Faculty Bibliography

Staphylococcus aureus subverts innate defenses during infection in part by killing host immune cells to exacerbate disease. This human pathogen intercepts host cues and activates a transcriptional response via the S. aureus exoprotein expression (SaeR/S) two-component system to secrete virulence factors critical for pathogenesis. We recently showed that the transcriptional repressor CodY adjusts nuclease (nuc) gene expression via SaeR/S but the mechanism remained unknown. Herein, we identified two CodY binding motifs upstream of the sae P1 promoter, which suggested direct regulation by this global regulator. We show that CodY shares a binding site with the positive activator SaeR and alleviating direct CodY repression at this site is sufficient to abrogate stochastic expression, suggesting that CodY represses sae expression by blocking SaeR binding. Epistasis experiments support a model that CodY also controls sae indirectly through Agr and Rot-mediated repression of the sae P1 promoter. We also demonstrate that CodY repression of sae restrains production of secreted cytotoxins that kill human neutrophils. We conclude that CodY plays a previously unrecognized role in controlling virulence gene expression via SaeR/S, and suggest a mechanism by which CodY acts as a master regulator of pathogenesis by tying nutrient availability to virulence gene expression.Importance Bacterial mechanisms that mediate the switch from a commensal to pathogenic lifestyle is one of the biggest unanswered questions in infectious disease. Since the expression of most virulence genes is often correlated with nutrient depletion, this implies that virulence is a response to the lack of nourishment in host tissues, and that pathogens like S. aureus produce virulence factors in order to gain access to nutrients in the host. Herein, we show that specific nutrient depletion signals appear to be funneled to the SaeR/S system through the global regulator CodY. Our findings reveal a strategy by which S. aureus delays the production of immune evasion and immune cell killing proteins until key nutrients are depleted.

Staphylococcus aureus has three types of cation/proton antiporters. The type three family includes two multi-subunit Na+/H+ (Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 in S. aureus Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter deficient KNabc E. coli strain. The catalytic properties of the antiporters were measured in everted vesicles (inside out). The Mnh1 antiporter exhibited significant exchange of Na+/H+ cations at pH 7.5. Mnh2 showed significant exchange of both Na+/H+ and K+/H+ cations, especially at pH 8.5. Under elevated salt conditions, deletion of the mnhA1 gene resulted in a significant reduction of the growth rate of S. aureus in the range of pH 7.5 to 9. Deletion of mnhA2 had similar effects but mainly in the range of pH 8.5 -- 9.5. Double deletion of mnhA1 and mnhA2 led to severe reduction of the S. aureus growth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters in S. aureus were also assessed via their support of virulence in the mouse in vivo infection model. Deletion of the mnhA1 gene led to a major loss of S. aureus virulence in mice, while deletion of mnh2 led to no change in virulence.Importance This study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters in S. aureus and their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+ antiport and it plays a significant role in maintaining halotolerance at pH 7.5 while, the Mnh2 antiporter has catalytic activities for Na+/H+ as well as K+/H+ antiport that have roles in both osmotolerance and halotolerance in S. aureus Study of S. aureus with a single deletion of either mnhA1 or mnhA2 was assessed in an infection model of mice. The result shows that mnhA1 but not mnhA2, plays a major role in S. aureus virulence.

The genome of Staphylococcus aureus has rapidly become one the most frequently sequenced among bacteria, with more than 40000 genome sequences uploaded to public databases. Computational resources required for analysis and quality assessment have lagged behind accumulation of sequence data. Improved analytic pipelines, in combination with the development of customized S. aureus reference databases, can be used to inform S. aureus biology and potentially predict clinical outcome. Here, we review the currently available data about S. aureus genome in public databases, and discuss their potential utility for understanding S. aureus evolution. Also discussed are ways to overcome challenges to the application of whole-genome sequencing data for prevention and management of S. aureus disease.

Staphylococcus aureus is a versatile bacterial pathogen that can cause significant disease burden and mortality. Like other pathogens, S. aureus must adapt to its environment to produce virulence factors to survive the immune responses evoked by infection. Despite the importance of environmental signals for S. aureus pathogenicity, only a limited number of these signals have been investigated in detail for their ability to modulate virulence. Here we show that pyruvate, a central metabolite, causes alterations in the overall metabolic flux of S. aureus and enhances its pathogenicity. We demonstrate that pyruvate induces the production of virulence factors such as the pore-forming leucocidins and that this induction results in increased virulence of community-acquired methicillin-resistant S. aureus (CA-MRSA) clone USA300. Specifically, we show that an efficient "pyruvate response" requires the activation of S. aureus master regulators AgrAC and SaeRS as well as the ArlRS two-component system. Altogether, our report further establishes a strong relationship between metabolism and virulence and identifies pyruvate as a novel regulatory signal for the coordination of the S. aureus virulon through intricate regulatory networks.IMPORTANCE Delineation of the influence of host-derived small molecules on the makeup of human pathogens is a growing field in understanding host-pathogen interactions. S. aureus is a prominent pathogen that colonizes up to one-third of the human population and can cause serious infections that result in mortality in ~15% of cases. Here, we show that pyruvate, a key nutrient and central metabolite, causes global changes to the metabolic flux of S. aureus and activates regulatory networks that allow significant increases in the production of leucocidins. These and other virulence factors are critical for S. aureus to infect diverse host niches, initiate infections, and effectively subvert host immune responses. Understanding how environmental signals, particularly ones that are essential to and prominent in the human host, affect virulence will allow us to better understand pathogenicity and consider more-targeted approaches to tackling the current S. aureus epidemic.

The hallmark of many bacterial infections is pain. The underlying mechanisms of pain during live pathogen invasion are not well understood. Here, we elucidate key molecular mechanisms of pain produced during live methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that spontaneous pain is dependent on the virulence determinant agr and bacterial pore-forming toxins (PFTs). The cation channel, TRPV1, mediated heat hyperalgesia as a distinct pain modality. Three classes of PFTs-alpha-hemolysin (Hla), phenol-soluble modulins (PSMs), and the leukocidin HlgAB-directly induced neuronal firing and produced spontaneous pain. From these mechanisms, we hypothesized that pores formed in neurons would allow entry of the membrane-impermeable sodium channel blocker QX-314 into nociceptors to silence pain during infection. QX-314 induced immediate and long-lasting blockade of pain caused by MRSA infection, significantly more than lidocaine or ibuprofen, two widely used clinical analgesic treatments.

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.