Alexander R. Horswill

Staphylococcus aureus is a formidable pathogen and the leading cause of skin and soft tissue infections. We recently showed that the highly conserved sasF gene, encoding for an uncharacterized sortase-anchored surface protein, is up-regulated when S. aureus colonizes skin. Here, we demonstrate that SasF is an enzyme that has glycoside epimerase activity, which is required for S. aureus resistance to skin fatty acids. Mutants of sasF are highly impaired in skin colonization and acute infection models. The SasF enzyme is crucial for maintaining the structure of wall teichoic acids and proper S. aureus surface chemistry. To our knowledge, this is the first report of a bacterial surface epimerase in this pathogen, suggesting that SasF could be a therapeutic target.

Staphylococcus aureus secretes a family of serine protease–like enzymes (SplA to SplF) that resemble eukaryotic granzymes, yet the mechanism by which amino-terminal processing activates this subclass has remained unresolved. Structural studies show insertion of the processed amino terminus without detectable changes in active-site geometry, creating a longstanding paradox as to how catalytic competence is achieved. Here, we identify SplB as the most highly expressed member of this family in a pathogenic methicillin-resistant S. aureus strain and use it to define the basis of activation. Solution nuclear magnetic resonance spectroscopy shows that precise amino-terminal processing triggers a long-range allosteric network coupling the amino terminus to the active site ∼20 angstroms away, unlocking global microsecond-to-millisecond dynamics that enable substrate engagement. Molecular dynamics simulations reveal the conformational ensembles underlying these motions. Last, mutational perturbation of this dynamic network modulates substrate engagement and catalytic activity in a manner consistent with dynamic control of binding competence. Together, these findings establish dynamic allostery as the mechanism of N-terminal activation in this subclass of serine proteases.

Diabetic wounds are often infected with microbes, which perpetuate inflammation, and stall wound healing. The bacterium Group B Streptococcus (GBS) is frequently isolated from diabetic wounds; however, little is known about how GBS adapts to survive in this niche. Previously, we found that GBS acquires stable mutations in the major two-component system CovRS during murine diabetic wound infection that result in increased pigmentation. Here, we further characterize these pigmented variants and determine the consequences on GBS survival. Using a murine model of wound infection, we find that covRS mutants arise specifically in diabetic hosts and are selected for across multiple GBS backgrounds. Whole genome sequencing of pigmented isolates revealed mutations in both covR and covS , with most isolates having a single nucleotide insertion or deletion in the covR promoter region. Phenotypic analysis of murine-acquired mutants reveals enhanced traits associated with virulence, including increased hemolytic activity, host cell cytotoxicity, and elevated nuclease activity. While our previous and current study indicated that engineered covR deletion mutants do not exhibit increased survival in the diabetic wound, we observe that a pigmented isolate survived better than wild-type during co-infection with Enterococcus faecalis , another frequently isolated wound pathogen. Finally, we find that depletion of neutrophils reduces the frequency of covRS mutant variants that arise in the population. Our work highlights the emergence of covRS mutations in GBS, and the consequences of these variations are associated with enhanced virulence and competitive fitness, underscoring the importance of these regulatory changes in the context of diabetic wounds.

Streptococcus pyogenes (group A streptococcus [GAS]) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum-sensing (QS) system, a cell-to-cell communication network, modifies the cell surface, resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon which is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wild type and operon mutants showed that QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like N -acetylglucosamine-linked ribitol (GlcNAc-Rbo) moiety. A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon QS. IMPORTANCE: Streptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections, and can lead to post-infection sequelae like rheumatic heart disease, whose prevalence on a global scale rivals that of the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long-term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host’s innate immune response. We find that modifications to the cell wall arise when this virulence system is activated, which has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

Skin cells secrete testosterone, with greater amounts secreted at the skin surface of males compared with females. Males are also more susceptible to skin infections than females. Here we report that mice engineered with testosterone-deficient skin are resistant to methicillin-resistant Staphylococcus aureus infections. Testosterone promoted the expression of S. aureus cytotoxic virulence factors by activating the accessory gene regulator (agr) quorum-sensing pathway in a concentration-dependent manner and independent of quorum-sensing-activating auto-inducing peptides. Mutational analysis revealed that a functional histidine kinase AgrC in S. aureus was required for testosterone to exert its effect, with in silico evidence indicating a direct interaction between testosterone and AgrC. An isomer of testosterone, enantiomer-testosterone, that blocked bacterial quorum sensing, inhibited S. aureus-induced cytotoxicity of human cells. These findings advance our understanding of how the skin regulates bacterial virulence and reveals a potential therapeutic strategy for the management of infections.

Staphylococcus aureus causes approximately 80% of skin and soft tissue infections (SSTIs). Collagen is the most abundant human extracellular matrix protein with critical roles in wound healing, and S. aureus encodes a collagen binding adhesin (Cna). The role of this protein during skin infections is unknown. Here we report that inability to bind collagen results in worsened pathology of intradermal Δcna S. aureus infection. WT/Cna+ S. aureus shows reduced infection severity, aggregate formation, and significantly improves clearance of bacteria. Cna binds to the collagen-like domain of serum C1q protein to reduce its opsonophagocytic functions. We demonstrate that infection of C1qKO mice with WT bacteria show results similar to the Δcna group. Conversely, inability to bind collagen results in an amplified inflammatory response caused in part by macrophage and neutrophil small molecule mediators released at the infection site (MMP-9, MMP-12, LTB₄), leading to increased immune cell infiltration and death.

SUMMARY: Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum sensing (QS) system, a cell-to-cell communication network, modifies the cell surface resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon that is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wildtype and operon mutants determined that the QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like moiety of N -acetylglucosamine-linked ribitol (GlcNAc-Rbo). A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon quorum sensing. IMPORTANCE: Streptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections and can lead to post-infection sequelae like rheumatic heart disease whose prevalence on a global scale rivals the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host innate immune response. We find that modifications to the cell wall arise when this virulence system is activated that has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

Coagulase-negative staphylococci are dominant human skin colonizers, producing natural products that shape the community and prevent pathogen colonization. The molecular mechanisms by which these natural products mediate interbacterial competition are not fully understood. Here, we identify a plasmid-borne daptide bacteriocin (hominicin) from a human skin isolate of Staphylococcus hominis, which features an unusual N₂-N₂-dimethyl-1,2-propanediamine C-terminus. Heterologous expression of the reconstituted biosynthetic loci yields a daptide product of the same molecular mass that exhibits antimicrobial activity against the skin pathogen Staphylococcus aureus, with amino-modified termini being essential for activity. Membrane permeability and voltage-clamp lipid bilayer experiments support a mechanism by which the daptide rapidly dissipates the transmembrane potential by forming peptidic channels. Additionally, we identify a cognate homI gene that confers resistance against membrane damage. Finally, the purified daptide effectively protects mouse skin from S. aureus-induced epicutaneous injury, as evidenced by reduced bacterial burden, inflammation, and transepithelial water loss, highlighting its therapeutic potential for treating bacterial skin infections. Our findings elucidate a mechanism of action, biosynthesis, and resistance for a staphylococcal bacteriocin belonging to a class of natural products called daptides.

Staphylococcus aureus is a Gram-positive opportunistic pathogen and a leading cause of skin and soft tissue infections (SSTIs). S. aureus colonizes approximately 20–30% of the population and transiently colonizes the skin. Although skin colonization is an SSTI risk factor, our understanding of S. aureus requirements to persist in this environment is still developing. The human skin is acidic, with a ubiquitous distribution of eccrine glands, which secrete metabolites, such as urea onto the skin surface. Many pathogens break down urea into ammonia and carbon dioxide by the Ni(II)-dependent enzyme urease as an acid resistance mechanism. S. aureus relies on urea metabolism for pH homeostasis in the murine kidney, but a role for this process on skin has not been shown. Using an in vitro model of the human skin surface environment, we investigated the role of urease in the methicillin-resistant USA300 strain S. aureus subsp. aureus (MRSA) LAC. We observe that urease contributes to an increase in extracellular pH and production of extracellular ammonium, which results in ammonium becoming a shared good in a mixed culture. We also demonstrate that the putative urea transporter SAUSA300_2237 ( ureT ) is required for optimal extracellular ammonia generation. Finally, we show that the urease inhibitor fluorofamide effectively inhibits S. aureus urease activity. This work identifies a role for urea metabolism during S. aureus skin colonization and offers the first published characterization of the S. aureus putative urea transporter, UreT. IMPORTANCE: The human skin is a unique microbial environment that has been challenging to faithfully model in the laboratory. Here, we utilize a recently developed media recipe incorporating metabolites measured from healthy human skin to assess the role of urease in Staphylococcus aureus pH homeostasis in the skin environment. These data provide new insights into the role of urea metabolism in S. aureus pH homeostasis during skin colonization. We also describe the first characterization of transporter SAUSA300_2237 ( ureT ) and demonstrate its role in urea metabolism. These insights advance our understanding of this metabolic pathway in S. aureus .

Mechanisms responsible for delayed wound repair are poorly understood despite the common impact of this disorder on health. To study how Staphylococcus aureus disrupts healing, mouse and human wound repair models were evaluated after exposure to S . aureus or commensal Staphylococcus . Quorum sensing by S . aureus , but not S . hominis , delayed repair and inhibited the expression of genes responsible for lipid metabolism in keratinocytes. S . aureus with inactive accessory gene regulator (agr) did not delay healing, and the inhibition of lipid metabolism was recapitulated in vitro by synthetic phenol soluble modulin α1 (psmα1) and psmα4, genes that are under agr control. However, S . aureus strains with single deletion of psmA , psmB , alpha-hemolysin ( hla ), or hld gene continued to delay repair, suggesting that S . aureus used multiple agr-dependent virulence factors to disrupt healing. These observations provide insight into mechanisms for delayed wound healing, identify quorum sensing as a critical event, and highlight the role of lipid biosynthesis in wound reepithelialization.