Faculty Bibliography

Staphylococcus aureus is a major human pathogen that imposes a great burden on the healthcare system. In the development of anti-staphylococcal modalities intended to reduce the burden of staphylococcal disease, it is imperative to select appropriate models of S. aureus strains when assessing the efficacy of novel agents. Here, using whole genome sequencing, we reveal that the commonly used strain Newman D2C from the American Type Culture Collection (ATCC) contains mutations that render the strain essentially avirulent. Importantly, Newman D2C is often inaccurately referred to as simply "Newman" in many publications, leading investigators to believe this is the well-described pathogenic strain Newman. This study reveals that Newman D2C carries a stop mutation in the open reading frame of the virulence gene regulator, agrA In addition, Newman D2C carries a single nucleotide polymorphism in the global virulence regulator saeR that results in loss of protein function. This loss of function is highlighted by complementation studies, whereby the saeR allele from Newman D2C is incapable of restoring functionality to an saeR null mutant. Additional functional assessment was achieved through the use of biochemical assays for protein secretion, ex vivo intoxications of human immune cells, and in vivo infections. Altogether, our study highlights the importance of judiciously screening for genetic changes in model S. aureus strains when assessing pathogenesis or the efficacy of novel agents. Moreover, we have identified a novel SNP in the virulence regulator saeR that directly affects the ability of the protein product to activate S. aureus virulence pathways.IMPORTANCEStaphylococcus aureus is a human pathogen that imposes an enormous burden on healthcare systems worldwide. This bacterium is capable of evoking a multitude of disease states that can range from self-limiting skin infections to life-threatening bacteremia. To combat these infections, numerous investigations are underway to develop therapeutics capable of thwarting the deadly effects of the bacterium. To generate successful treatments, it is of paramount importance that investigators use suitable models for examining the efficacy of drugs under study. Here, we demonstrate that a commonly used strain of S. aureus for drug efficacy studies is severely mutated and displays markedly reduced pathogenicity. As such, this organism is an inappropriate model for disease studies.

Diet, and specifically dietary metals, can modify the risk of infection. However, the mechanisms by which manganese (Mn), a common dietary supplement, alters infection remain unexplored. We report that dietary Mn levels dictate the outcome of systemic infections caused by Staphylococcus aureus, a leading cause of bacterial endocarditis. Mice fed a high Mn diet display alterations in Mn levels and localization within infected tissues, and S. aureus virulence and infection of the heart are enhanced. Although the canonical mammalian Mn-sequestering protein calprotectin surrounds staphylococcal heart abscesses, calprotectin is not released into the abscess nidus and does not limit Mn in this organ. Consequently, excess Mn is bioavailable to S. aureus in the heart. Bioavailable Mn is utilized by S. aureus to detoxify reactive oxygen species and protect against neutrophil killing, enhancing fitness within the heart. Therefore, a single dietary modification overwhelms vital host antimicrobial strategies, leading to fatal staphylococcal infection.

Bacterial superantigens (SAgs) cause Vbeta-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 Vbeta-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 aureus is a major bacterial pathogen that causes disease worldwide. The emergence of strains that are resistant to commonly used antibiotics and the failure of vaccine development have resulted in a renewed interest in the pathophysiology of this bacterium. Staphylococcal leukocidins are a family of bi-component pore-forming toxins that are important virulence factors. During the past five years, cellular receptors have been identified for all of the bi-component leukocidins. The identification of the leukocidin receptors explains the cellular tropism and species specificity that is exhibited by these toxins, which has important biological consequences. In this Review, we summarize the recent discoveries that have reignited interest in these toxins and provide an outlook for future research.

Staphylococcus aureus (S. aureus) produces many different exotoxins including the gamma-toxins, HlgAB and HlgCB. Gamma-toxins form pores in both leukocyte and erythrocyte membranes, resulting in cell lysis. The genes encoding gamma-toxins are present in most strains of S. aureus, and are commonly expressed in clinical isolates recovered from menstrual Toxic Shock Syndrome (mTSS) patients. This study set out to investigate the cytotoxic and proinflammatory effects of gamma-toxins on vaginal epithelial surfaces. We found that both HlgAB and HlgCB were cytotoxic to cultured human vaginal epithelial cells (HVECs) and induced cytokine production at sub-cytotoxic doses. Cytokine production induced by gamma-toxin treatment of HVECs was found to involve epidermal growth factor receptor (EGFR) signaling and mediated by shedding of EGFR ligands from the cell surface. The gamma-toxin subunits displayed differential binding to HVECs (HlgA 93%, HlgB 97% and HlgC 28%) with both components (HlgAB or HlgCB) required for maximum detectable binding and significant stimulation of cytokine production. In studies using full thickness ex vivo porcine vaginal mucosa, HlgAB or HlgCB stimulated a dose-dependent cytokine response, which was reduced significantly by inhibition of EGFR signaling. The effects of gamma-toxins on porcine vaginal tissue and cultured HVECs were validated using ex vivo human ectocervical tissue. Collectively, these studies have identified the EGFR-signaling pathway as a key component in gamma-toxin-induced proinflammatory changes at epithelial surfaces and highlight a potential therapeutic target to diminish toxigenic effects of S. aureus infections.

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is, in part, a result of antimicrobial resistance highlighting the need to uncover novel antibiotic targets and discover new therapeutic agents. In the present study we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization) dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and suf transcription was positively influenced by sigma factor B. We characterized a S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*) resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. These included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the TCA cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular non-chelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.

Staphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is due to its ability to adapt to the environment by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the secreted Sa proteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties and genetic lineages, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains.