u003c/h2\u003e\u003cp\u003eThe ability of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains to resist phagocytosis was evaluated using both murine macrophages and primary liver Kupffer cells. For infection assays, macrophages or Kupffer cells were seeded into 24-well plates at a density of 1 \u0026times; 10⁵ cells/well and infected with \u003cem\u003eK. pneumoniae\u003c/em\u003e strains at a multiplicity of infection (MOI) of 50. Plates were centrifuged at 200 \u0026times; g for 5 min to facilitate bacterial contact and incubated at 37\u0026deg;C with 5% CO₂ for 1.5 h to allow phagocytosis.\u003c/p\u003e\u003cp\u003eAfter infection, cells were washed three times with phosphate-buffered saline (PBS) and incubated for an additional 1.5 h in medium containing meropenem (100 \u0026micro;g/mL) to eliminate extracellular bacteria. Following three further washes with PBS, cells were lysed with 0.2% Triton X-100 for 20 min. Serial dilutions of the lysates were plated on LB agar to enumerate intracellular bacteria. All assays were performed in triplicate using three independent biological replicates per strain.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eβ-galactosidase assays\u003c/h2\u003e\u003cp\u003eTo investigate the regulation of ArgR on the promoter activity of genes within the \u003cem\u003ecps\u003c/em\u003e operon, promoter regions of \u003cem\u003ermpA\u003c/em\u003e, \u003cem\u003egalF\u003c/em\u003e, and \u003cem\u003ewzi\u003c/em\u003e were amplified from \u003cem\u003eK. pneumoniae\u003c/em\u003e strains NTUH-K2044 and FK3009 and cloned upstream of the \u003cem\u003elacZ\u003c/em\u003e reporter gene. Promoter activity was quantified by measuring β-galactosidase activity using a modified Miller assay in bacterial cultures harvested at mid-logarithmic growth phase. Enzyme inhibitors were added during the final step, and cell lysates were prepared using an ultrasonic disruptor to ensure complete release of β-galactosidase. All assays were performed in duplicate\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eProtein Purification and Electrophoretic Mobility Shift Assay (EMSA)\u003c/h2\u003e\u003cp\u003eThe ArgR expression plasmid was commercially constructed by GenScript (Nanjing, China). Briefly, the \u003cem\u003eargR\u003c/em\u003e gene was synthesized and inserted into the pET-30a(+) vector using \u003cem\u003eNdeI\u003c/em\u003e and \u003cem\u003eHindIII\u003c/em\u003e restriction sites, generating a kanamycin-resistant construct. The recombinant plasmid was verified by restriction digestion and confirmed by sequencing.\u003c/p\u003e\u003cp\u003eFor recombinant protein production, the expression plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). Protein expression and purification were performed by GenScript using a standard Ni-IDA affinity chromatography system. In brief, expression was induced with IPTG at low temperature, bacterial cells were lysed by sonication, and the clarified lysate was applied to a Ni-IDA column. Bound protein was eluted with imidazole and dialyzed into storage buffer (10 mM HEPES, pH 7.8, 150 mM NaCl, 5% glycerol, 1 mM DTT). The purified ArgR protein was filtered through a 0.22 \u0026micro;m membrane, aliquoted, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Protein purity was confirmed by SDS-PAGE.\u003c/p\u003e\u003cp\u003eFor EMSA, DNA probes corresponding to the promoter regions of target genes were amplified by PCR with primers listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, purified, and end-labeled with [γ-\u0026sup3;\u0026sup2;P]ATP using T4 polynucleotide kinase (New England Biolabs). Binding reactions were performed by incubating purified ArgR protein with labeled probes in binding buffer (10 mM Tris-HCl, pH 7.5; 50 mM KCl; 1 mM DTT; 5 mM MgCl₂; 0.05% Nonidet\u0026reg; P-40; 2.5% glycerol; 50 mM acetyl phosphate) at 37\u0026deg;C for 30 min. Samples were separated on 4% native polyacrylamide gels in 0.5\u0026times; Tris-borate-EDTA (TBE) buffer at 90 V for 40 min at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003eFollowing electrophoresis, DNA\u0026ndash;protein complexes were transferred to a nylon membrane in 0.5\u0026times; TBE buffer at 4\u0026deg;C using a constant current of 380 mA for 1 h. Membranes were UV-crosslinked, blocked, and incubated with stabilized streptavidin\u0026ndash;HRP conjugate (1:200 in blocking buffer). After washing, signals were detected by chemiluminescence using a digital imaging system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMurine Intestinal Colonization Model\u003c/h2\u003e\u003cp\u003eTo assess gut colonization and translocation, mice were pretreated with a combination of ampicillin, metronidazole, neomycin, and vancomycin (AMNV; each at 0.5 g/L, put in the drinking water) for three days prior to \u003cem\u003eK. pneumoniae\u003c/em\u003e inoculation, and then flush out with the combination of vancomycin and metronidazole (each at 0.5 g/L) for two days to deplete endogenous microbiota. A 200 \u0026micro;L of the bacterial suspension (10\u003csup\u003e4\u003c/sup\u003e CFU for NTUH-K2044;10\u003csup\u003e5\u003c/sup\u003e CFU for FK3009) was administered to mice via oral gavage. Following oral inoculation, mice received drinking water containing vancomycin (0.5 g/L) and metronidazole (0.5 g/L) either alone or in combination with the L-arginine throughout the study period. Vancomycin and metronidazole were included to suppress competing microbiota and maintain \u003cem\u003eK. pneumoniae\u003c/em\u003e colonization. Fecal samples were collected on days 1, 3, 7, and 14 post-infections to monitor intestinal colonization. At corresponding time point, mice were euthanized, and bacterial burdens in key organs, including the liver and spleen, were quantified by plating serial dilutions of tissue homogenates on LB agar to assess systemic dissemination from the intestinal tract.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMetabolomic analysis of cecal contents in the AMNV antibiotics treated mice\u003c/h2\u003e\u003cp\u003eTo characterize amino acid alterations induced by the antibiotic-treated colonization model used in this study, eight SPF female BALB/c mice (6\u0026ndash;8 weeks old) were subjected to microbiota depletion following the previously described AMNV protocol. Mice were then provided sterile PBS in drinking water for an additional 24 hours to remove residual antibiotics. All animals were euthanized thereafter, and cecal contents were immediately collected under sterile conditions, flash-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Targeted amino acid metabolomic profiling was performed to determine the baseline luminal amino acid distribution associated with the antibiotic-treated model employed throughout this study.\u003c/p\u003e\u003cp\u003eTargeted amino acid metabolomic profiling of mouse cecal contents was conducted using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC\u0026ndash;MS/MS; Agilent 1290 Infinity II system and QTRAP 6500\u0026thinsp;+\u0026thinsp;mass spectrometer, SCIEX). Approximately 20 mg of frozen cecal content was extracted with prechilled methanol/water (4:1, v/v) containing internal standards, followed by homogenization, sonication, derivatization with FDAA, and centrifugation. Metabolites were separated on an ACQUITY UPLC BEH C18 column (2.1 \u0026times; 150 mm, 1.7 \u0026micro;m) using a 5 mM ammonium acetate\u0026ndash;acetonitrile gradient. Data were acquired in multiple reaction monitoring (MRM) mode, and analyte quantification was achieved using isotope-labeled internal standards and external calibration curves (R\u0026sup2; \u0026gt;0.99). The limits of detection and quantification were established according to FDA bioanalytical method validation guidelines, ensuring analytical precision and reproducibility (RSD\u0026thinsp;\u0026lt;\u0026thinsp;20%). All the original metabolomic data were listed in data-set-4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eIntestinal Colonization Competition Assay in Mice\u003c/h2\u003e\u003cp\u003eTo evaluate the role of arginine metabolism in intestinal colonization by \u003cem\u003eK. pneumoniae\u003c/em\u003e, we performed colonization competition assays using the hypervirulent strain NTUH-K2044, along with isogenic deletion mutants lacking \u003cem\u003eartP\u003c/em\u003e (a critical transporter) or \u003cem\u003eargR\u003c/em\u003e (a key transcriptional regulator). The intestinal colonization model was established as described previously. Briefly, mice were inoculated by gavage with a 1:1 mixture of the wild-type strain and the corresponding mutant. Fecal samples were collected on days 1, 3, and 7 post-inoculation, and the competitive index was calculated by comparing mutant to wild-type bacterial counts to assess dynamic changes over time.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGalleria mellonella\u003c/b\u003e \u003cb\u003eInfection Model\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eGalleria mellonella\u003c/em\u003e infection assays also applied to assess the bacterial virulence. Briefly, ten larvae weighing approximately 300 mg were randomly assigned to each experimental group. For infection, 10 \u0026micro;L of bacterial suspension (10⁶ CFU/mL in PBS) was injected into the last left proleg using a Hamilton syringe. Control larvae received 10 \u0026micro;L of sterile PBS. Following infection, larvae were incubated at 37\u0026deg;C in the dark and monitored for 96 h. Larvae were considered dead if they failed to respond to repeated physical stimulation. Survival was recorded at regular intervals, and data were analyzed to assess differences in virulence among strains.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eMurine Bloodstream Infection Model\u003c/h2\u003e\u003cp\u003eTo evaluate \u003cem\u003eK. pneumoniae\u003c/em\u003e virulence under different experimental conditions, murine infection models were established to assess host survival and bacterial dissemination to key organs.\u003c/p\u003e\u003cp\u003eFor systemic infection, \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were cultured overnight in LB broth at 37\u0026deg;C, washed, and resuspended in sterile PBS. Female BALB/c mice (6\u0026ndash;8 weeks old; specific pathogen-free) were injected via the tail vein with 100 \u0026micro;L of bacterial suspension (10\u003csup\u003e4\u003c/sup\u003e CFU for NTUH-K2044;10\u003csup\u003e6\u003c/sup\u003e CFU for FK3009). For experiments involving exogenous amino acids, bacteria were pre-incubated with the indicated amino acid in PBS for 3 hours prior to injection and keep drinking water supplemented with corresponding amino acids. Survival was monitored over 7 days, and bacterial loads in the liver and spleen were quantified at designated time points by plating serial dilutions of tissue homogenates on LB agar.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eShort-term (12-hour) intestinal colonization model to assess early mucosal invasion and systemic dissemination\u003c/h2\u003e\u003cp\u003eTo quantify the early impact of intestinal arginine enrichment on mucosal invasion and systemic spread, a short-term colonization model was established. Mice were first treated with the AMNV antibiotic protocol to deplete commensal microbiota, followed by vancomycin/metronidazole washout as described above. Animals were then orally inoculated with 1 \u0026times; 10⁵ CFU of NTUH-K2044 (wild-type, ΔartP, or ΔargR strains). Immediately after gavage, mice were provided either sterile drinking water or water supplemented with 10mM L-arginine.\u003c/p\u003e\u003cp\u003eAt 12 h post-inoculation, mice were euthanized, and tissues were aseptically harvested, including small intestinal segments (ileum), cecal tissue, liver, and spleen. Intestinal tissues were thoroughly washed to remove luminal bacteria, homogenized, and plated to determine tissue-associated bacterial burdens. Liver and spleen homogenates were similarly plated to quantify early systemic dissemination. This model allowed discrimination between arginine-driven enhancement of mucosal penetration and downstream systemic expansion, and enabled assessment of whether these effects require ArtP-mediated arginine uptake and ArgR-dependent transcriptional regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo arginase treatment of cecal contents to assess arginine-dependent\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e \u003cb\u003eexpansion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate whether enzymatic depletion of luminal arginine limits \u003cem\u003eK. pneumoniae\u003c/em\u003e expansion in the gut environment, an ex vivo cecal content assay was established. Cecal contents were collected from three antibiotic-treated mice (AMNV protocol as described above), pooled, and resuspended in sterile PBS at a ratio of 100 mg/mL to mimic the intestinal milieu. The suspension was homogenized, briefly centrifuged to remove large debris, and aliquoted into equal volumes. Recombinant L-arginase (MCE) was added to the treatment group at a final total amount of 1 U per tube, while control tubes received an equal volume of PBS.\u003c/p\u003e\u003cp\u003eAfter a 30-min pre-incubation at 37\u0026deg;C to allow enzymatic depletion of free arginine, each tube was inoculated with 1 \u0026times; 10⁶ CFU of NTUH-K2044 wild-type or the Δ\u003cem\u003eartP\u003c/em\u003e mutant. Samples were incubated at 37\u0026deg;C for 6h, 12h, and 24h under anaerobic environment, after which bacterial counts were determined by serial dilution and plating on LB agar.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA) and R (version 4.2.2). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) as indicated in figure legends. For comparisons between two groups, a two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for normally distributed data, and a Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test was applied for nonparametric data. Comparisons among multiple groups were analyzed using a one-way or two-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s or Sidak\u0026rsquo;s multiple-comparison test. Competitive index assays were evaluated using a one-sample t-test or Wilcoxon signed-rank test to determine whether the mean log₁₀(CI) differed from zero.\u003c/p\u003e\u003cp\u003eRNA-seq differential expression analysis was performed using DESeq2, with significance defined as adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log₂(fold change)| \u0026ge; 1. All experiments were performed in at least three independent biological replicates, and statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Exact \u003cem\u003ep\u003c/em\u003e-values and statistical tests used for each dataset are reported in the corresponding figure legends.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributors\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFY, LC, and SB contributed to conceptualization, supervision, and designed the study. YZ contributed to funding acquisition. YZ, and HG performed data analysis, including methodology, software, and interpretation. YZ, HG, SB, LC, and FY verified the underlying data. YZ, FY, LC, and SB wrote the original draft. PZ, CW, CC, BW, HZ, JF, and JZ contributed to the interpretation of data. All the authors contributed to the manuscript drafting. All authors read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw RNA sequencing data have been deposited in the NCBI database (SAMN51231398-SAMN51231401). https://www.ncbi.nlm.nih.gov/bioproject/1328593\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the authority of NTUH-K2044 by Professor Jin-Town Wang from Department of Internal Medicine, National Taiwan University Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe informed consent was obtained from all subjects and/or their legal guardian. The research protocol was approved by the Ethics Committee of Shanghai Pulmonary Hospital(K24-096Y).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report that there are no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the [National Natural Science Foundation of China] under Grant number [82472287, 82202564], and [Shanghai \u0026ldquo;Chen Guang\u0026rdquo; project] under Grant number [23CGA22].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal burden of bacterial antimicrobial (2024) resistance 1990\u0026ndash;2021: a systematic analysis with forecasts to 2050. Lancet 404:1199\u0026ndash;1226\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGlobal burden of bacterial antimicrobial resistance (2022) in 2019: a systematic analysis. 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Int J Antimicrob Agents 64:107361\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Klebsiella pneumoniae, intestinal colonization, arginine metabolism, Capsular polysaccharide, ArgR regulatory pathway","lastPublishedDoi":"10.21203/rs.3.rs-8289577/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8289577/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntestinal colonization is a critical precursor to invasive \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infection, yet the host-derived metabolic cues that license this transition remain unclear. Here, we identify arginine as a consistently enriched amino acid across inflammation-, diet-, and antibiotic-induced gut perturbations. Arginine markedly enhances capsular polysaccharide production and virulence in both hypervirulent ST23-KL1 and carbapenem-resistant ST11-KL64 lineages under conditions reflective of the gut microenvironment. Mechanistically, arginine must be transported by the ArtP ATP-binding transporter to activate the regulator ArgR, which directly binds capsular polysaccharide operon promoters and indirectly upregulates \u003cem\u003ermpA\u003c/em\u003e, forming a conserved ArtP\u0026ndash;ArgR\u0026ndash;capsule signaling axis. In mouse models, elevated intestinal arginine increases gut colonization, accelerates mucosal invasion, and promotes systemic dissemination, whereas arginase-mediated arginine depletion or loss of ArtP/ArgR abrogates these effects. Together, our findings reveal intestinal arginine as a key host-derived signal that drives \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenic progression and identify arginine metabolism as a tractable therapeutic target.\u003c/p\u003e","manuscriptTitle":"Host-derived arginine promotes Klebsiella pneumoniae colonization and dissemination through ArtP–ArgR–capsule signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 05:58:44","doi":"10.21203/rs.3.rs-8289577/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"793d56d8-f7c9-4f6c-8393-7cec3ce9f8ee","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59697987,"name":"Biological sciences/Microbiology/Pathogens"},{"id":59697988,"name":"Biological sciences/Microbiology/Clinical microbiology"}],"tags":[],"updatedAt":"2026-02-02T17:26:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 05:58:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8289577","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8289577","identity":"rs-8289577","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}