Transcriptional plasticity enables Staphylococcus aureus adaptation to polymicrobial interactions

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Nirmala, Yogendra Pratap Mathuria, Balram Ji Omar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6909814/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the microbial world, survival is not solitary. Staphylococcus aureus thrives or falters depending on its neighbors. This opportunistic pathogen frequently inhabits polymicrobial environments such as chronic wounds, implanted devices, and mucosal surfaces, where interspecies interactions shape its behavior and complicate treatment outcomes. Focusing on the Type VII Secretion System (T7SS), this study explores how S. aureus transcriptionally and functionally adapts during co-culture with three clinically relevant organisms: Pseudomonas aeruginosa , Candida albicans , and Enterococcus faecalis . RNA sequencing revealed distinct ecological responses: P. aeruginosa induced a strongly antagonistic interaction, causing global transcriptional repression, including silencing of virulence genes and T7SS; C. albicans promoted a synergistic response, activating virulence, stress, and metabolic genes despite T7SS repression; and E. faecalis elicited a competitive interaction marked by robust activation of T7SS, cytotoxic effectors, and biosynthetic programs. Western blotting of EsxA validated condition-specific T7SS expression. These findings reveal how S. aureus transcriptionally adapts to microbial neighbors, positioning interspecies signaling as a key driver of precision microbiology and potential target for managing polymicrobial infections. Polymicrobial interactions Transcriptomics Type VII Secretion System Staphylococcus aureus Virulence modulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Microbes rarely live alone. The idea that microbes exist in isolation has been overturned by the discoveries revealing that most pathogens thrive in complex, polymicrobial communities, a concept dating back to the 17th century, when Antonie van Leeuwenhoek first described diverse ‘animalcules’ coexisting in dental plaque( 1 ). Polymicrobial infections represent a major clinical challenge due to the complex interspecies interactions that influence pathogen behavior, host response, and treatment outcomes( 2 ). When two microbes meet, they engage in molecular crosstalk through secreted factors, contact-dependent signaling, and competition for nutrients. These interactions can reprogram gene expression, modulate virulence, and reshape microbial physiology, in often unpredictable ways ( 3 ). Among the diverse microbial species implicated in such infections, Staphylococcus aureus is a prominent opportunistic pathogen frequently encountered in biofilm-associated and chronic infections( 4 ). Its ability to modulate virulence, evade host immunity, and develop antimicrobial resistance is well-documented( 5 , 6 ). Yet, the extent to which these traits are shaped by cohabiting microbial species remains unexplored. Polymicrobial environments, such as chronic wounds( 7 ), cystic fibrosis lungs( 8 ), and implanted medical devices( 9 ), often harbour diverse microbial communities where S. aureus coexists with bacterial and fungal species ( 10 ). Interactions with Gram-negative bacteria ( Pseudomonas aeruginosa )( 11 ), fungi ( Candida albicans )( 12 ), and other Gram-positive bacteria such as Enterococcus faecalis ( 13 ) can significantly alter S. aureus physiology and pathogenic potential. Depending on the partner organism and the ecological niche, these interactions may be competitive, synergistic, or suppressive( 14 , 15 ). One mechanism by which S. aureus engages in interbacterial competition is through the Type VII Secretion System (T7SS), a specialized protein export apparatus implicated in bacterial antagonism, virulence, and niche adaptation( 16 ). While the role of T7SS in mono-species infection models is increasingly recognized( 17 , 18 ), its regulation and functional relevance in polymicrobial contexts remain poorly defined. This study explores how S. aureus adapts its transcriptome during co-culture with P. aeruginosa , C. albicans , and E. faecalis . Using a clinical isolate confirmed to express T7SS, we cultivated defined dual-species communities and performed scanning electron microscopy (SEM) to confirm polymicrobial interactions. Whole transcriptome sequencing and differential expression analysis were then employed to characterize S. aureus 's gene expression shifts in each co-culture condition. By integrating gene ontology and pathway enrichment analyses, we reveal condition-specific virulence regulation, metabolic reprogramming, and the differential deployment of T7SS during polymicrobial growth in S. aureus . These findings were further validated by Western blotting for EsxA protein expression, representing a systems-level investigation into the transcriptional logic of S. aureus during ecologically diverse microbial interactions. Methods Bacterial Strain, T7SS Confirmation, and Polymicrobial Co-Culture A clinical isolate of S. aureus , collected from the Bacteriology Laboratory at AIIMS Rishikesh, was identified using the VITEK2 automated system and MALDI-TOF mass spectrometry. The isolate was confirmed to harbor an active T7SS through PCR and RT-qPCR targeting the esxA gene, using primers listed in Supplementary Table 1. This validated isolate was used in all subsequent experiments. For polymicrobial co-culture, S. aureus was individually incubated with clinical isolates of P. aeruginosa , C. albicans , and E. faecalis in LB broth for 24 hours, and a S. aureus monoculture was included as the control. The physical association between microbial partners was confirmed by scanning electron microscopy (SEM), following the protocol described by B et al.( 19 ), which provided morphological evidence of co-culture. Ethical approval for the study (September 2023 to March 2025) was obtained from the Institutional Ethics Committee, AIIMS Rishikesh (Letter No- AIIMS/IEC/23/294). RNA Sequencing Total RNA was extracted from each polymicrobial co-culture and the S. aureus monoculture using the HiMedia RNA isolation kit. RNA integrity was confirmed by 1% agarose gel electrophoresis, and concentrations were measured with a Qubit® 4.0 Fluorometer. High-quality RNA was reverse-transcribed to cDNA (G-Biosciences, USA), and the presence of the T7SS was verified by PCR amplification of the esxA gene. Libraries were then prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus, quality-checked on the Agilent TapeStation, and sequenced on the Illumina NovaSeq X Plus platform to generate high-depth, paired-end reads for transcriptomic analysis. Data Analysis Raw reads from each condition were quality-checked using FASTQC (v0.12.1) and preprocessed with fastp (v0.23.4). De novo transcriptome assembly was performed using Trinity (v2.15.1), and assembly quality was assessed with assembly-stats (v1.0.1). Transcript abundance was estimated with RSEM (v1.3.3), and differential gene expression (DGE) analysis was conducted using edgeR (v3.42.4). Comparisons were made between each polymicrobial condition and the S. aureus monoculture: S + P ( P. aeruginosa ), S + C ( C. albicans ), and S + E ( E. faecalis ). Protein-coding regions were predicted using TransDecoder (v5.5.0) and aligned against the UniProt reviewed database using BLAST (v2.14.1) with a ≥ 50% similarity cutoff. Annotated DEGs were subjected to GO and KEGG enrichment analyses to identify key biological processes and pathways. Data visualization of differentially expressed genes (DEGs) was generated using Python (v3.11.12). Western Blotting for EsxA Validation Western blotting was performed in three independent replicates on S. aureus monoculture and co-cultures to validate EsxA protein expression. After 24-hour incubation, whole-cell lysates were prepared according to the protocol described by Anderson et al.( 20 ) Equal amounts of protein (25 µg) were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. Membranes were incubated overnight at 4°C with a primary rabbit anti-EsxA antibody in a 1:1000 dilution (Santa Cruz Biotechnology). After washing, membranes were incubated with anti-rabbit HRP-conjugated secondary antibody using a 1:5000 dilution (Santa Cruz Biotechnology), and signals were visualized using ECL substrate on a chemiDoc imaging system (ChemiDoc XRS+, Bio-Rad)( 21 ). Results & Discussion The clinical isolate of S. aureus used in this study was first confirmed to harbor the T7SS through PCR amplification targeting the esxA gene (Supplementary Fig. 1), a well-established core component of the T7SS machinery( 22 ). Subsequent RT-qPCR analysis verified active expression of esxA (Supplementary Fig. 2), indicating transcriptional competence of the secretion system under monoculture conditions. This characterized strain was then employed in polymicrobial co-culture experiments with P. aeruginosa , C. albicans , and E. faecalis . In parallel, scanning electron microscopy (SEM) confirmed the physical coexistence of S. aureus with each co-culture partner, validating the establishment of polymicrobial associations before transcriptomic profiling (Fig. 1 ). Following co-culture, total RNA was isolated from each condition and subjected to rigorous quality control. RNA integrity was assessed using a 1% agarose gel, and concentrations were measured with a Qubit® 4.0 Fluorometer. The Agilent TapeStation 4150 system with High Sensitivity D1000 ScreenTape®, further confirmed high-quality, non-degraded RNA suitable for transcriptomic analysis. cDNA was synthesized from the purified RNA, and subsequent PCR amplification again confirmed the presence of esxA transcripts, reinforcing that the T7SS remained transcriptionally detectable across all experimental contexts (Fig. 2 a). Pseudomonas aeruginosa induces global transcriptional suppression in Staphylococcus aureus during co-culture P. aeruginosa co-culture induced a global transcriptional shutdown in S. aureus , with 230 genes significantly downregulated and only one, hly (alpha-hemolysin), upregulated (log₂FC = + 0.48) (Fig. 3 a). This repression was supported by Venn and barplot analyses (Fig. 3 b-c). STRING network visualization (Fig. 3 e) revealed strong interconnectivity among the downregulated proteins, implicating core processes such as energy metabolism, translation, and regulatory pathways. In contrast, hly appeared as a solitary, unconnected node (Fig. 3 d), suggesting an independent, stress-induced regulatory response. Functional enrichment analyses (Fig. 3 f-g) identified suppression across critical metabolic and cellular pathways, including amino acid biosynthesis, carbohydrate metabolism, ribosome assembly, and membrane-associated functions. Genes encoding ribosomal proteins ( rplX, rpsD, rpsL )( 23 ) and translation factors ( tuf, efp )( 24 ) were downregulated, indicating a protein synthesis shutdown likely driven by nutrient limitation or quorum-sensing interference from P. aeruginosa . Repression of TCA cycle enzymes ( mqo2 )( 25 ) and glycolytic genes ( ackA, ptsG, fba )( 26 ) reflects metabolic quiescence, potentially induced by P. aeruginosa -derived respiratory inhibitors such as HQNO( 27 ). Amino acid biosynthetic genes ( ilvA, ilvC, leuB, tyrA )( 28 ) were suppressed, consistent with reduced anabolic demand and competition-driven nutrient scarcity. Downregulation of stress response and chaperone genes ( dnaK, groEL )( 29 ) suggests impaired ability to respond to oxidative or envelope stress, possibly exacerbated by P. aeruginosa redox-active metabolites. Suppressed expression of staphyloxanthin biosynthesis genes ( crtQ, crtO, crtM )( 30 ) likely compromises antioxidant defense, rendering S. aureus more susceptible to oxidative stress induced by P. aeruginosa -derived ROS. Key virulence factors ( spa, lukEv, sak, isdB, isdI, clfA, atl, sdrC, sdrD )( 31 ) were silenced, suggesting diminished pathogenicity under competitive pressure. Transcriptional regulators ( sarZ, sarX, sigS, mgrA, spx, agrB )( 32 , 33 ) were repressed, indicating upstream disruption of quorum-sensing and global regulatory networks. Downregulation of cell wall genes ( murG, femX, lytN, lytM, sle1 )( 34 ) may weaken envelope integrity, a known consequence of interspecies interactions. Genes involved in nucleotide and cofactor metabolism ( purD, purN, pyrB, queF, bioW, bioD, pdxS, moaE )( 35 , 36 ) were also suppressed, aligning with metabolic restraint. Importantly, S. aureus downregulated metal ion transporters ( copA, nikD, norB )( 37 ), possibly in response to P. aeruginosa 's siderophore (e.g., pyoverdine) production and iron sequestration, which disrupts S. aureus iron acquisition systems. Finally, the absence of T7SS gene expression indicates complete suppression of interbacterial antagonism( 38 ), suggesting P. aeruginosa outcompetes and functionally disarms S. aureus . Despite this global repression, hly was uniquely upregulated in the P. aeruginosa co-culture. This gene encodes a stress-induced, independently regulated cytotoxin( 39 ), enabling S. aureus to retain minimal cytolytic potential even under widespread transcriptional silencing. Rather than indicating a full virulence program, this selective activation likely represents a compensatory survival response to polymicrobial stress. Gene-level visualizations (Fig. 4 a–e) demonstrated consistent repression across major functional modules, highlighting hly as a unique outlier. Heatmaps (Fig. 4 a–b) showed widespread downregulation across functional categories, while the circular barplot (Fig. 4 c) emphasized the predominance of suppressed transcripts. UMAP analysis (Fig. 6 e) revealed clustering of downregulated genes into distinct regulatory groups, with hly appearing as an isolated node. The Sankey diagram (Fig. 6 d), depicting the top differentially expressed genes involved in metabolism, stress response, and cell wall biogenesis, showed that all were directed into the downregulated category, further highlighting the extent of P. aeruginosa -mediated suppression. These findings indicate that P. aeruginosa enforces a coordinated and multifaceted transcriptional silencing of S. aureus , disrupting key metabolic, virulence, and stress response pathways. This repression reflects a competitive dominance strategy in polymicrobial environments, functionally disarming S. aureus and diminishing its ecological competitiveness. Elucidating the molecular basis of this suppression may inform the development of therapeutic approaches that harness or mimic interspecies antagonism. For example, P. aeruginosa -derived molecules such as 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), pyocyanin, or siderophore analogs could be repurposed or engineered to target S. aureus persistence mechanisms without promoting broad-spectrum resistance. Alternatively, disrupting S. aureus quorum sensing or stress resilience pathways revealed to be suppressed in co-culture may offer synergistic strategies when combined with biofilm-targeted therapies. Such approaches represent a paradigm shift, from pathogen eradication to ecological modulation, offering precision tools for managing chronic, device-associated, or polymicrobial infections. Candida albicans induces transcriptional reprogramming and virulence activation in S. aureus during co-culture In contrast to the repressive effects of P. aeruginosa , co-culturing S. aureus with C. albicans elicited a bidirectional transcriptional response, with 64 genes significantly upregulated and 54 downregulated (Fig. 5 a), as confirmed by barplot and Venn analyses (Fig. 5 b-c). STRING-based interaction networks (Fig. 5 d-e) revealed that both gene sets formed interconnected modules, suggesting coordinated regulatory shifts. Functional enrichment analyses highlighted activation of amino acid biosynthesis, cofactor metabolism, carbohydrate processing, and siderophore pathways (KEGG, Fig. 5 f). GO terms (Fig. 5 g) emphasized ATP binding, metal ion binding, ribosomal structure, and membrane-associated components, indicating broad physiological adaptation. Upregulated genes reflected enhanced virulence, metabolic reprogramming, and stress tolerance. Virulence factors ( clfA , spa , lukEv , atl , cidA , hly )( 40 ) suggested increased adhesion, immune evasion, and cytotoxicity, consistent with prior reports of S. aureus–C. albicans synergy during mucosal invasion. Oxidative stress defense genes ( sodM , sodA , ahpC , rex , perR )( 41 ) were elevated, likely counteracting ROS generated by fungal metabolism or host immune activity. The induction of fermentative metabolism genes ( mqo2 , ldh1 , ptsG , glcT , pflA , adh )( 42 ) indicates a shift toward anaerobic energy production, reflecting hypoxic and nutrient-depleted biofilm conditions often shaped by C. albicans . Upregulation of membrane remodeling genes ( lytN )( 34 ) may facilitate adaptation to altered physical environments, including hyphal invasion. Anabolic genes ( ilvC , aspS , argR , argG , metK , pyrR )( 28 ) and translational components ( rpsL , rplX , rplK , spsA , engB )( 23 ) were also upregulated, supporting biosynthesis and active growth. Regulatory genes ( mgrA , saeS , vraR , spx )( 43 , 44 ) suggest global transcriptional activation. Notably, metal ion transporters ( copA , copB )( 37 ) were induced, likely in response to iron limitation imposed by fungal siderophores. In contrast, downregulated genes indicated suppression of core metabolic, stress response, and proliferative functions. Repression of central metabolic enzymes ( ackA , glcU )( 45 , 46 ) suggested reduced energy flux, likely due to glucose depletion or oxygen limitation in shared niches. Downregulation of amino acid biosynthesis genes ( tyrS , glnA , moaE )( 28 ) reflected nutrient competition, while stress response genes ( hslO , ctsR )( 47 , 48 ) were also suppressed, potentially compromising adaptability under oxidative stress. Virulence and regulatory genes ( clfA , isaB , sarX )( 49 – 51 ) were downregulated, possibly reflecting immune evasion or interkingdom tolerance. Suppression of DNA replication and repair ( recU , recX , dnaA )( 52 , 53 ) and cell division genes ( parC )( 54 ) suggests a shift toward a low-proliferative or quiescent state. Notably, repression of norB and norG, components of the Nor efflux pump regulatory network, suggests reduced efflux capacity, potentially sensitizing S. aureus to antimicrobial stress or toxic metabolites during co-culture. Gene-level visualizations supported the observed transcriptional reprogramming. Heatmaps (Fig. 6 a-b) illustrated a clear contrast between upregulated and downregulated genes across key functional categories, indicating strong condition-specific expression shifts. The circular barplot (Fig. 6 c) emphasized the balanced distribution of regulatory changes, while UMAP clustering (Fig. 6 e) distinctly separated gene expression patterns into two polarized groups. The Sankey diagram (Fig. 6 d) mapped the top differentially expressed genes into major biological categories, highlighting the coordinated nature of the bidirectional response in S. aureus . Together, these results demonstrate that C. albicans induces a finely orchestrated transcriptional reprogramming in S. aureus , activating virulence, stress tolerance, and metabolic flexibility, while repressing growth-associated, biosynthetic, and oxidative defense pathways. This dual modulation likely reflects a survival strategy tailored to the polymicrobial niche, enabling S. aureus to persist under cooperative or competitive pressures imposed by C. albicans . The observed plasticity aligns with prior evidence of fungal–bacterial synergy in mucosal colonization, biofilms, and disseminated infections. Importantly, the selective silencing of replication and redox defenses, alongside virulence activation, suggests a shift toward a quiescent yet pathogenic phenotype. These findings offer mechanistic insight into interkingdom interactions and identify potential vulnerabilities for therapeutic targeting in chronic or device-associated infections. Enterococcus faecalis activates type VII secretion and virulence expression in Staphylococcus aureus during co-culture Global transcriptomic profiling identified 1,576 differentially expressed genes (DEGs) in S. aureus during co-culture with E. faecalis , comprising 890 upregulated and 686 downregulated genes (Fig. 7 a). The volcano plot (Fig. 1 ) highlights the distribution of significantly altered genes across a broad range of fold changes and p-values, while the bar and bubble plots (Fig. 7 b-c) confirm the relative proportions of gene regulation categories. STRING-based protein–protein interaction networks (Fig. 7 d-e) revealed tightly interconnected clusters among up- and downregulated DEGs, suggesting coordinated regulatory control during polymicrobial stress. Functional enrichment analysis (Fig. 7 f-g) showed that upregulated genes were enriched in amino acid biosynthesis, cofactor metabolism, and carbohydrate degradation pathways. GO molecular functions included ATP binding, metal ion binding, and stress response activities. At the same time, enriched cellular components such as membrane-associated and extracellular regions suggest enhanced environmental sensing, structural adaptation, and nutrient acquisition. S. aureus underwent extensive transcriptional reprogramming in response to E. faecalis , characterized by the upregulation of virulence, stress adaptation, metabolic plasticity, and interbacterial competition mechanisms. Prominent virulence factors such as spa, clfA, fnbB, fib, bbp, sdrC, and sraP ( 40 , 55 )were significantly upregulated, promoting adhesion, immune evasion, and biofilm establishment features crucial for persistence in polymicrobial infections. Increasing lukDv, lukEv, hlgB, and hly( 39 , 40 ) suggested a cytolytic phenotype, enhancing host tissue damage and competitive fitness. Notably, the activation of essA and splF indicated the T7SS engagement( 56 ), a contact-dependent pathway in S. aureus that translocates toxic effectors into Gram-positive competitors to establish niche dominance. T7SS is selectively activated against structurally compatible bacteria such as E. faecalis , whose thick peptidoglycan layer enables effector delivery and physical sensing( 57 ). In contrast, P. aeruginosa , a Gram-negative bacterium, possesses an outer membrane that impedes T7SS activity and secretes diffusible inhibitors (e.g., HQNO, pyocyanin)( 27 ) that suppress S. aureus transcriptionally, including T7SS components. Likewise, C. albicans lacks the structural features required for T7SS targeting and does not pose direct antibacterial aggression. Instead, S. aureus exhibits a synergistic transcriptional response to C. albicans , upregulating virulence and stress pathways without T7SS induction, consistent with their known mutual persistence in biofilms and mucosal niches( 58 ). This context-dependent specificity emphasizes a finely tuned defense system in S. aureus , selectively deployed against compatible bacterial threats. Stress adaptation pathways were also markedly elevated. Oxidative and DNA damage responses were reflected by upregulation of ahpC, uvrA, recO, lexA, and trxA,( 53 , 59 , 60 ) likely countering peroxide and reactive oxygen species produced by E. faecalis . The induction of clpP, cap5A, and cpfC, supported structural stabilization,( 61 , 62 ), promoting envelope integrity and redox control. While adhesion- and remodeling-associated genes ( atl , sle1 , spa , sdrC )( 31 , 63 ) were upregulated, the concurrent upregulation of icaR ( 63 ), a repressor of the icaADBC operon, and downregulation of icaD ( 63 ) suggest attenuation of PIA-mediated biofilm synthesis. This indicates a shift toward a proteinaceous, rather than polysaccharide-driven, biofilm phenotype in response to E. faecalis . Metabolic reprogramming was evident through increased expression of adh, ackA, glcU, pyk, pflA, and qoxC,( 45 , 46 , 64 ) consistent with a metabolic switch between fermentation and respiration. Upregulated biosynthetic genes, including tyrA, proC, leuS, and thrS,( 65 , 66 ) pointed to enhanced anabolic activity to support cellular demands under competitive conditions. Induction of cntL, nikD, and kdpA( 67 , 68 ) further indicated active acquisition of trace elements, likely in response to metal competition within the shared niche. Mobile element genes such as tnpR and tnpC,( 69 ) alongside transcriptional regulators rpoB, rpoE, rpsJ, spx, and spxH,( 70 ) were also upregulated, suggesting increased genomic plasticity and stress-adaptive transcriptional remodeling. Conversely, substantial transcriptional repression was observed across key biosynthetic, metabolic, and virulence functions. Downregulated metabolic genes included core glycolytic and TCA cycle components gapA2, glkA, gpsA, pckA, ldh1, odhA, fumC, pta, and mqo2,( 25 , 71 , 72 ) indicating reduced energy flux and metabolic conservation. Suppressed amino acid and nucleotide biosynthesis gene purD,( 73 ) reflected a shift away from growth and proliferation. Decreased expression of atpF and ctaB( 74 ) further signaled respiratory downregulation. Ribosomal proteins (rplU, rplV, rpsL)( 23 ), elongation factor efp,( 75 ) and tRNA synthetases (pheS, ileS, lysS)( 76 ) were repressed, indicating global translational silencing. DNA replication and repair elements dnaA and recR( 52 , 53 ) were downregulated, indicating reduced cell cycle progression. Key virulence genes, including fnbA, sdrD, ssl10, sak, hlgA, and secA2( 77 – 79 ) were suppressed, as were capsule and cell wall components mprF, ebhA, lytM, and cls2,( 34 , 80 ) indicating a downshift in structural virulence and immune evasion. In addition, staphyloxanthin biosynthesis genes crtM and crtP ( 30 ) were repressed, suggesting diminished antioxidant capacity and stress resistance. Regulatory downregulation of ctsR, rex, and srrB,( 81 ) implied diminished environmental sensing. Finally, division proteins ftsA, ezrA,( 82 ) were suppressed, supporting a metabolically restrained, quiescent phenotype under antagonistic stress. Gene-level visualizations validated these findings. Heatmaps (Fig. 8 a-b) revealed distinct clusters of upregulated and downregulated genes, reflecting a coordinated transcriptional shift in response to polymicrobial co-culture. UMAP clustering and radial barplots (Fig. 8 c and 8 e) further illustrated a clear polarization of gene expression profiles, with minimal overlap between expression states. The Sankey diagram (Fig. 8 d) mapped major transcriptional changes across key functional categories, emphasizing the organized reprogramming of S. aureus under competitive stress. These data reveal that S. aureus mounts a dualistic response to E. faecalis co-culture, activating T7SS, virulence pathways, and biosynthesis, while concurrently silencing growth, translation, and energetically costly processes. This ecological trade-off prioritizes stress endurance, competitive fitness, and sessile adaptation over proliferation. Given the known secretion of bacteriocins, hydrogen peroxide, and biofilm-promoting enzymes by E. faecalis , the transcriptional reprogramming in S. aureus likely reflects an integrated response to direct and indirect microbial antagonism. These findings emphasize the importance of interbacterial signaling in modulating pathogen behavior and suggest that disrupting communication pathways such as T7SS or quorum sensing may provide therapeutic avenues for managing polymicrobial infections. Western blotting was performed to validate transcriptomic findings on T7SS regulation and detect EsxA protein across all co-culture conditions. A strong EsxA band (~ 22–35 kDa) was observed in S. aureus monoculture (S) and co-culture with E. faecalis (S + E), supporting active T7SS expression. In contrast, the band was weak in the P. aeruginosa co-culture (S + P) and absent or faint in the C. albicans co-culture (S + C), consistent with RNA-seq data showing esxA repression under these conditions (Fig. 2 b). Although EsxA is predicted to be ~ 11 kDa, the observed higher molecular weight may result from dimerization or polymerization (Fig. 2 d-e), as reported by Sundaramoorthy et al ( 22 ). These results confirm that EsxA expression is context-dependent, active against Gram-positive competitors like E. faecalis and silenced during interaction with C. albicans and P. aeruginosa . The primary limitation of this study is that it provides a single-time-point, snapshot view of the transcriptomic landscape, without capturing dynamic transitions or temporal changes in gene expression. Nevertheless, key findings were validated through complementary assays, including SEM, PCR, and Western blotting, supporting the reliability of the observed condition-specific responses. A graphical summary of the distinct interaction modes, antagonism with P. aeruginosa , synergy with C. albicans , and competitive response to E. faecalis , is depicted in Fig. 9 , highlighting condition-specific modulation of virulence, metabolism, adhesion, biofilm, and T7SS expression in S. aureus . This schematic illustrates the distinct gene expression profiles of Staphylococcus aureus when co-cultured with Candida albicans , Pseudomonas aeruginosa , and Enterococcus faecalis . In the left panel, representing co-culture with C. albicans , S. aureus exhibits a synergistic interaction marked by upregulation of virulence and cell adhesion genes, while genes involved in T7SS secretion and metabolism are downregulated. In the centre panel, during co-culture with P. aeruginosa , an antagonistic response is observed, with global transcriptional repression affecting metabolism, biofilm formation, adhesion, and T7SS genes, and only minimal virulence activation, indicating suppression of S. aureus pathogenic potential. In the right panel, co-culture with E. faecalis leads to a competitive interaction characterized by upregulation of T7SS, virulence factors, metabolic pathways, and adhesion genes, while biofilm-related genes are downregulated. Upregulated and downregulated functions are indicated by green ( ▲ ) and red ( ▼ ) arrows, respectively. Icons represent core functional themes such as T7SS, metabolism, biofilm, virulence, and adhesins. The central test tubes depict S. aureus co-cultured with each partner organism. Conclusion This study demonstrates that S. aureus exhibits distinct, context-dependent transcriptional responses when co-cultured with P. aeruginosa , C. albicans , and E. faecalis . While P. aeruginosa imposes a dominant suppressive effect, silencing metabolism, stress response, and virulence networks, C. albicans elicits a synergistic reprogramming marked by co-activation of virulence, metabolic flexibility, and redox tolerance. In contrast, E. faecalis triggers a defensive, antagonistic response characterized by activation of the T7SS, virulence regulators, and anabolic machinery, reflecting a competitive interbacterial strategy. These divergent expression profiles reflect tailored ecological adaptations: suppression under Gram-negative stress, cooperation with fungal partners, and aggression against Gram-positive competitors. These findings provide mechanistic insight into how S. aureus dynamically modulates its transcriptome to balance competition, coexistence, and persistence in polymicrobial environments, laying the foundation for precision microbiology strategies that target interspecies signaling and microbial crosstalk in complex infections. Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of the manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution Nirmala B designed and performed the experiments, analyzed the data, and wrote the manuscript. Yogendra P Mathuria and Balram Ji Omar supervised the study and validated the findings. All authors reviewed and approved the final manuscript. All authors read and approved the final manuscript. Acknowledgement We acknowledge the Department of Biotechnology, Ministry of Science and Technology, Government of India, for providing a DBT-JRF scholarship to B Nirmala. We also acknowledge the All India Institute of Medical Sciences (AIIMS) Rishikesh for providing infrastructural support for this study. Data Availability The raw and processed RNA-seq data generated in this study will be deposited in the NCBI Sequence Read Archive (SRA) under a BioProject accession number, which will be provided upon manuscript acceptance. An Excel file containing all differentially expressed genes (DEG) data, metadata, and gene expression matrices is provided in the Supplementary Information for transparency. Python scripts used for analysis will be made available on GitHub. References Zhang M, Whiteley M, Lewin GR. 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Expression of Staphylococcus aureus translation elongation factor P is regulated by a stress-inducible promotor. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology. 2024 Dec 1;117(1). Giegé R, Springer M. Aminoacyl-tRNA Synthetases in the Bacterial World. EcoSal Plus. 2012 Dec 31;5(1). Rahman S, Das AK. Staphylococcal superantigen-like protein 10 enhances the amyloidogenic biofilm formation in Staphylococcus aureus. BMC Microbiol. 2023 Dec 1;23(1). Zhu Z, Hu Z, Li S, Fang R, Ono HK, Hu DL. Molecular Characteristics and Pathogenicity of Staphylococcus aureus Exotoxins. Vol. 25, International Journal of Molecular Sciences. Multidisciplinary Digital Publishing Institute (MDPI); 2024. Rodrigues RA, Pizauro LJL, Varani A de M, de Almeida CC, Silva SR, Cardozo MV, et al. Comparative genomics study of Staphylococcus aureus isolated from cattle and humans reveals virulence patterns exclusively associated with bovine clinical mastitis strains. Front Microbiol. 2022 Nov 7;13. Yamanashi Y, Shimamura Y, Sasahara H, Komuro M, Sasaki K, Morimitsu Y, et al. Effects of Growth Stage on the Characterization of Enterotoxin A-Producing Staphylococcus aureus‐Derived Membrane vesicles. Microorganisms. 2022 Mar 1;10(3). Dmitriev A, Chen X, Paluscio E, Stephens AC, Banerjee SK, Vitko NP, et al. The Intersection of the Staphylococcus aureus Rex and SrrAB Regulons: an Example of Metabolic Evolution That Maximizes Resistance to Immune Radicals. 2021; Available from: https://doi.org/10.1128/mBio Myrbråten IS, Stamsås GA, Chan H, Angeles DM, Knutsen TM, Salehian Z, et al. SmdA is a Novel Cell Morphology Determinant in Staphylococcus aureus. mBio. 2022 Apr 1;13(2). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.pdf SAPvsSA.xlsx SACvsSA.xlsx SAEvsSA.xlsx Cite Share Download PDF Status: Posted 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6909814","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486445068,"identity":"05ce92c2-be11-40d7-a3b7-6597b18a6cb2","order_by":0,"name":"B. Nirmala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACxgY2ECUBYjY++ACk2NiJ18LcbDgDpIWZoD1sMAZ7mzQPiCakhbm9LfFx5Q6LaN0ZiW3SNr+2yfMxMzB++JiDx2E9xw4bnj0jkbvtRmKzdW7fbcM2ZgZmyZnb8GiZkd4m2dgG1tJ4O7fnNiNQCxszL34t7T+hWhqkLXtu2xOhJe0YI1RLkzTDj9uJhLX0HEuGOOzMw2bD3obbyW3MjM14/WLY3mb4sbGtLnfb8fSHD378uW07v7354IeP+LQ0wFgCCUA728A2N+BQDAHycBb/ASDxB6/iUTAKRsEoGKEAALgWVyN0MgkyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0005-9306-260X","institution":"Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, Uttarakhand, India","correspondingAuthor":true,"prefix":"","firstName":"B.","middleName":"","lastName":"Nirmala","suffix":""},{"id":486445069,"identity":"e90bab05-c59b-4530-bb75-ac3392e30b2f","order_by":1,"name":"Yogendra Pratap Mathuria","email":"","orcid":"","institution":"Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, Uttarakhand, India","correspondingAuthor":false,"prefix":"","firstName":"Yogendra","middleName":"Pratap","lastName":"Mathuria","suffix":""},{"id":486445073,"identity":"c8cb36af-b962-433e-ac27-3b38e9eebc4e","order_by":2,"name":"Balram Ji Omar","email":"data:image/png;base64,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","orcid":"","institution":"Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, Uttarakhand, India","correspondingAuthor":true,"prefix":"","firstName":"Balram","middleName":"Ji","lastName":"Omar","suffix":""}],"badges":[],"createdAt":"2025-06-17 03:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6909814/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6909814/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86957754,"identity":"f42b9e04-ad7d-42cb-9574-d2dff39cad80","added_by":"auto","created_at":"2025-07-17 15:28:00","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":650162,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs confirming the polymicrobial association of Staphylococcus aureus with co-culture microbes. (a) \u003cem\u003eS. aureus + Candida albicans\u003c/em\u003e: yeast and hyphal forms of \u003cem\u003eC. albicans\u003c/em\u003eembedded with coccoid S. aureus cells, forming dense polymicrobial clusters.\u003cbr\u003e\n(b) \u003cem\u003eS. aureus + Pseudomonas aeruginosa\u003c/em\u003e: rod-shaped \u003cem\u003eP. aeruginosa\u003c/em\u003einterspersed with S. aureus cells in mixed microcolonies.\u003cbr\u003e\n(c) \u003cem\u003eS. aureus + Enterococcus faecalis\u003c/em\u003e: compact aggregates of cocci indicating dual Gram-positive biofilm organization.\u003cbr\u003e\n(d) \u003cem\u003eS. aureus\u003c/em\u003e monoculture as control, showing characteristic coccoid clustering.\u003cbr\u003e\nScale bars: 2–5 μm.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/0b9b8b89f3cf402edd672266.jpeg"},{"id":86958248,"identity":"83864a21-f996-4e09-b507-71bcf62d86f8","added_by":"auto","created_at":"2025-07-17 15:36:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation and structural representation of EsxA, a core effector of the Type VII Secretion System (T7SS).\u003c/strong\u003e\u003cbr\u003e\n(a) cDNA PCR amplification of \u003cem\u003eesxA\u003c/em\u003efrom \u003cem\u003eStaphylococcus aureus\u003c/em\u003emonoculture (S) and co-cultures with \u003cem\u003eE. faecalis\u003c/em\u003e(S+E), \u003cem\u003eP. aeruginosa\u003c/em\u003e(S+P), and \u003cem\u003eC. albicans\u003c/em\u003e(S+C), confirming the presence of transcripts before RNA-seq. The expected amplicon size (~200 bp) is visible in all lanes. M: 100 bp DNA ladder.\u003cbr\u003e\n(b) Western blot analysis of EsxA protein (~22–35 kDa), showing strong expression in S and S+E, weak expression in S+P, and absent or faint in S+C.\u003cbr\u003e\n(c) STRING protein–protein interaction network centered on EsxA, revealing functional associations with core T7SS components including EssA, EssB, and EsxB.\u003cbr\u003e\n(d) Molecular structure of EsxA generated using PyMOL from a Protein Data Bank (PDB) model, highlighting α-helical domains.\u003cbr\u003e\n(e) Polymeric assembly model of EsxA based on PDB structural data, illustrating potential higher-order organization.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/fc5518cb5459d54ea90bf21f.jpeg"},{"id":86958249,"identity":"77e5200d-6684-4a9c-bd60-edeaf6e4d4ea","added_by":"auto","created_at":"2025-07-17 15:36:00","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":267482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStaphylococcus aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e during co-culture with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Volcano plot showing differentially expressed genes (DEGs); green dot indicates the single upregulated gene (\u003cem\u003ehly\u003c/em\u003e), while red dots represent downregulated genes.\u003cbr\u003e\n(b) Venn diagram summarizing DEG distribution across upregulated, downregulated, and nonsignificant genes.\u003cbr\u003e\n(c) Bar plot depicting counts of upregulated and downregulated genes.\u003cbr\u003e\n(d) The STRING network view shows \u003cem\u003ehly\u003c/em\u003e as a disconnected, solitary node.\u003cbr\u003e\n(e) STRING interaction network of significantly downregulated genes, highlighting dense protein–protein interactions.\u003cbr\u003e\n(f) KEGG pathway enrichment analysis of downregulated genes; bars indicate gene count per pathway.\u003cbr\u003e\n(g) GO enrichment analysis categorized into biological processes (BP), molecular functions (MF), and cellular components (CC), with gene counts plotted per term.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/6bbc65bc97b6139e484fe0d0.jpeg"},{"id":86959527,"identity":"104ae508-7a00-4bc9-af51-e536103f2687","added_by":"auto","created_at":"2025-07-17 15:52:00","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":377997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene-level visualization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStaphylococcus aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcriptional response during co-culture with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (S+P).\u003c/strong\u003e\u003cbr\u003e\n(a) Heatmap of normalized expression values for all differentially expressed genes (DEGs), showing widespread downregulation.\u003cbr\u003e\n(b) Focused heatmap of selected significantly downregulated genes involved in metabolism, stress response, and virulence.\u003cbr\u003e\n(c) A circular barplot highlights the dominance of transcriptional repression, with downregulated genes shown in pink and \u003cem\u003ehly\u003c/em\u003e as the only upregulated gene in green.\u003cbr\u003e\n(d) Sankey diagram mapping top-downregulated genes into functional categories such as nucleotide metabolism, energy production, and stress adaptation.\u003cbr\u003e\n(e) UMAP clustering of DEG expression profiles, showing coherent grouping of downregulated genes and \u003cem\u003ehly\u003c/em\u003e as a distant outlier.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/bf52f008fda264885d808b1d.jpeg"},{"id":86959293,"identity":"ccae5d03-3ea8-492e-bcb5-d878da18d724","added_by":"auto","created_at":"2025-07-17 15:44:00","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":199612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStaphylococcus aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e during co-culture with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida albicans (S+C)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Volcano plot showing differentially expressed genes (DEGs), with upregulated genes in green and downregulated genes in red.\u003cbr\u003e\n(b) Bar plot showing the number of upregulated and downregulated genes.\u003cbr\u003e\n(c) Venn diagram summarizing DEG distribution across upregulated, downregulated, and nonsignificant categories.\u003cbr\u003e\n(d) STRING network of upregulated genes highlighting functional modules involved in virulence, stress response, and metabolism.\u003cbr\u003e\n(e) STRING network of downregulated genes showing scattered and loosely connected nodes.\u003cbr\u003e\n(f) KEGG pathway enrichment analysis shows major functional categories including amino acid biosynthesis, cofactor biosynthesis, and carbohydrate metabolism.\u003cbr\u003e\n(g) GO enrichment analysis of DEGs across biological processes (BP), molecular functions (MF), and cellular components (CC), revealing significant enrichment in ATP binding, DNA binding, and extracellular region components.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/b981ae0dc6452ead47c65c44.jpeg"},{"id":86957762,"identity":"cd960a86-427d-40fb-835b-fa2c9b1ab823","added_by":"auto","created_at":"2025-07-17 15:28:00","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":367347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene-level visualization of Staphylococcus aureus transcriptomic reprogramming during co-culture with Candida albicans.\u003c/strong\u003e\u003cbr\u003e\n(a) Heatmap of log₂ fold change values for all differentially expressed genes (DEGs), showing bidirectional expression shifts.\u003cbr\u003e\n(b) Heatmap of the top 50 most upregulated and top 50 most downregulated genes, highlighting functional responses in virulence, metabolism, and stress adaptation.\u003cbr\u003e\n(c) Circular barplot displaying the relative proportion and magnitude of upregulated (green) and downregulated (pink) genes.\u003cbr\u003e\n(d) The Sankey diagram shows the top upregulated and downregulated genes mapped by expression category.\u003cbr\u003e\n(e) UMAP clustering of S. aureus gene expression profiles, showing clear separation between upregulated and downregulated gene groups during co-culture.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/c5f0fa9a0567acd3878d9c28.jpeg"},{"id":86957768,"identity":"ae63dc70-cc96-4cf0-964d-d8e7995d0727","added_by":"auto","created_at":"2025-07-17 15:28:00","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":271140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStaphylococcus aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e during co-culture with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Volcano plot of differentially expressed genes (DEGs), with upregulated genes in green and downregulated genes in red.\u003cbr\u003e\n(b) Venn diagram summarizing the distribution of DEGs across upregulated, downregulated, and nonsignificant categories.\u003cbr\u003e\n(c) Bar plot displaying the number of genes in each DEG category.\u003cbr\u003e\n(d) STRING interaction network of upregulated genes, showing functional clustering in pathways related to virulence, stress adaptation, and interbacterial competition.\u003cbr\u003e\n(e) STRING network of downregulated genes, depicting dense protein–protein connectivity associated with translation, metabolism, and cell division.\u003cbr\u003e\n(f) KEGG pathway enrichment analysis of all DEGs, showing the most significantly regulated biological processes, including amino acid biosynthesis, carbohydrate metabolism, and cofactor biosynthesis.\u003cbr\u003e\n(g) GO enrichment analysis categorized into biological processes (BP), molecular functions (MF), and cellular components (CC), highlighting enrichment in ATP binding, DNA binding, and membrane-associated components.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/ea874523c0823cc20be38dad.jpeg"},{"id":86958258,"identity":"b04e98d6-86dc-4df5-875a-4259ac3134a6","added_by":"auto","created_at":"2025-07-17 15:36:00","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":368733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene-level visualization of Staphylococcus aureus transcriptomic response during co-culture with Enterococcus faecalis.\u003c/strong\u003e\u003cbr\u003e\n(a) Heatmap of log₂ fold change values for all differentially expressed genes (DEGs), highlighting a broad range of transcriptional modulation.\u003cbr\u003e\n(b) Heatmap of the top 50 most upregulated and top 50 most downregulated genes, revealing strong differential expression in stress response, virulence, and metabolic pathways.\u003cbr\u003e\n(c) Circular barplot representing the distribution and magnitude of gene regulation, with upregulated genes in green and downregulated genes in pink.\u003cbr\u003e\n(d) Sankey diagram mapping top DEGs into upregulated and downregulated categories.\u003cbr\u003e\n(e) UMAP clustering of gene expression profiles showing clear segregation between upregulated and downregulated gene groups under S+E condition.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/e5b14874de8edd17877e43a4.jpeg"},{"id":86957764,"identity":"5b39f282-83bd-4f67-9fa4-11991d01bdc4","added_by":"auto","created_at":"2025-07-17 15:28:00","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":197485,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical summary of condition-specific transcriptional responses of Staphylococcus aureusduring polymicrobial co-culture.\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/a858a7e1bbe01fc38041a5e8.jpeg"},{"id":89117057,"identity":"833cc3a1-e455-44ad-aec1-76b11b5d17ec","added_by":"auto","created_at":"2025-08-14 23:46:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3875470,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/b883dda4-2761-4318-b1a8-4ddab8c09c8a.pdf"},{"id":86957757,"identity":"07ceb471-105a-4b6f-8381-2db420bd0c65","added_by":"auto","created_at":"2025-07-17 15:28:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":262003,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/420dce9529fc50d3d1163495.pdf"},{"id":86959295,"identity":"8461f642-77ce-4dc0-89e4-d6852dfc9c89","added_by":"auto","created_at":"2025-07-17 15:44:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":488619,"visible":true,"origin":"","legend":"","description":"","filename":"SAPvsSA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/0276508c6cb28caf72d7436f.xlsx"},{"id":86958253,"identity":"e75b9776-c376-41c6-b921-465021150d7a","added_by":"auto","created_at":"2025-07-17 15:36:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":318512,"visible":true,"origin":"","legend":"","description":"","filename":"SACvsSA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/40ead950eb5f45f00b5548fb.xlsx"},{"id":86958251,"identity":"72f4ca38-6ee6-4b25-8d65-8c0ea7856a32","added_by":"auto","created_at":"2025-07-17 15:36:00","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":494294,"visible":true,"origin":"","legend":"","description":"","filename":"SAEvsSA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6909814/v1/108a9936d24935fc36492dd7.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptional plasticity enables Staphylococcus aureus adaptation to polymicrobial interactions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobes rarely live alone. The idea that microbes exist in isolation has been overturned by the discoveries revealing that most pathogens thrive in complex, polymicrobial communities, a concept dating back to the 17th century, when Antonie van Leeuwenhoek first described diverse ‘animalcules’ coexisting in dental plaque(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePolymicrobial infections represent a major clinical challenge due to the complex interspecies interactions that influence pathogen behavior, host response, and treatment outcomes(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). When two microbes meet, they engage in molecular crosstalk through secreted factors, contact-dependent signaling, and competition for nutrients. These interactions can reprogram gene expression, modulate virulence, and reshape microbial physiology, in often unpredictable ways (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Among the diverse microbial species implicated in such infections, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a prominent opportunistic pathogen frequently encountered in biofilm-associated and chronic infections(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Its ability to modulate virulence, evade host immunity, and develop antimicrobial resistance is well-documented(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Yet, the extent to which these traits are shaped by cohabiting microbial species remains unexplored.\u003c/p\u003e\u003cp\u003ePolymicrobial environments, such as chronic wounds(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), cystic fibrosis lungs(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and implanted medical devices(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), often harbour diverse microbial communities where \u003cem\u003eS. aureus\u003c/em\u003e coexists with bacterial and fungal species (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Interactions with Gram-negative bacteria (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e)(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), fungi (\u003cem\u003eCandida albicans\u003c/em\u003e)(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and other Gram-positive bacteria such as \u003cem\u003eEnterococcus faecalis\u003c/em\u003e(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) can significantly alter \u003cem\u003eS. aureus\u003c/em\u003e physiology and pathogenic potential. Depending on the partner organism and the ecological niche, these interactions may be competitive, synergistic, or suppressive(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne mechanism by which \u003cem\u003eS. aureus\u003c/em\u003e engages in interbacterial competition is through the Type VII Secretion System (T7SS), a specialized protein export apparatus implicated in bacterial antagonism, virulence, and niche adaptation(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). While the role of T7SS in mono-species infection models is increasingly recognized(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), its regulation and functional relevance in polymicrobial contexts remain poorly defined.\u003c/p\u003e\u003cp\u003eThis study explores how \u003cem\u003eS. aureus\u003c/em\u003e adapts its transcriptome during co-culture with \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e. Using a clinical isolate confirmed to express T7SS, we cultivated defined dual-species communities and performed scanning electron microscopy (SEM) to confirm polymicrobial interactions. Whole transcriptome sequencing and differential expression analysis were then employed to characterize \u003cem\u003eS. aureus\u003c/em\u003e's gene expression shifts in each co-culture condition. By integrating gene ontology and pathway enrichment analyses, we reveal condition-specific virulence regulation, metabolic reprogramming, and the differential deployment of T7SS during polymicrobial growth in \u003cem\u003eS. aureus\u003c/em\u003e. These findings were further validated by Western blotting for EsxA protein expression, representing a systems-level investigation into the transcriptional logic of \u003cem\u003eS. aureus\u003c/em\u003e during ecologically diverse microbial interactions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eBacterial Strain, T7SS Confirmation, and Polymicrobial Co-Culture\u003c/p\u003e\u003cp\u003eA clinical isolate of \u003cem\u003eS. aureus\u003c/em\u003e, collected from the Bacteriology Laboratory at AIIMS Rishikesh, was identified using the VITEK2 automated system and MALDI-TOF mass spectrometry. The isolate was confirmed to harbor an active T7SS through PCR and RT-qPCR targeting the \u003cem\u003eesxA\u003c/em\u003e gene, using primers listed in Supplementary Table\u0026nbsp;1. This validated isolate was used in all subsequent experiments. For polymicrobial co-culture, \u003cem\u003eS. aureus\u003c/em\u003e was individually incubated with clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e in LB broth for 24 hours, and a \u003cem\u003eS. aureus\u003c/em\u003e monoculture was included as the control. The physical association between microbial partners was confirmed by scanning electron microscopy (SEM), following the protocol described by B et al.(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), which provided morphological evidence of co-culture. Ethical approval for the study (September 2023 to March 2025) was obtained from the Institutional Ethics Committee, AIIMS Rishikesh (Letter No- AIIMS/IEC/23/294).\u003c/p\u003e\u003cp\u003eRNA Sequencing\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from each polymicrobial co-culture and the \u003cem\u003eS. aureus\u003c/em\u003e monoculture using the HiMedia RNA isolation kit. RNA integrity was confirmed by 1% agarose gel electrophoresis, and concentrations were measured with a Qubit® 4.0 Fluorometer. High-quality RNA was reverse-transcribed to cDNA (G-Biosciences, USA), and the presence of the T7SS was verified by PCR amplification of the \u003cem\u003eesxA\u003c/em\u003e gene. Libraries were then prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus, quality-checked on the Agilent TapeStation, and sequenced on the Illumina NovaSeq X Plus platform to generate high-depth, paired-end reads for transcriptomic analysis.\u003c/p\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eRaw reads from each condition were quality-checked using FASTQC (v0.12.1) and preprocessed with fastp (v0.23.4). De novo transcriptome assembly was performed using Trinity (v2.15.1), and assembly quality was assessed with assembly-stats (v1.0.1). Transcript abundance was estimated with RSEM (v1.3.3), and differential gene expression (DGE) analysis was conducted using edgeR (v3.42.4). Comparisons were made between each polymicrobial condition and the \u003cem\u003eS. aureus\u003c/em\u003e monoculture: S + P (\u003cem\u003eP. aeruginosa\u003c/em\u003e), S + C (\u003cem\u003eC. albicans\u003c/em\u003e), and S + E (\u003cem\u003eE. faecalis\u003c/em\u003e). Protein-coding regions were predicted using TransDecoder (v5.5.0) and aligned against the UniProt reviewed database using BLAST (v2.14.1) with a ≥ 50% similarity cutoff. Annotated DEGs were subjected to GO and KEGG enrichment analyses to identify key biological processes and pathways. Data visualization of differentially expressed genes (DEGs) was generated using Python (v3.11.12).\u003c/p\u003e\u003cp\u003eWestern Blotting for EsxA Validation\u003c/p\u003e\u003cp\u003eWestern blotting was performed in three independent replicates on \u003cem\u003eS. aureus\u003c/em\u003e monoculture and co-cultures to validate EsxA protein expression. After 24-hour incubation, whole-cell lysates were prepared according to the protocol described by Anderson et al.(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) Equal amounts of protein (25 µg) were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. Membranes were incubated overnight at 4°C with a primary rabbit anti-EsxA antibody in a 1:1000 dilution (Santa Cruz Biotechnology). After washing, membranes were incubated with anti-rabbit HRP-conjugated secondary antibody using a 1:5000 dilution (Santa Cruz Biotechnology), and signals were visualized using ECL substrate on a chemiDoc imaging system (ChemiDoc XRS+, Bio-Rad)(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results \u0026 Discussion","content":"\u003cp\u003eThe clinical isolate of \u003cem\u003eS. aureus\u003c/em\u003e used in this study was first confirmed to harbor the T7SS through PCR amplification targeting the \u003cem\u003eesxA\u003c/em\u003e gene (Supplementary Fig.\u0026nbsp;1), a well-established core component of the T7SS machinery(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Subsequent RT-qPCR analysis verified active expression of \u003cem\u003eesxA\u003c/em\u003e (Supplementary Fig.\u0026nbsp;2), indicating transcriptional competence of the secretion system under monoculture conditions. This characterized strain was then employed in polymicrobial co-culture experiments with \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e. In parallel, scanning electron microscopy (SEM) confirmed the physical coexistence of \u003cem\u003eS. aureus\u003c/em\u003e with each co-culture partner, validating the establishment of polymicrobial associations before transcriptomic profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFollowing co-culture, total RNA was isolated from each condition and subjected to rigorous quality control. RNA integrity was assessed using a 1% agarose gel, and concentrations were measured with a Qubit® 4.0 Fluorometer. The Agilent TapeStation 4150 system with High Sensitivity D1000 ScreenTape®, further confirmed high-quality, non-degraded RNA suitable for transcriptomic analysis. cDNA was synthesized from the purified RNA, and subsequent PCR amplification again confirmed the presence of \u003cem\u003eesxA\u003c/em\u003e transcripts, reinforcing that the T7SS remained transcriptionally detectable across all experimental contexts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e induces global transcriptional suppression in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e during co-culture\u003c/p\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e co-culture induced a global transcriptional shutdown in \u003cem\u003eS. aureus\u003c/em\u003e, with 230 genes significantly downregulated and only one, \u003cem\u003ehly\u003c/em\u003e (alpha-hemolysin), upregulated (log₂FC = + 0.48) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This repression was supported by Venn and barplot analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c). STRING network visualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) revealed strong interconnectivity among the downregulated proteins, implicating core processes such as energy metabolism, translation, and regulatory pathways. In contrast, \u003cem\u003ehly\u003c/em\u003e appeared as a solitary, unconnected node (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), suggesting an independent, stress-induced regulatory response.\u003c/p\u003e\u003cp\u003eFunctional enrichment analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-g) identified suppression across critical metabolic and cellular pathways, including amino acid biosynthesis, carbohydrate metabolism, ribosome assembly, and membrane-associated functions. Genes encoding ribosomal proteins (\u003cem\u003erplX, rpsD, rpsL\u003c/em\u003e)(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) and translation factors (\u003cem\u003etuf, efp\u003c/em\u003e)(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) were downregulated, indicating a protein synthesis shutdown likely driven by nutrient limitation or quorum-sensing interference from \u003cem\u003eP. aeruginosa\u003c/em\u003e. Repression of TCA cycle enzymes (\u003cem\u003emqo2\u003c/em\u003e)(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and glycolytic genes (\u003cem\u003eackA, ptsG, fba\u003c/em\u003e)(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) reflects metabolic quiescence, potentially induced by \u003cem\u003eP. aeruginosa\u003c/em\u003e-derived respiratory inhibitors such as HQNO(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Amino acid biosynthetic genes (\u003cem\u003eilvA, ilvC, leuB, tyrA\u003c/em\u003e)(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) were suppressed, consistent with reduced anabolic demand and competition-driven nutrient scarcity. Downregulation of stress response and chaperone genes (\u003cem\u003ednaK, groEL\u003c/em\u003e)(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) suggests impaired ability to respond to oxidative or envelope stress, possibly exacerbated by \u003cem\u003eP. aeruginosa\u003c/em\u003e redox-active metabolites. Suppressed expression of staphyloxanthin biosynthesis genes (\u003cem\u003ecrtQ, crtO, crtM\u003c/em\u003e)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) likely compromises antioxidant defense, rendering \u003cem\u003eS. aureus\u003c/em\u003e more susceptible to oxidative stress induced by \u003cem\u003eP. aeruginosa\u003c/em\u003e-derived ROS. Key virulence factors (\u003cem\u003espa, lukEv, sak, isdB, isdI, clfA, atl, sdrC, sdrD\u003c/em\u003e)(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) were silenced, suggesting diminished pathogenicity under competitive pressure. Transcriptional regulators (\u003cem\u003esarZ, sarX, sigS, mgrA, spx, agrB\u003c/em\u003e)(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) were repressed, indicating upstream disruption of quorum-sensing and global regulatory networks. Downregulation of cell wall genes (\u003cem\u003emurG, femX, lytN, lytM, sle1\u003c/em\u003e)(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) may weaken envelope integrity, a known consequence of interspecies interactions. Genes involved in nucleotide and cofactor metabolism (\u003cem\u003epurD, purN, pyrB, queF, bioW, bioD, pdxS, moaE\u003c/em\u003e)(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) were also suppressed, aligning with metabolic restraint. Importantly, \u003cem\u003eS. aureus\u003c/em\u003e downregulated metal ion transporters (\u003cem\u003ecopA, nikD, norB\u003c/em\u003e)(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), possibly in response to \u003cem\u003eP. aeruginosa\u003c/em\u003e's siderophore (e.g., pyoverdine) production and iron sequestration, which disrupts \u003cem\u003eS. aureus\u003c/em\u003e iron acquisition systems. Finally, the absence of T7SS gene expression indicates complete suppression of interbacterial antagonism(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), suggesting \u003cem\u003eP. aeruginosa\u003c/em\u003e outcompetes and functionally disarms \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eDespite this global repression, hly was uniquely upregulated in the \u003cem\u003eP. aeruginosa\u003c/em\u003e co-culture. This gene encodes a stress-induced, independently regulated cytotoxin(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), enabling \u003cem\u003eS. aureus\u003c/em\u003e to retain minimal cytolytic potential even under widespread transcriptional silencing. Rather than indicating a full virulence program, this selective activation likely represents a compensatory survival response to polymicrobial stress.\u003c/p\u003e\u003cp\u003eGene-level visualizations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea–e) demonstrated consistent repression across major functional modules, highlighting \u003cem\u003ehly\u003c/em\u003e as a unique outlier. Heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea–b) showed widespread downregulation across functional categories, while the circular barplot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) emphasized the predominance of suppressed transcripts. UMAP analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) revealed clustering of downregulated genes into distinct regulatory groups, with \u003cem\u003ehly\u003c/em\u003e appearing as an isolated node. The Sankey diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), depicting the top differentially expressed genes involved in metabolism, stress response, and cell wall biogenesis, showed that all were directed into the downregulated category, further highlighting the extent of \u003cem\u003eP. aeruginosa\u003c/em\u003e-mediated suppression.\u003c/p\u003e\u003cp\u003eThese findings indicate that \u003cem\u003eP. aeruginosa\u003c/em\u003e enforces a coordinated and multifaceted transcriptional silencing of \u003cem\u003eS. aureus\u003c/em\u003e, disrupting key metabolic, virulence, and stress response pathways. This repression reflects a competitive dominance strategy in polymicrobial environments, functionally disarming \u003cem\u003eS. aureus\u003c/em\u003e and diminishing its ecological competitiveness. Elucidating the molecular basis of this suppression may inform the development of therapeutic approaches that harness or mimic interspecies antagonism. For example, \u003cem\u003eP. aeruginosa\u003c/em\u003e-derived molecules such as 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), pyocyanin, or siderophore analogs could be repurposed or engineered to target \u003cem\u003eS. aureus\u003c/em\u003e persistence mechanisms without promoting broad-spectrum resistance. Alternatively, disrupting \u003cem\u003eS. aureus\u003c/em\u003e quorum sensing or stress resilience pathways revealed to be suppressed in co-culture may offer synergistic strategies when combined with biofilm-targeted therapies. Such approaches represent a paradigm shift, from pathogen eradication to ecological modulation, offering precision tools for managing chronic, device-associated, or polymicrobial infections.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e induces transcriptional reprogramming and virulence activation in \u003cem\u003eS. aureus\u003c/em\u003e during co-culture\u003c/p\u003e\u003cp\u003eIn contrast to the repressive effects of \u003cem\u003eP. aeruginosa\u003c/em\u003e, co-culturing \u003cem\u003eS. aureus\u003c/em\u003e with \u003cem\u003eC. albicans\u003c/em\u003e elicited a bidirectional transcriptional response, with 64 genes significantly upregulated and 54 downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), as confirmed by barplot and Venn analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-c). STRING-based interaction networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e) revealed that both gene sets formed interconnected modules, suggesting coordinated regulatory shifts.\u003c/p\u003e\u003cp\u003eFunctional enrichment analyses highlighted activation of amino acid biosynthesis, cofactor metabolism, carbohydrate processing, and siderophore pathways (KEGG, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). GO terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg) emphasized ATP binding, metal ion binding, ribosomal structure, and membrane-associated components, indicating broad physiological adaptation.\u003c/p\u003e\u003cp\u003eUpregulated genes reflected enhanced virulence, metabolic reprogramming, and stress tolerance. Virulence factors (\u003cem\u003eclfA\u003c/em\u003e, \u003cem\u003espa\u003c/em\u003e, \u003cem\u003elukEv\u003c/em\u003e, \u003cem\u003eatl\u003c/em\u003e, \u003cem\u003ecidA\u003c/em\u003e, \u003cem\u003ehly\u003c/em\u003e)(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) suggested increased adhesion, immune evasion, and cytotoxicity, consistent with prior reports of \u003cem\u003eS. aureus–C. albicans\u003c/em\u003e synergy during mucosal invasion. Oxidative stress defense genes (\u003cem\u003esodM\u003c/em\u003e, \u003cem\u003esodA\u003c/em\u003e, \u003cem\u003eahpC\u003c/em\u003e, \u003cem\u003erex\u003c/em\u003e, \u003cem\u003eperR\u003c/em\u003e)(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) were elevated, likely counteracting ROS generated by fungal metabolism or host immune activity. The induction of fermentative metabolism genes (\u003cem\u003emqo2\u003c/em\u003e, \u003cem\u003eldh1\u003c/em\u003e, \u003cem\u003eptsG\u003c/em\u003e, \u003cem\u003eglcT\u003c/em\u003e, \u003cem\u003epflA\u003c/em\u003e, \u003cem\u003eadh\u003c/em\u003e)(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) indicates a shift toward anaerobic energy production, reflecting hypoxic and nutrient-depleted biofilm conditions often shaped by \u003cem\u003eC. albicans\u003c/em\u003e. Upregulation of membrane remodeling genes (\u003cem\u003elytN\u003c/em\u003e)(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) may facilitate adaptation to altered physical environments, including hyphal invasion. Anabolic genes (\u003cem\u003eilvC\u003c/em\u003e, \u003cem\u003easpS\u003c/em\u003e, \u003cem\u003eargR\u003c/em\u003e, \u003cem\u003eargG\u003c/em\u003e, \u003cem\u003emetK\u003c/em\u003e, \u003cem\u003epyrR\u003c/em\u003e)(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and translational components (\u003cem\u003erpsL\u003c/em\u003e, \u003cem\u003erplX\u003c/em\u003e, \u003cem\u003erplK\u003c/em\u003e, \u003cem\u003espsA\u003c/em\u003e, \u003cem\u003eengB\u003c/em\u003e)(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) were also upregulated, supporting biosynthesis and active growth. Regulatory genes (\u003cem\u003emgrA\u003c/em\u003e, \u003cem\u003esaeS\u003c/em\u003e, \u003cem\u003evraR\u003c/em\u003e, \u003cem\u003espx\u003c/em\u003e)(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) suggest global transcriptional activation. Notably, metal ion transporters (\u003cem\u003ecopA\u003c/em\u003e, \u003cem\u003ecopB\u003c/em\u003e)(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) were induced, likely in response to iron limitation imposed by fungal siderophores.\u003c/p\u003e\u003cp\u003eIn contrast, downregulated genes indicated suppression of core metabolic, stress response, and proliferative functions. Repression of central metabolic enzymes (\u003cem\u003eackA\u003c/em\u003e, \u003cem\u003eglcU\u003c/em\u003e)(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) suggested reduced energy flux, likely due to glucose depletion or oxygen limitation in shared niches. Downregulation of amino acid biosynthesis genes (\u003cem\u003etyrS\u003c/em\u003e, \u003cem\u003eglnA\u003c/em\u003e, \u003cem\u003emoaE\u003c/em\u003e)(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) reflected nutrient competition, while stress response genes (\u003cem\u003ehslO\u003c/em\u003e, \u003cem\u003ectsR\u003c/em\u003e)(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) were also suppressed, potentially compromising adaptability under oxidative stress. Virulence and regulatory genes (\u003cem\u003eclfA\u003c/em\u003e, \u003cem\u003eisaB\u003c/em\u003e, \u003cem\u003esarX\u003c/em\u003e)(\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e–\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) were downregulated, possibly reflecting immune evasion or interkingdom tolerance. Suppression of DNA replication and repair (\u003cem\u003erecU\u003c/em\u003e, \u003cem\u003erecX\u003c/em\u003e, \u003cem\u003ednaA\u003c/em\u003e)(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) and cell division genes (\u003cem\u003eparC\u003c/em\u003e)(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) suggests a shift toward a low-proliferative or quiescent state. Notably, repression of norB and norG, components of the Nor efflux pump regulatory network, suggests reduced efflux capacity, potentially sensitizing \u003cem\u003eS. aureus\u003c/em\u003e to antimicrobial stress or toxic metabolites during co-culture.\u003c/p\u003e\u003cp\u003eGene-level visualizations supported the observed transcriptional reprogramming. Heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b) illustrated a clear contrast between upregulated and downregulated genes across key functional categories, indicating strong condition-specific expression shifts. The circular barplot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) emphasized the balanced distribution of regulatory changes, while UMAP clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) distinctly separated gene expression patterns into two polarized groups. The Sankey diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) mapped the top differentially expressed genes into major biological categories, highlighting the coordinated nature of the bidirectional response in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTogether, these results demonstrate that \u003cem\u003eC. albicans\u003c/em\u003e induces a finely orchestrated transcriptional reprogramming in \u003cem\u003eS. aureus\u003c/em\u003e, activating virulence, stress tolerance, and metabolic flexibility, while repressing growth-associated, biosynthetic, and oxidative defense pathways. This dual modulation likely reflects a survival strategy tailored to the polymicrobial niche, enabling \u003cem\u003eS. aureus\u003c/em\u003e to persist under cooperative or competitive pressures imposed by \u003cem\u003eC. albicans\u003c/em\u003e. The observed plasticity aligns with prior evidence of fungal–bacterial synergy in mucosal colonization, biofilms, and disseminated infections. Importantly, the selective silencing of replication and redox defenses, alongside virulence activation, suggests a shift toward a quiescent yet pathogenic phenotype. These findings offer mechanistic insight into interkingdom interactions and identify potential vulnerabilities for therapeutic targeting in chronic or device-associated infections.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e activates type VII secretion and virulence expression in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e during co-culture\u003c/p\u003e\u003cp\u003eGlobal transcriptomic profiling identified 1,576 differentially expressed genes (DEGs) in \u003cem\u003eS. aureus\u003c/em\u003e during co-culture with \u003cem\u003eE. faecalis\u003c/em\u003e, comprising 890 upregulated and 686 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) highlights the distribution of significantly altered genes across a broad range of fold changes and p-values, while the bar and bubble plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c) confirm the relative proportions of gene regulation categories. STRING-based protein–protein interaction networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-e) revealed tightly interconnected clusters among up- and downregulated DEGs, suggesting coordinated regulatory control during polymicrobial stress.\u003c/p\u003e\u003cp\u003eFunctional enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef-g) showed that upregulated genes were enriched in amino acid biosynthesis, cofactor metabolism, and carbohydrate degradation pathways. GO molecular functions included ATP binding, metal ion binding, and stress response activities. At the same time, enriched cellular components such as membrane-associated and extracellular regions suggest enhanced environmental sensing, structural adaptation, and nutrient acquisition.\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e underwent extensive transcriptional reprogramming in response to \u003cem\u003eE. faecalis\u003c/em\u003e, characterized by the upregulation of virulence, stress adaptation, metabolic plasticity, and interbacterial competition mechanisms. Prominent virulence factors such as spa, clfA, fnbB, fib, bbp, sdrC, and sraP (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e)were significantly upregulated, promoting adhesion, immune evasion, and biofilm establishment features crucial for persistence in polymicrobial infections. Increasing lukDv, lukEv, hlgB, and hly(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) suggested a cytolytic phenotype, enhancing host tissue damage and competitive fitness. Notably, the activation of \u003cem\u003eessA\u003c/em\u003e and \u003cem\u003esplF\u003c/em\u003e indicated the T7SS engagement(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e), a contact-dependent pathway in \u003cem\u003eS. aureus\u003c/em\u003e that translocates toxic effectors into Gram-positive competitors to establish niche dominance. T7SS is selectively activated against structurally compatible bacteria such as \u003cem\u003eE. faecalis\u003c/em\u003e, whose thick peptidoglycan layer enables effector delivery and physical sensing(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). In contrast, \u003cem\u003eP. aeruginosa\u003c/em\u003e, a Gram-negative bacterium, possesses an outer membrane that impedes T7SS activity and secretes diffusible inhibitors (e.g., HQNO, pyocyanin)(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) that suppress \u003cem\u003eS. aureus\u003c/em\u003e transcriptionally, including T7SS components. Likewise, \u003cem\u003eC. albicans\u003c/em\u003e lacks the structural features required for T7SS targeting and does not pose direct antibacterial aggression. Instead, \u003cem\u003eS. aureus\u003c/em\u003e exhibits a synergistic transcriptional response to \u003cem\u003eC. albicans\u003c/em\u003e, upregulating virulence and stress pathways without T7SS induction, consistent with their known mutual persistence in biofilms and mucosal niches(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). This context-dependent specificity emphasizes a finely tuned defense system in \u003cem\u003eS. aureus\u003c/em\u003e, selectively deployed against compatible bacterial threats.\u003c/p\u003e\u003cp\u003eStress adaptation pathways were also markedly elevated. Oxidative and DNA damage responses were reflected by upregulation of ahpC, uvrA, recO, lexA, and trxA,(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) likely countering peroxide and reactive oxygen species produced by \u003cem\u003eE. faecalis\u003c/em\u003e. The induction of clpP, cap5A, and cpfC, supported structural stabilization,(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e), promoting envelope integrity and redox control. While adhesion- and remodeling-associated genes (\u003cem\u003eatl\u003c/em\u003e, \u003cem\u003esle1\u003c/em\u003e, \u003cem\u003espa\u003c/em\u003e, \u003cem\u003esdrC\u003c/em\u003e)(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) were upregulated, the concurrent upregulation of \u003cem\u003eicaR\u003c/em\u003e(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), a repressor of the \u003cem\u003eicaADBC\u003c/em\u003e operon, and downregulation of \u003cem\u003eicaD\u003c/em\u003e(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) suggest attenuation of PIA-mediated biofilm synthesis. This indicates a shift toward a proteinaceous, rather than polysaccharide-driven, biofilm phenotype in response to \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eMetabolic reprogramming was evident through increased expression of adh, ackA, glcU, pyk, pflA, and qoxC,(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e) consistent with a metabolic switch between fermentation and respiration. Upregulated biosynthetic genes, including tyrA, proC, leuS, and thrS,(\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) pointed to enhanced anabolic activity to support cellular demands under competitive conditions. Induction of cntL, nikD, and kdpA(\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) further indicated active acquisition of trace elements, likely in response to metal competition within the shared niche. Mobile element genes such as tnpR and tnpC,(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e) alongside transcriptional regulators rpoB, rpoE, rpsJ, spx, and spxH,(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e) were also upregulated, suggesting increased genomic plasticity and stress-adaptive transcriptional remodeling.\u003c/p\u003e\u003cp\u003eConversely, substantial transcriptional repression was observed across key biosynthetic, metabolic, and virulence functions. Downregulated metabolic genes included core glycolytic and TCA cycle components gapA2, glkA, gpsA, pckA, ldh1, odhA, fumC, pta, and mqo2,(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e) indicating reduced energy flux and metabolic conservation. Suppressed amino acid and nucleotide biosynthesis gene purD,(\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e) reflected a shift away from growth and proliferation. Decreased expression of atpF and ctaB(\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) further signaled respiratory downregulation. Ribosomal proteins (rplU, rplV, rpsL)(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), elongation factor efp,(\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e) and tRNA synthetases (pheS, ileS, lysS)(\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e) were repressed, indicating global translational silencing. DNA replication and repair elements dnaA and recR(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) were downregulated, indicating reduced cell cycle progression.\u003c/p\u003e\u003cp\u003eKey virulence genes, including fnbA, sdrD, ssl10, sak, hlgA, and secA2(\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e–\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e) were suppressed, as were capsule and cell wall components mprF, ebhA, lytM, and cls2,(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e) indicating a downshift in structural virulence and immune evasion. In addition, staphyloxanthin biosynthesis genes \u003cem\u003ecrtM\u003c/em\u003e and \u003cem\u003ecrtP\u003c/em\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) were repressed, suggesting diminished antioxidant capacity and stress resistance. Regulatory downregulation of ctsR, rex, and srrB,(\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e) implied diminished environmental sensing. Finally, division proteins ftsA, ezrA,(\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e) were suppressed, supporting a metabolically restrained, quiescent phenotype under antagonistic stress.\u003c/p\u003e\u003cp\u003eGene-level visualizations validated these findings. Heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-b) revealed distinct clusters of upregulated and downregulated genes, reflecting a coordinated transcriptional shift in response to polymicrobial co-culture. UMAP clustering and radial barplots (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee) further illustrated a clear polarization of gene expression profiles, with minimal overlap between expression states. The Sankey diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed) mapped major transcriptional changes across key functional categories, emphasizing the organized reprogramming of \u003cem\u003eS. aureus\u003c/em\u003e under competitive stress.\u003c/p\u003e\u003cp\u003eThese data reveal that \u003cem\u003eS. aureus\u003c/em\u003e mounts a dualistic response to \u003cem\u003eE. faecalis\u003c/em\u003e co-culture, activating T7SS, virulence pathways, and biosynthesis, while concurrently silencing growth, translation, and energetically costly processes. This ecological trade-off prioritizes stress endurance, competitive fitness, and sessile adaptation over proliferation. Given the known secretion of bacteriocins, hydrogen peroxide, and biofilm-promoting enzymes by \u003cem\u003eE. faecalis\u003c/em\u003e, the transcriptional reprogramming in \u003cem\u003eS. aureus\u003c/em\u003e likely reflects an integrated response to direct and indirect microbial antagonism. These findings emphasize the importance of interbacterial signaling in modulating pathogen behavior and suggest that disrupting communication pathways such as T7SS or quorum sensing may provide therapeutic avenues for managing polymicrobial infections.\u003c/p\u003e\u003cp\u003eWestern blotting was performed to validate transcriptomic findings on T7SS regulation and detect EsxA protein across all co-culture conditions. A strong EsxA band (~ 22–35 kDa) was observed in \u003cem\u003eS. aureus\u003c/em\u003e monoculture (S) and co-culture with \u003cem\u003eE. faecalis\u003c/em\u003e (S + E), supporting active T7SS expression. In contrast, the band was weak in the \u003cem\u003eP. aeruginosa\u003c/em\u003e co-culture (S + P) and absent or faint in the \u003cem\u003eC. albicans\u003c/em\u003e co-culture (S + C), consistent with RNA-seq data showing \u003cem\u003eesxA\u003c/em\u003e repression under these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Although EsxA is predicted to be ~ 11 kDa, the observed higher molecular weight may result from dimerization or polymerization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e), as reported by Sundaramoorthy et al (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). These results confirm that EsxA expression is context-dependent, active against Gram-positive competitors like \u003cem\u003eE. faecalis\u003c/em\u003e and silenced during interaction with \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe primary limitation of this study is that it provides a single-time-point, snapshot view of the transcriptomic landscape, without capturing dynamic transitions or temporal changes in gene expression. Nevertheless, key findings were validated through complementary assays, including SEM, PCR, and Western blotting, supporting the reliability of the observed condition-specific responses.\u003c/p\u003e\u003cp\u003eA graphical summary of the distinct interaction modes, antagonism with \u003cem\u003eP. aeruginosa\u003c/em\u003e, synergy with \u003cem\u003eC. albicans\u003c/em\u003e, and competitive response to \u003cem\u003eE. faecalis\u003c/em\u003e, is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, highlighting condition-specific modulation of virulence, metabolism, adhesion, biofilm, and T7SS expression in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis schematic illustrates the distinct gene expression profiles of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e when co-cultured with \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e. In the left panel, representing co-culture with \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e exhibits a synergistic interaction marked by upregulation of virulence and cell adhesion genes, while genes involved in T7SS secretion and metabolism are downregulated. In the centre panel, during co-culture with \u003cem\u003eP. aeruginosa\u003c/em\u003e, an antagonistic response is observed, with global transcriptional repression affecting metabolism, biofilm formation, adhesion, and T7SS genes, and only minimal virulence activation, indicating suppression of \u003cem\u003eS. aureus\u003c/em\u003e pathogenic potential. In the right panel, co-culture with \u003cem\u003eE. faecalis\u003c/em\u003e leads to a competitive interaction characterized by upregulation of T7SS, virulence factors, metabolic pathways, and adhesion genes, while biofilm-related genes are downregulated. Upregulated and downregulated functions are indicated by green (\u003cspan style=\"font-size: 16px; line-height: 200%; font-family: Arial, sans-serif; color: rgb(0, 176, 80); font-style: normal;\"\u003e▲\u003c/span\u003e) and red (\u003cspan style=\"font-size: 16px; line-height: 200%; font-family: Arial, sans-serif; color: rgb(192, 0, 0); font-style: normal;\"\u003e▼\u003c/span\u003e) arrows, respectively. Icons represent core functional themes such as T7SS, metabolism, biofilm, virulence, and adhesins. The central test tubes depict \u003cem\u003eS. aureus\u003c/em\u003e co-cultured with each partner organism.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eS. aureus\u003c/em\u003e exhibits distinct, context-dependent transcriptional responses when co-cultured with \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e. While \u003cem\u003eP. aeruginosa\u003c/em\u003e imposes a dominant suppressive effect, silencing metabolism, stress response, and virulence networks, \u003cem\u003eC. albicans\u003c/em\u003e elicits a synergistic reprogramming marked by co-activation of virulence, metabolic flexibility, and redox tolerance. In contrast, \u003cem\u003eE. faecalis\u003c/em\u003e triggers a defensive, antagonistic response characterized by activation of the T7SS, virulence regulators, and anabolic machinery, reflecting a competitive interbacterial strategy. These divergent expression profiles reflect tailored ecological adaptations: suppression under Gram-negative stress, cooperation with fungal partners, and aggression against Gram-positive competitors. These findings provide mechanistic insight into how \u003cem\u003eS. aureus\u003c/em\u003e dynamically modulates its transcriptome to balance competition, coexistence, and persistence in polymicrobial environments, laying the foundation for precision microbiology strategies that target interspecies signaling and microbial crosstalk in complex infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of the manuscript.\u003c/p\u003e\u003cp\u003eCompeting Interests\u003c/p\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNirmala B designed and performed the experiments, analyzed the data, and wrote the manuscript. Yogendra P Mathuria and Balram Ji Omar supervised the study and validated the findings. All authors reviewed and approved the final manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the Department of Biotechnology, Ministry of Science and Technology, Government of India, for providing a DBT-JRF scholarship to B Nirmala. We also acknowledge the All India Institute of Medical Sciences (AIIMS) Rishikesh for providing infrastructural support for this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw and processed RNA-seq data generated in this study will be deposited in the NCBI Sequence Read Archive (SRA) under a BioProject accession number, which will be provided upon manuscript acceptance. An Excel file containing all differentially expressed genes (DEG) data, metadata, and gene expression matrices is provided in the Supplementary Information for transparency. Python scripts used for analysis will be made available on GitHub.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang M, Whiteley M, Lewin GR. Polymicrobial Interactions of Oral Microbiota: a Historical Review and Current Perspective. Vol. 13, mBio. American Society for Microbiology; 2022. \u003c/li\u003e\n\u003cli\u003eMariani F, Galvan EM. Staphylococcus aureus in Polymicrobial Skinand Soft Tissue Infections: Impact of Inter-Species Interactionsin Disease Outcome. Vol. 12, Antibiotics. Multidisciplinary Digital Publishing Institute (MDPI); 2023. \u003c/li\u003e\n\u003cli\u003eYu G, Ge X, Li W, Ji L, Yang S. Interspecific cross-talk: The catalyst driving microbial biosynthesis of secondary metabolites. Vol. 76, Biotechnology Advances. Elsevier Inc.; 2024. \u003c/li\u003e\n\u003cli\u003eRupp M, Kern S, Weber T, Menges TD, Schnettler R, Hei\u0026szlig; C, et al. 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Microorganisms. 2022 Mar 1;10(3). \u003c/li\u003e\n\u003cli\u003eDmitriev A, Chen X, Paluscio E, Stephens AC, Banerjee SK, Vitko NP, et al. The Intersection of the Staphylococcus aureus Rex and SrrAB Regulons: an Example of Metabolic Evolution That Maximizes Resistance to Immune Radicals. 2021; Available from: https://doi.org/10.1128/mBio\u003c/li\u003e\n\u003cli\u003eMyrbr\u0026aring;ten IS, Stams\u0026aring;s GA, Chan H, Angeles DM, Knutsen TM, Salehian Z, et al. SmdA is a Novel Cell Morphology Determinant in Staphylococcus aureus. mBio. 2022 Apr 1;13(2). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polymicrobial interactions, Transcriptomics, Type VII Secretion System, Staphylococcus aureus, Virulence modulation","lastPublishedDoi":"10.21203/rs.3.rs-6909814/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6909814/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the microbial world, survival is not solitary. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e thrives or falters depending on its neighbors. This opportunistic pathogen frequently inhabits polymicrobial environments such as chronic wounds, implanted devices, and mucosal surfaces, where interspecies interactions shape its behavior and complicate treatment outcomes. Focusing on the Type VII Secretion System (T7SS), this study explores how \u003cem\u003eS. aureus\u003c/em\u003e transcriptionally and functionally adapts during co-culture with three clinically relevant organisms: \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e. RNA sequencing revealed distinct ecological responses: \u003cem\u003eP. aeruginosa\u003c/em\u003e induced a strongly antagonistic interaction, causing global transcriptional repression, including silencing of virulence genes and T7SS; \u003cem\u003eC. albicans\u003c/em\u003e promoted a synergistic response, activating virulence, stress, and metabolic genes despite T7SS repression; and \u003cem\u003eE. faecalis\u003c/em\u003e elicited a competitive interaction marked by robust activation of T7SS, cytotoxic effectors, and biosynthetic programs. Western blotting of EsxA validated condition-specific T7SS expression. These findings reveal how \u003cem\u003eS. aureus\u003c/em\u003e transcriptionally adapts to microbial neighbors, positioning interspecies signaling as a key driver of precision microbiology and potential target for managing polymicrobial infections.\u003c/p\u003e","manuscriptTitle":"Transcriptional plasticity enables Staphylococcus aureus adaptation to polymicrobial interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 15:27:55","doi":"10.21203/rs.3.rs-6909814/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9240885-1b69-4068-9c17-c982522b003f","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-14T23:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 15:27:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6909814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6909814","identity":"rs-6909814","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}