Kyler B. Pallister

Staphylococcus aureus Nuclease Is an SaeRS-Dependent Virulence Factor

ABSTRACT Several prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies of Staphylococcus aureus Nuc attributed its regulation to the agr quorum-sensing system. However, recent microarray data have indicated that nuc is under the control of the SaeRS two-component system, which is a major regulator of S. aureus virulence determinants. Here we report that the nuc gene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to the agr system. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representative S. aureus isolates. Moreover, with community-associated methicillin-resistant S. aureus (CA MRSA) in a mouse model of peritonitis, we observed in vivo expression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important for in vivo survival, confirming the enzymes role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an important S. aureus virulence factor and part of the SaeRS regulon.

Alpha-Toxin Induces Programmed Cell Death of Human T cells, B cells, and Monocytes during USA300 Infection

This investigation examines the influence of alpha-toxin (Hla) during USA300 infection of human leukocytes. Survival of an USA300 isogenic deletion mutant of hla (USA300Δhla) in human blood was comparable to the parental wild-type strain and polymorphonuclear leukocyte (PMN) plasma membrane permeability caused by USA300 did not require Hla. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) following infection by USA300, USA300Δhla, and USA300Δhla transformed with a plasmid over-expressing Hla (USA300Δhla Comp) demonstrated this toxin plays a significant role inducing plasma membrane permeability of CD14+, CD3+, and CD19+ PBMCs. Rapid plasma membrane permeability independent of Hla was observed for PMNs, CD14+ and CD19+ PBMCs following intoxication with USA300 supernatant while the majority of CD3+ PBMC plasma membrane permeability induced by USA300 required Hla. Addition of recombinant Hla to USA300Δhla supernatant rescued CD3+ and CD19+ PBMC plasma membrane permeability generated by USA300 supernatant. An observed delay in plasma membrane permeability caused by Hla in conjunction with Annexin V binding and ApoBrdU Tunel assays examining PBMCs intoxicated with recombinant Hla or infected with USA300, USA300Δhla, USA300Δhla Comp, and USA300ΔsaeR/S suggest Hla induces programmed cell death of monocytes, B cells, and T cells that results in plasma membrane permeability. Together these findings underscore the importance of Hla during S. aureus infection of human tissue and specifically demonstrate Hla activity during USA300 infection triggers programmed cell death of human monocytes, T cells and B cells that leads to plasma membrane permeability.

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli

Significance Staphylococcus aureus is a ubiquitous pathogen that causes disease in a variety of tissues. Our studies provide insight into how this pathogen uses the SaeS sensor kinase to recognize innate immunity signals and induce a transcriptional response tailored for its environment. Our results demonstrate that the specificity of these responses is determined by individual amino acids predicted to be in an extracellular domain. These amino acids include aromatic anchors and a methionine residue essential for activation of target gene transcription and virulence. Our findings provide a putative mechanism for the ability of bacterial sensory systems to integrate and diversify the responses to host stimuli. They underscore the exquisite nature of bacterial signaling and the complexity of host-pathogen interactions. Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the histidine kinase SaeS recognizes specific host stimuli is unknown. After mutagenizing the predicted extracellular loop of SaeS, we discovered one methionine residue (M31) was essential for the ability of S. aureus to transcribe sae target genes, including hla, lukAB/lukGH, and hlgA. This single M31A mutation also significantly reduced cytotoxicity in human neutrophils to levels observed in cells following interaction with ΔsaeS. Another important discovery was that mutation of two aromatic anchor residues (W32A and F33A) disrupted the normal basal signaling of SaeS in the absence of inducing signals, yet both mutant kinases had appropriate activation of effector genes following exposure to neutrophils. Although the transcriptional profile of aromatic mutation W32A was consistent with that of WT in response to human α-defensin 1, mutant kinase F33A did not properly transcribe the γ-toxin genes in response to this stimulus. Taken together, our results provide molecular evidence for how SaeS recognizes host signals and triggers activation of select virulence factors to facilitate evasion of innate immunity. These findings have important implications for signal transduction in prokaryotes and eukaryotes due to conservation of aromatic anchor residues across both of these domains and the important role they play in sensor protein structure and function.

The SaeR/S Gene Regulatory System Induces a Pro-Inflammatory Cytokine Response during Staphylococcus aureus Infection

Community-associated methicillin-resistant Staphylococcus aureus accounts for a large portion of the increased staphylococcal disease incidence and can cause illness ranging from mild skin infections to rapidly fatal sepsis syndromes. Currently, we have limited understanding of S. aureus-derived mechanisms contributing to bacterial pathogenesis and host inflammation during staphylococcal disease. Herein, we characterize an influential role for the saeR/S two-component gene regulatory system in mediating cytokine induction using mouse models of S. aureus pathogenesis. Invasive S. aureus infection induced the production of localized and systemic pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin (IL)-6 and IL-2. In contrast, mice infected with an isogenic saeR/S deletion mutant demonstrated significantly reduced pro-inflammatory cytokine levels. Additionally, secreted factors influenced by saeR/S elicited pro-inflammatory cytokines in human blood ex vivo. Our study further demonstrated robust saeR/S-mediated IFN-γproduction during both invasive and subcutaneous skin infections. Results also indicated a critical role for saeR/S in promoting bacterial survival and enhancing host mortality during S. aureus peritonitis. Taken together, this study provides insight into specific mechanisms used by S. aureus during staphylococcal disease and characterizes a relationship between a bacterial global regulator of virulence and the production of pro-inflammatory mediators.

The Role of Innate Immunity in Promoting SaeR/S-Mediated Virulence in Staphylococcus aureus

The ability of Staphylococcus aureus to infect tissues is dependent on precise control of virulence through gene-regulatory systems. While the SaeR/S two-component system has been shown to be a major regulator of S. aureus virulence, the influence of the host environment on SaeR/S-regulated genes (saeR/S targets) remains incompletely defined. Using QuantiGene 2.0 transcriptional assays, we examined expression of genes with the SaeR binding site in USA300 exposed to human and mouse neutrophils and host-derived peptides and during subcutaneous skin infection. We found that only some of the saeR/S targets, as opposed to the entire SaeR/S virulon, were activated within 5 and 10 min of interacting with human neutrophils as well as α-defensin. Furthermore, mouse neutrophils promoted transcription of saeR/S targets despite lacking α-defensin, and the murine skin environment elicited a distinctive expression profile of saeR/S targets. These findings indicate that saeR/S-mediated transcription is unique to and dependent on specific host stimuli. By using isogenic USA300ΔsaeR/S and USA300Δagr knockout strains, we also determined that SaeR/S is the major regulator of virulence factors, while Agr, a quorum-sensing two-component system, has moderate influence on transcription of the saeR/S targets under the tested physiological conditions.

Virus-Like Particle-Induced Protection Against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function

The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms.

Increased Pilus Production Conferred by a Naturally Occurring Mutation Alters Host-Pathogen Interaction in Favor of Carriage in Streptococcus pyogenes

ABSTRACT Studies of the human pathogen group A Streptococcus (GAS) define the carrier phenotype to be an increased ability to adhere to and persist on epithelial surfaces and a decreased ability to cause disease. We tested the hypothesis that a single amino acid change (Arg135Gly) in a highly conserved sensor kinase (LiaS) of a poorly defined GAS regulatory system contributes to a carrier phenotype through increased pilus production. When introduced into an emm serotype-matched invasive strain, the carrier allele (the gene encoding the LiaS protein with an arginine-to-glycine change at position 135 [liaSR135G]) recapitulated a carrier phenotype defined by an increased ability to adhere to mucosal surfaces and a decreased ability to cause disease. Gene transcript analyses revealed that the liaS mutation significantly altered transcription of the genes encoding pilus in the presence of bacitracin. Elimination of pilus production in the isogenic carrier mutant decreased its ability to colonize the mouse nasopharynx and to adhere to and be internalized by cultured human epithelial cells and restored the virulence phenotype in a mouse model of necrotizing fasciitis. We also observed significantly reduced survival of the isogenic carrier mutant compared to that of the parental invasive strain after exposure to human neutrophils. Elimination of pilus in the isogenic carrier mutant increased the level of survival after exposure to human neutrophils to that for the parental invasive strain. Together, our data demonstrate that the carrier mutation (liaSR135G) affects pilus expression. Our data suggest new mechanisms of pilus gene regulation in GAS and that the invasiveness associated with pilus gene regulation in GAS differs from the enhanced invasiveness associated with increased pilus production in other bacterial pathogens.

The impact of α-toxin on host cell plasma membrane permeability and cytokine expression during human blood infection by CA-MRSA USA300

This investigation examines the influence of α‐toxin (Hla) expression by CA‐MRSA on host immune cell integrity and cytokine expression during infection of human blood. Flow cytometry analysis of human blood infected by Staphylococcus aureus PFGE type USA300 or a USA300Δhla demonstrated that Hla expression significantly increased plasma membrane permeability of human CD14+ monocytes. The increased susceptibility of human CD14+ monocytes to Hla toxicity paralleled the high cell‐surface expression on these cell types of ADAM10. USA300 rapidly associated with PMNs and monocytes but not T cells following inoculation of human blood. Transcription analysis indicated a strong up‐regulation of proinflammatory cytokine transcription following infection of human blood by USA300 and USA300Δhla. CBAs and ELISAs determined that IL‐6, IL‐10, TNF‐α, IFN‐γ, IL‐1β, IL‐8, and IL‐4 are significantly up‐regulated during the initial phases of human blood infection by USA300 relative to mock‐infected blood but failed to distinguish any significant differences in secreted cytokine protein concentrations during infection by USA300Δhla relative to USA300. Collectively, these findings demonstrate that expression of Hla by USA300 has a significant impact on human CD14+ monocyte plasma membrane integrity but is not exclusively responsible for the proinflammatory cytokine profile induced by USA300 during the initial stages of human blood infection.

Staphylococcus aureus Inhibits Neutrophil-derived IL-8 to Promote Cell Death

While Staphylococcus aureus accelerates human neutrophil cell death, the underlying host- and pathogen-derived mechanisms remain incompletely defined. Previous studies demonstrated that the S. aureus SaeR/S sensory system is essential for pathogen survival following neutrophil phagocytosis. Herein, we demonstrate that the SaeR/S system promoted accelerated cell death, suppressed phosphorylation of nuclear factor-κB, and reduced interleukin-8 (IL-8) production in human neutrophils. Treatment of neutrophils with recombinant IL-8 significantly reduced bacterial burden and apoptosis. Our findings demonstrate a mechanism by which S. aureus suppresses the early neutrophil-derived IL-8 response to disrupt cell fate and promote disease.

Staphylococcus aureus SaeR/S-regulated factors reduce human neutrophil reactive oxygen species production

Neutrophils are the first line of defense after a pathogen has breached the epithelial barriers, and unimpaired neutrophil functions are essential to clear infections. Staphylococcus aureus is a prevalent human pathogen that is able to withstand neutrophil killing, yet the mechanisms used by S. aureus to inhibit neutrophil clearance remain incompletely defined. The production of reactive oxygen species (ROS) is a vital neutrophil antimicrobial mechanism. Herein, we test the hypothesis that S. aureus uses the SaeR/S two‐component gene regulatory system to produce virulence factors that reduce neutrophil ROS production. With the use of ROS probes, the temporal and overall production of neutrophil ROS was assessed during exposure to the clinically relevant S. aureus USA300 (strain LAC) and its isogenic mutant LACΔsaeR/S. Our results demonstrated that SaeR/S‐regulated factors do not inhibit neutrophil superoxide (O2−) production. However, subsequent neutrophil ROS production was significantly reduced during exposure to LAC compared with LACΔsaeR/S. In addition, neutrophil H2O2 production was reduced significantly by SaeR/S‐regulated factors by a mechanism independent of catalase. Consequently, the reduction in neutrophil H2O2 resulted in decreased production of the highly antimicrobial agent hypochlorous acid/hypochlorite anion (HOCl/−OCl). These findings suggest a new evasion strategy used by S. aureus to diminish a vital neutrophil antimicrobial mechanism.