Autolysin-mediated peptidoglycan hydrolysis is required for the surface display of Staphylococcus aureus cell wall-anchored proteins.

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.

Superantigens promote Staphylococcus aureus bloodstream infection by eliciting pathogenic interferon-gamma production.

Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of exotoxins from S. aureus that bind directly to major histocompatibility complex (MHC) class II and T cell receptors to drive extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease, including toxic shock syndrome, the specific pathological mechanisms remain unclear. Herein, we aimed to elucidate how SAgs contribute to pathogenesis during bloodstream infections and utilized transgenic mice encoding human MHC class II to render mice susceptible to SAg activity. We demonstrate that SAgs contribute to S. aureus bacteremia by massively increasing bacterial burden in the liver, and this was mediated by CD4+ T cells that produced interferon gamma (IFN-γ) to high levels in a SAg-dependent manner. Bacterial burdens were reduced by blocking IFN-γ, phenocopying SAg-deletion mutant strains, and inhibiting a proinflammatory response. Infection kinetics and flow cytometry analyses suggested that this was a macrophage-driven mechanism, which was confirmed through macrophage-depletion experiments. Experiments in human cells demonstrated that excessive IFN-γ allowed S. aureus to replicate efficiently within macrophages. This indicates that SAgs promote bacterial survival by manipulating the immune response to inhibit effective clearing of S. aureus Altogether, this work implicates SAg toxins as critical therapeutic targets for preventing persistent or severe S. aureus disease.

The Purine Biosynthesis Repressor, PurR, Contributes to Vancomycin Susceptibility of Methicillin-resistant Staphylococcus aureus in Experimental Endocarditis.

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.

MRSA Isolates from Patients with Persistent Bacteremia Generate Nonstable Small Colony Variants In Vitro within Macrophages and Endothelial Cells during Prolonged Vancomycin Exposure.

Staphylococcus aureus (especially methicillin-resistant S. aureus [MRSA]) is frequently associated with persistent bacteremia (PB) during vancomycin therapy despite consistent susceptibility in vitro. Strategic comparisons of PB strains versus those from vancomycin-resolving bacteremia (RB) would yield important mechanistic insights into PB outcomes. Clinical PB versus RB isolates were assessed in vitro for intracellular replication and small colony variant (SCV) formation within macrophages and endothelial cells (ECs) in the presence or absence of exogenous vancomycin. In both macrophages and ECs, PB and RB isolates replicated within lysosome-associated membrane protein-1 (LAMP-1)-positive compartments. PB isolates formed nonstable small colony variants (nsSCVs) in vancomycin-exposed host cells at a significantly higher frequency than matched RB isolates (in granulocyte-macrophage colony-stimulating factor [GM-CSF], human macrophages PB versus RB, P < 0.0001 at 48 h; in ECs, PB versus RB, P < 0.0001 at 24 h). This phenotype could represent one potential basis for the unique ability of PB isolates to adaptively resist vancomycin therapy and cause PB in humans. Elucidating the molecular mechanism(s) by which PB strains form nsSCVs could facilitate the discovery of novel treatment strategies to mitigate PB due to MRSA.

Mutations in a Membrane Permease or hpt Lead to 6-Thioguanine Resistance in Staphylococcus aureus.

We recently discovered that 6-thioguanine (6-TG) is an antivirulence compound that is produced by a number of coagulase-negative staphylococci. In Staphylococcus aureus, it inhibits de novo purine biosynthesis and ribosomal protein expression, thus inhibiting growth and abrogating toxin production. Mechanisms by which S. aureus may develop resistance to this compound are currently unknown. Here, we show that 6-TG-resistant S. aureus mutants emerge spontaneously when the bacteria are subjected to high concentrations of 6-TG in vitro. Whole-genome sequencing of these mutants revealed frameshift and missense mutations in a xanthine-uracil permease family protein (stgP [six thioguanine permease]) and single nucleotide polymorphisms in hypoxanthine phosphoribosyltransferase (hpt). These mutations engender S. aureus the ability to resist both the growth inhibitory and toxin downregulation effects of 6-TG. While prophylactic administration of 6-TG ameliorates necrotic lesions in subcutaneous infection of mice with methicillin-resistant S. aureus (MRSA) strain USA300 LAC, the drug did not reduce lesion size formed by the 6-TG-resistant strains. These findings identify mechanisms of 6-TG resistance, and this information can be leveraged to inform strategies to slow the evolution of resistance.

De Novo Purine Biosynthesis Is Required for Intracellular Growth of Staphylococcus aureus and for the Hypervirulence Phenotype of a purR Mutant.

Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.

The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function.

Bacterial superantigens (SAgs) cause Vß-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vß-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis.

Staphylococcus pseudintermedius Surface Protein L (SpsL) Is Required for Abscess Formation in a Murine Model of Cutaneous Infection.

Staphylococcus pseudintermedius is the leading cause of pyoderma in dogs and is often associated with recurrent skin infections that require prolonged antibiotic therapy. High levels of antibiotic use have led to multidrug resistance, including the emergence of epidemic methicillin-resistant clones. Our understanding of the pathogenesis of S. pseudintermedius skin infection is very limited, and the identification of the key host-pathogen interactions underpinning infection could lead to the design of novel therapeutic or vaccine-based approaches for controlling disease. Here, we employ a novel murine cutaneous-infection model of S. pseudintermedius and investigate the role of the two cell wall-associated proteins (SpsD and SpsL) in skin disease pathogenesis. Experimental infection with wild-type S. pseudintermedius strain ED99 or a gene-deletion derivative deficient in expression of SpsD led to a focal accumulation of neutrophils and necrotic debris in the dermis and deeper tissues of the skin characteristic of a classical cutaneous abscess. In contrast, mice infected with mutants deficient in SpsL or both SpsD and SpsL developed larger cutaneous lesions with distinct histopathological features of regionally extensive cellulitis rather than focal abscessation. Furthermore, comparison of the bacterial loads in S. pseudintermedius-induced cutaneous lesions revealed a significantly increased burden of bacteria in the mice infected with SpsL-deficient mutants. These findings reveal a key role for SpsL in murine skin abscess formation and highlight a novel function for a bacterial surface protein in determining the clinical outcome and pathology of infection caused by a major canine pathogen.

Protocol for studying co-infection between SARS-CoV-2 and Staphylococcus aureus in vitro.

Bacterial co-infection is one of the most common complications of SARS CoV-2 infection. Here, we present a protocol for the in vitro study of co-infection between SARS CoV-2 and Staphylococcus aureus. We describe steps for quantifying viral and bacterial replication kinetics in the same sample, with the optional extraction of host RNA and proteins. This protocol is applicable to many viral and bacterial strains and can be performed in different cell types. For complete details on the use and execution of this protocol, please refer to Goncheva et al.1.

The Staphylococcus aureus protein IsdA increases SARS CoV-2 replication by modulating JAK-STAT signaling.

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.

Nucleotide biosynthesis: the base of bacterial pathogenesis.

Most free-living organisms require the synthesis and/or acquisition of purines and pyrimidines, which form the basis of nucleotides, to survive. In most bacteria, the nucleotides are synthesized de novo and the products are used in many cell functions, including DNA replication, energy storage, and as signaling molecules. Due to their central role in the metabolism of bacteria, both nucleotide biosynthesis pathways have strong links with the virulence of opportunistic and bona fide bacterial pathogens. Recent findings have established a new, shared link in the control of nucleotide biosynthesis and the production of virulence factors. Furthermore, targeting of these pathways forms the basis of interspecies competition and can provide an open source for new antimicrobial compounds. Here, we highlight the contribution of nucleotide biosynthesis to bacterial pathogenesis in a plethora of different diseases and speculate on how they can be targeted by intervention strategies.

Coagulase-negative staphylococci release a purine analog that inhibits Staphylococcus aureus virulence.

Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.

Evolutionary and Functional Analysis of Coagulase Positivity among the Staphylococci.

The bacterial genus Staphylococcus comprises a large group of pathogenic and nonpathogenic species associated with an array of host species. Staphylococci are differentiated into coagulase-positive or coagulase-negative groups based on the capacity to promote clotting of plasma, a phenotype historically associated with the ability to cause disease. However, the genetic basis of this important diagnostic and pathogenic trait across the genus has not been examined to date. Here, we selected 54 representative staphylococcal species and subspecies to examine coagulation of plasma derived from six representative host species. In total, 13 staphylococcal species mediated coagulation of plasma from at least one host species including one previously identified as coagulase negative (Staphylococcus condimenti). Comparative genomic analysis revealed that coagulase activity correlated with the presence of a gene (vwb) encoding the von Willebrand binding protein (vWbp) whereas only the Staphylococcus aureus complex contained a gene encoding staphylocoagulase (Coa), the classical mediator of coagulation. Importantly, S. aureus retained vwb-dependent coagulase activity in an S. aureus strain deleted for coa whereas deletion of vwb in Staphylococcus pseudintermedius resulted in loss of coagulase activity. Whole-genome-based phylogenetic reconstruction of the Staphylococcus genus revealed that the vwb gene has been acquired on at least four different occasions during the evolution of the Staphylococcus genus followed by allelic diversification via mutation and recombination. Allelic variants of vWbp from selected coagulase-positive staphylococci mediated coagulation in a host-dependent manner indicative of host-adaptive evolution. Taken together, we have determined the genetic and evolutionary basis of staphylococcal coagulation, revealing vWbp to be its archetypal determinant. IMPORTANCE The ability of some species of staphylococci to promote coagulation of plasma is a key pathogenic and diagnostic trait. Here, we provide a comprehensive analysis of the coagulase positivity of the staphylococci and its evolutionary genetic basis. We demonstrate that the von Willebrand binding protein rather than staphylocoagulase is the archetypal coagulation factor of the staphylococci and that the vwb gene has been acquired several times independently during the evolution of the staphylococci. Subsequently, vwb has undergone adaptive diversification to facilitate host-specific functionality. Our findings provide important insights into the evolution of pathogenicity among the staphylococci and the genetic basis for a defining diagnostic phenotype.

Staphylococcus aureus secreted lipases do not inhibit innate immune killing mechanisms.

Background: Staphylococcus aureus causes an array of diseases in both humans and livestock. Pathogenesis is mediated by a plethora of proteins secreted by S. aureus, many of which remain incompletely characterised. For example, S. aureus abundantly secretes two isoforms of the enzyme lipase into the extracellular milieu, where they scavenge upon polymeric triglycerides. It has previously been suggested that lipases may interfere with the function of innate immune cells, such as macrophages and neutrophils, but the impact of lipases on phagocytic killing mechanisms remains unknown. Methods: We employed the epidemic S. aureus clone USA300 strain LAC and its lipase deficient isogenic mutant, along with recombinant lipase proteins, in in vitro experimental infection assays. To determine if lipases can inhibit innate immune killing mechanisms, the bactericidal activity of whole blood, human neutrophils, and macrophages was analysed. In addition, gentamycin protection assays were carried out to examine the influence of lipases on S. aureus innate immune cell escape. Results: There were no differences in the survival of S. aureus USA300 LAC wild type and its lipase-deficient isogenic mutant after incubation with human whole blood or neutrophils. Furthermore, there was no detectable lipase-dependent effect on phagocytosis, intracellular survival, or escape from both human primary and immortalised cell line macrophages, even upon supplementation with exogenous recombinant lipases. Conclusions: S. aureus lipases do not inhibit bacterial killing mechanisms of human macrophages, neutrophils, or whole blood. These findings broaden our understanding of the interaction of S. aureus with the innate immune system.

Staphylococcus aureus Lipase 1 Enhances Influenza A Virus Replication.

Influenza A virus (IAV) causes annual epidemics of respiratory disease in humans, often complicated by secondary coinfection with bacterial pathogens such as Staphylococcus aureus Here, we report that the S. aureus secreted protein lipase 1 enhances IAV replication in vitro in primary cells, including human lung fibroblasts. The proviral activity of lipase 1 is dependent on its enzymatic function, acts late in the viral life cycle, and results in increased infectivity through positive modulation of virus budding. Furthermore, the proviral effect of lipase 1 on IAV is exhibited during in vivo infection of embryonated hen's eggs and, importantly, increases the yield of a vaccine strain of IAV by approximately 5-fold. Thus, we have identified the first S. aureus protein to enhance IAV replication, suggesting a potential role in coinfection. Importantly, this activity may be harnessed to address global shortages of influenza vaccines.IMPORTANCE Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease. Secondary bacterial coinfection by organisms such as Staphylococcus aureus is the most common complication of primary IAV infection and is associated with high levels of morbidity and mortality. Here, we report the first identified S. aureus factor (lipase 1) that enhances IAV replication during infection via positive modulation of virus budding. The effect is observed in vivo in embryonated hen's eggs and greatly enhances the yield of a vaccine strain, a finding that could be applied to address global shortages of influenza vaccines.

Stress-induced inactivation of the Staphylococcus aureus purine biosynthesis repressor leads to hypervirulence.

Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.

Immunological Homeostasis at the Ovine Placenta May Reflect the Degree of Maternal Fetal Interaction.

Successful mammalian pregnancies are a result of complex physiological, endocrinological, and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents.