Staphylococcus aureus is a globally prevalent gram-positive pathogen that can cause numerous types of infection. Due to host nutritional immunity and iron (Fe) sequestration, S. aureus experiences Fe limitation during infection. To overcome this, S. aureus expresses an arsenal of Fe acquisition systems whose expression is coordinated through the Fe-binding transcriptional regulator, Fur. Here, from a screen to identify S. aureus mutants defective for Fe-restricted growth, we identified several with mutations in perR, encoding a transcriptional regulator involved in resistance to oxidative stress. RNA-seq identified that the most downregulated genes in a perR mutant growing in Fe-restriction are those from the sbn operon that encodes staphyloferrin B biosynthesis. In agreement, perR mutants grew poorly in Fe-deficient media due to deficient staphyloferrin B production. In a subcutaneous model of S. aureus skin infection, S. aureus perR caused significantly smaller lesions, consistent with our finding that this mutant had decreased alpha-hemolysin expression during Fe-restricted growth. These findings are consistent with the hypothesis that PerR acts to fine-tune access to Fe ostensibly to avoid Fe-dependent toxicity. The importance of the PerR function to S. aureus was further highlighted by examination of over 8,000 human bloodstream isolates of S. aureus, showing that the PerR sequence was highly conserved. Together, these findings demonstrate the importance of PerR to S. aureus in providing an additional level of regulation of Fe homeostasis beyond Fur-dependent Fe sensing.IMPORTANCEStaphylococcus aureus is a notoriously antibiotic-resistant human pathogen and has the potential to cause a myriad of potentially life-threatening infections. Understanding the intricacies of S. aureus pathogenesis in the host will underpin the development of novel therapeutic approaches that target bacterial virulence mechanisms rather than essential cellular processes. The significance of our research lies in identifying how S. aureus regulates Fe acquisition in response to environmental signals, providing further insight into nutrient sensing and acquisition at the host-pathogen interface to facilitate the development of therapeutics that may target this process.

Antibiotics were loaded into a poly(ester amide) coating with a T g just above 37 °C. Induction heating of a coated titanium implant triggered localized antibiotic release, leading to synergistic killing of S. aureus biofilms by heat and antibiotics.

Staphylococcus aureus is a leading cause of skin and soft tissue infections, endocarditis, and bloodstream infections worldwide. The emergence of methicillin-resistant S. aureus (MRSA) and growing resistance to last-resort antibiotics like vancomycin have created an urgent need for new antimicrobials with distinct mechanisms of action. In this study, we characterize DB10, a planar, fluorene-based compound identified in a high-throughput screen for MRSA growth inhibitors. Upon UVA exposure, DB10 undergoes photoconversion from a red-colored form (DB10-R) to a yellow-colored form (DB10-Y). In comparison with DB10-R, DB10-Y exhibits reduced hydrophobicity, lower cytotoxicity, and modestly improved minimum inhibitory concentrations toward several Gram-positive bacteria. DB10-Y intercalates into DNA and induces double-strand breaks within bacterial cells, and resistance emerged only at low levels after prolonged serial passaging. To optimize this scaffold, we screened a panel of fluorene analogs and identified the photoconverting analog DB33, which in its yellow form (DB33-Y) is nontoxic and retained DNA intercalating activity. DB33-Y was effective against intracellular S. aureus in macrophages and endothelial cells and significantly reduced bacterial burden and lesion size in a murine skin infection model. DB10-Y and DB33-Y both also suppressed expression of α-hemolysin at sub-minimum inhibitory concentrations, indicating an additional antivirulence effect. Together, these findings highlight the therapeutic potential of fluorene-based DNA intercalators as a new class of antimicrobial and antivirulence agents against MRSA.

As one of the leading global causes of death associated with antimicrobial resistance, Staphylococcus aureus frequently colonizes the human nasal cavity and adheres to keratinized skin, establishing reservoirs that drive subsequent infections and emphasize the need for new decolonization strategies. Using a high-throughput, whole-cell screening platform, here we identify geranylgeranoic acid (GGA), a naturally occurring polyunsaturated, branched-chain fatty acid, as having dual activity against methicillin-resistant S. aureus (MRSA). At elevated concentrations, GGA exhibits microbicidal effects, whereas at sub‑microbicidal doses, it effectively inhibits MRSA adhesion to keratin, fibronectin, fibrinogen, and immunoglobulins. GGA possesses anti-adhesive activity against a panel of multidrug-resistant S. aureus clinical isolates and demonstrates efficacy against MRSA skin and soft tissue infections in mice, offering a promising new avenue for combating this challenging pathogen. The anti-adhesive activity of GGA is attributed to the transcriptional modulation of S. aureus cell wall-anchored (CWA) proteins. For the fibronectin-binding proteins, this effect is mediated by inhibition of the SaeRS two-component system, which, in turn, down-regulates a variety of S. aureus virulence determinants. Our data also suggest that GGA targets additional transcriptional regulators beyond SaeRS. These insights expand the translational relevance of our findings and underscore the potential of GGA as an anti‑MRSA therapeutic.

Staphylococcus aureus is a major cause of infections, ranging from mild skin conditions to severe life-threatening systemic diseases. Despite decades of effort, vaccine development has been unsuccessful, highlighting the need for alternative approaches. One promising candidate is MEDI4893 (Suvratoxumab), a monoclonal antibody that neutralizes α-toxin (AT), a key virulence factor with diverse functions including cellular lysis and induction of platelet aggregation during sepsis. MEDI4893 has demonstrated efficacy in various preclinical models of infection and showed encouraging results in a phase IIb clinical trial for ventilator-associated pneumonia. However, as an exogenous antibody, MEDI4893 is limited by its short half-life, high production costs, and restricted availability. To overcome these challenges, we investigated vectored immunoprophylaxis as a novel strategy for long-term protection. This approach uses an adeno-associated viral vector (AAV) to deliver the MEDI4893 gene, enabling sustained in vivo antibody expression. Following AAV-MEDI4893 administration, mice developed high and stable antibody levels in plasma and detectable titers at mucosal surfaces. Using intravital microscopy, we observed that vector-mediated MEDI4893 effectively prevented AT-induced platelet aggregation and microvascular thrombosis, thereby protecting animals from the lethal effects of intravenous toxin challenge. Infection studies confirmed that this protective effect extended to clinically relevant models. In murine pneumonia models, treated mice showed improved survival and reduced sickness behavior despite similar bacterial burdens, while in skin infection models, they were protected from dermal necrosis and exhibited lower bacterial loads. These findings highlight that AT is a major driver of pathology across multiple tissues and that its neutralization can mitigate disease severity. Together, our results demonstrate that AAV-mediated delivery of MEDI4893 provides durable, protective antibody levels and effectively neutralizes AT in vivo. This strategy represents a cost-effective, long-lasting alternative to traditional monoclonal antibody therapy and offers a promising prophylactic approach to mitigate S. aureus infections in the absence of a vaccine.

[This corrects the article DOI: 10.1371/journal.ppat.1013291.].

Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability in vivo limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (Sequence enTAngLEd Multi lAyered geneTic buffEring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudoessentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganuclease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement simultaneously inactivated both biocontainment layers, leading to cell death by the ColE9 nuclease and the elimination of escape mutants. By lengthening the entangled region, refining ColE9 expression, and optimizing the TSM sequence against IS911 insertion, we achieved escape rates below 10-10 as compared to rates of 10-5 with the nonentangled TSM. The STALEMATE system contained plasmids in E. coli Nissle 1917 for over a week in the mouse gastrointestinal tract with nearly undetectable escape rates upon excretion. STALEMATE offers a modular and simple biocontainment approach to buffer against mutational inactivation in the mammalian gut without a requirement for engineered bacteria or exogenous signaling ligands.

Staphylococcus aureus teichoic acids are anionic glycopolymers covalently attached to peptidoglycan (wall teichoic acids, WTAs) or anchored to the phospholipid membrane (lipoteichoic acids, LTAs). The post-synthetic addition of D-alanine (D-Ala) residues to these polymers modulates surface charge and contributes to pathogen survival in the host environment. Despite this importance, the underlying mechanisms controlling WTA D-alanylation remain a significant area for further investigation. Here, we demonstrate that the teichoic acid D-Ala esterase, FmtA, is essential for WTA D-alanylation. The inactivation of fmtA results in a more negative net surface charge, impacting host adhesion, biofilm formation, and cell aggregation. We found that LTA from fmtA-deficient strains retains normal D-alanylation levels, while WTA is almost devoid of D-Ala. These data support the notion that LTA provides the D-Ala for WTA modification, a process dependent on FmtA.IMPORTANCEThe D-alanine (D-Ala) modification of Staphylococcus aureus teichoic acids influences bacterial interactions and survival under stress. While this modification is important for host survival, the mechanisms underlying wall teichoic acid (WTA) D-alanylation remain unclear. A deeper understanding of this process could lead to the development of targeted therapies to combat S. aureus infections. We have identified FmtA as essential for this process, supporting the idea that lipoteichoic acid (LTA) provides the D-Ala used to modify WTAs. Our findings highlight a critical gap in understanding this mechanism: an acyltransferase must incorporate the D-Ala released from LTAs by FmtA into WTAs.

Staphylococcus aureus is a notorious human pathogen that thrives in macrophages. It resides in mature phagolysosomes, where a subset of the bacteria eventually begin to proliferate. How S. aureus acquires essential nutrients, such as amino acids, for growth in this niche is poorly understood. Using a long-term primary human macrophage infection model, we show that branched-chain amino acid (BCAA) uptake mediated by the major transporter BrnQ1 is required by S. aureus for intracellular replication in macrophages and we provide mechanistic insight into the role of BCAAs in the success of intracellular S. aureus. Loss of BrnQ1 function renders intracellular S. aureus non-replicative and non-cytotoxic. The defective intracellular growth of S. aureus brnQ1 mutants can be rescued by supplementation with BCAAs or by overexpression of the BCAA transporters BrnQ1 or BcaP. Inactivation of the CodY repressor rescues the ability of S. aureus brnQ1 mutants to proliferate intracellularly independent of endogenous BCAA synthesis but dependent on BcaP expression. Non-replicating brnQ1 mutants in primary human macrophages become metabolically quiescent and display aberrant gene expression marked by failure to respond to intraphagosomal iron starvation. The bacteria remain, however, viable for an inordinate length of time. This dormant, yet viable bacterial state is distinct from classical persisters and small colony variants.

Staphylococcus aureus is a leading cause of skin and soft tissue infections, endocarditis, and bloodstream infections worldwide. The emergence of methicillin-resistant S. aureus (MRSA) and growing resistance to last-resort antibiotics like vancomycin have created an urgent need for new antimicrobials with distinct mechanisms of action. In this study, we characterize DB10, a planar, fluorene-based compound identified in a high-throughput screen for MRSA inhibitors. Upon UVA exposure, DB10 undergoes photoconversion from a red-colored form (DB10-R) to a yellow-colored form (DB10-Y). In comparison with DB10-R, DB10-Y exhibits reduced hydrophobicity, lower cytotoxicity, and modestly improved minimum inhibitory concentrations (MICs) towards a number of Gram-positive bacteria. DB10-Y intercalates into DNA and induces double-stranded breaks, yet resistance emerged only at low levels after prolonged serial passaging. To optimize this scaffold, we screened a panel of fluorene analogs and identified the photoconverting analog DB33, which in its yellow form (DB33-Y) is non-toxic and retained DNA intercalation activity. DB33-Y was effective against intracellular S. aureus in macrophages and epithelial cells and significantly reduced bacterial burden and lesion size in a murine skin infection model. DB10-Y and DB33-Y both also suppressed expression of α-toxin at sub-MIC concentrations, indicating an additional anti-virulence effect. Together, these findings highlight the therapeutic potential of fluorene-based DNA intercalators as a new class of antimicrobial and anti-virulence agents against MRSA. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, PJT-183848 Natural Sciences and Engineering Research Council, https://ror.org/01h531d29, RGPIN-2021-03950

ABSTRACT Staphylococcus aureus is a predominant cause of post-operative surgical site infections and persistent bacteremia. Here, we describe a patient who experienced three episodes of S. aureus infection over a period of 4 months following a total knee arthroplasty. The initial bloodstream isolate (SAB-0429) was a clonal complex 5 (CC5) and methicillin-resistant S. aureus (MRSA), whereas two subsequent isolates (SAB-0485 and SAB-0495) were CC5 isolates but methicillin-sensitive S. aureus . The two latter isolates harbored a plasmid encoding three superantigen genes that were not present in the primary MRSA isolate. SAB-0485 and SAB-0495 both expressed the plasmid-encoded staphylococcal enterotoxin R exotoxin and demonstrated increased superantigen activity compared with SAB-0429. Compared to SAB-0429, the latter isolates also demonstrated an increased bacterial burden in a mouse bacteremia model that was dependent on increased interferon-γ production. Curing of the plasmid from SAB-0485 reduced this virulence phenotype. These findings suggest that the superantigen exotoxins may provide a selective advantage in chronic post-surgical infections. IMPORTANCE In this study, we investigated bacterial isolates from a patient who experienced three recurrent S. aureus infections over a 4 month period following total knee arthroplasty. Genomic and phenotypic characterization of these isolates revealed that they all belonged to clonal complex 5, yet the latter two strains contained an additional plasmid encoding superantigen exotoxins. Subsequent experimental infection experiments in mice demonstrated that the plasmid-encoded superantigens exacerbated bacteremia by promoting liver abscess formation. These experiments suggest that despite appropriate antibiotic therapy, bacterial superantigens may be able to promote persistent infection following post-surgery.

AbstractStaphylococcus aureusis a predominant cause of postoperative surgical site infections and persistent bacteremia. Here we describe a patient that following a total knee arthroplasty subsequently experienced three episodes ofS. aureusbacteremia over a period of 4 months. The initial blood stream isolate (SAB-0429) was a clonal complex (CC) 5 and methicillin resistantS. aureus(MRSA), whereas two subsequent blood stream isolates (SAB-0485 and SAB-0495) were CC5 isolates but methicillin sensitiveS. aureus(MSSA). The two latter isolates harbored a plasmid encoding three superantigen genes not present in the primary MRSA isolate. SAB-0485 and SAB-0495 expressed the plasmid encoded staphylococcal enterotoxin R (SER) exotoxin and demonstrated increased superantigen activity compared with SAB-0429. Compared to SAB-0429, the latter isolates also demonstrated an increased bacterial burden in a mouse bacteremia model that was dependent on increased IFNγ production. Curing of the plasmid from SAB-0485 reduced this virulence phenotype. These findings suggest that the superantigen exotoxins may provide a selective advantage in chronic postsurgical infections.

Staphylococcus aureus poses a significant threat in both community and hospital settings due to its infective and pathogenic nature combined with its ability to resist the action of chemotherapeutic agents. Methicillin-resistant S. aureus (MRSA) represents a critical challenge. Metal-chelating thiosemicarbazones (TSCs) have shown promise in combating MRSA and while previous studies hinted at the antimicrobial potential of TSCs, their mechanisms of action against MRSA are still under investigation. We screened a chemical library for anti-staphylococcal compounds and identified a potent molecule named R91 that contained the NNSN structural motif found within TSCs. We identified that R91 and several structural analogs exhibited antimicrobial activity against numerous S. aureus isolates as well as other Gram-positive bacteria. RNAseq analysis revealed that R91 induces copper and oxidative stress responses. Checkerboard assays demonstrated synergy of R91 with copper, nickel, and zinc. Mutation of the SrrAB two-component regulatory system sensitizes S. aureus to R91 killing, further linking the oxidative stress response to R91 resistance. Moreover, R91 was found to induce hydrogen peroxide production, which contributed to its antimicrobial activity. Remarkably, no mutants with elevated R91 resistance were identified, despite extensive attempts. We further demonstrate that R91 can be used to effectively treat an intracellular reservoir of S. aureus in cell culture and can reduce bacterial burdens in a murine skin infection model. Combined, these data position R91 as a potent TSC effective against MRSA and other Gram-positive bacteria, with implications for future therapeutic development.

BACKGROUND:Staphylococcus aureus is the most common cause of life-threatening endovascular infections, including infective endocarditis (IE). These infections, especially when caused by methicillin-resistant strains (MRSA), feature limited therapeutic options and high morbidity and mortality rates. METHODS:Herein, we investigated the role of the purine biosynthesis repressor, PurR, in virulence factor expression and vancomycin (VAN) treatment outcomes in experimental IE due to MRSA. RESULTS:The PurR-mediated repression of purine biosynthesis was confirmed by enhanced purF expression and production of an intermediate purine metabolite in purR mutant strain. In addition, enhanced expression of the transcriptional regulators, sigB and sarA, and their key downstream virulence genes (eg, fnbA, and hla) was demonstrated in the purR mutant in vitro and within infected cardiac vegetations. Furthermore, purR deficiency enhanced fnbA/fnbB transcription, translating to increased fibronectin adhesion versus the wild type and purR-complemented strains. Notably, the purR mutant was refractory to significant reduction in target tissues MRSA burden following VAN treatment in the IE model. CONCLUSIONS:These findings suggest that the purine biosynthetic pathway intersects the coordination of virulence factor expression and in vivo persistence during VAN treatment, and may represent an avenue for novel antimicrobial development targeting MRSA.

Aims:The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against Staphylococcus aureus. Methods:We performed in vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10 µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated its sensitivity to killing, comparing wild type (WT) and CopAZB-deficient USA300 strains. Results:UAMS-1 demonstrated a greater sensitivity to SF compared to USA300 WT at 12 hours (p = 0.001) and 24 hours (p = 0.027). UAMS-1 died in statistically significant quantities at 24 hours (p = 0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at four (p = 0.001), 12 (p = 0.005), and 24 hours (p = 0.006). We confirmed a high sensitivity to killing with the addition of exogenous copper on both strains at four (p = 0.011), 12 (p = 0.011), and 24 hours (p = 0.011). WT and CopAZB-deficient USA300 strains significantly died in SF, demonstrating a MBC-Cu of 50 µM against USA300 WT (p = 0.011). Conclusion:SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. Adding 10 µM of copper was highly toxic, confirming its bactericidal effect. We found CopAZB proteins to be involved in copper effluxion by demonstrating the high sensitivity of mutant strains to lower copper concentrations. Thus, we propose CopAZB proteins as potential targets and use exogenous copper as a treatment alternative against S. aureus.

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia.

Bacteria continue to develop resistance against antibiotics, including last-resort ones, reinforcing the urgent need for new antimicrobial strategies. Chemicals at infection sites in the host often influence microbial virulence and antibiotic response; such interactions may offer new antimicrobial targets. Polyamines are cationic small molecules bacteria may encounter at infection sites. They are overproduced during infection, modulating host immune responses. The ability of bacteria to detoxify polyamines such as by a spermine/spermidine acetyltransferase (SpeG) correlated with hypervirulence of pathogens, including Salmonella Typhimurium, Enterococcus faecium, and the community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain USA300. Polyamines may also influence bacterial antibiotic response. For example, we found that USA300 uses exogenous polyamines to resist antibiotics, including vancomycin, a phenotype lost in the ΔspeG mutant. Therefore, we aimed to uncover inhibitors of polyamine detoxification. A high-throughput chemical screen against S. aureus USA300 identified OES2-0017, which showed potent synergy with polyamines and growth-inhibitory effects at the low micromolar range. We revealed a dual mode of action of OES2-0017, where low concentrations inhibited SpeG and other polyamine detoxification enzymes, and higher concentrations perturbed the bacterial membrane. Eukaryotic cell membranes were not impacted at the same concentration range, as observed in a hemolysis assay. OES2-0017 abolished the polyamine-mediated antibiotic resistance in MRSA USA300, suggesting its potential utility as an antibiotic adjuvant. Notably, OES2-0017 showed similar polyamine synergy and growth inhibitory activities against other Gram-positive (e.g., E. faecium and E. faecalis) and Gram-negative (e.g., Klebsiella pneumoniae, S. Typhimurium, and Burkholderia cenocepacia) pathogens. OES2-0017 prevented S. Typhimurium from replicating in murine macrophages, which also suggests its potential application as an antivirulence agent. Together, this work exploits understudied aspects of chemically mediated host-pathogen interactions, offering a potential new antimicrobial strategy with a novel mode of action for multidrug-resistant priority pathogens. ### Competing Interest Statement The authors have declared no competing interest.

Peptidoglycan hydrolases, or autolysins, play a critical role in cell wall remodeling and degradation, facilitating bacterial growth, cell division, and cell separation. In Staphylococcus aureus, the so-called “major” autolysin, Atl, has long been associated with host adhesion; however, the molecular basis underlying this phenomenon remains understudied. To investigate, we used the type V glycopeptide antibiotic complestatin, which binds to peptidoglycan and blocks the activity of autolysins, as a chemical probe of autolysin function. We also generated a chromosomally encoded, catalytically inactive variant of the Atl enzyme. Autolysin-mediated peptidoglycan hydrolysis, in particular Atl-mediated daughter cell separation, was shown to be critical for maintaining optimal surface levels of S. aureus cell wall-anchored proteins, including the fibronectin-binding proteins (FnBPs) and protein A (Spa). As such, disrupting autolysin function reduced the affinity of S. aureus for host cell ligands, and negatively impacted early stages of bacterial colonization in a systemic model of S. aureus infection. Phenotypic studies revealed that Spa was sequestered at the septum of complestatin-treated cells, highlighting that autolysins are required to liberate Spa during cell division. In summary, we reveal the hydrolytic activities of autolysins are associated with the surface display of S. aureus cell wall-anchored proteins. We demonstrate that by blocking autolysin function, type V glycopeptide antibiotics are promising antivirulence agents for the development of strategies to control S. aureus infections.

The Severe Acute Respiratory Syndrome Coronavirus 2 (CoV-2) pandemic has affected millions globally. A significant complication of CoV-2 infection is secondary bacterial co-infection, as seen in approximately 25% of severe cases. The most common organism isolated during co-infection is Staphylococcus aureus. Here, we describe the development of an in vitro co-infection model where both viral and bacterial replication kinetics may be examined. We demonstrate CoV-2 infection does not alter bacterial interactions with host epithelial cells. In contrast, S. aureus enhances CoV-2 replication by 10- to 15-fold. We identify this pro-viral activity is due to the S. aureus iron-regulated surface determinant A (IsdA) protein and demonstrate IsdA modifies host transcription. We find that IsdA alters Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) signaling, by affecting JAK2-STAT3 levels, ultimately leading to increased viral replication. These findings provide key insight into the molecular interactions between host cells, CoV-2 and S. aureus during co-infection.