Faculty Bibliography

This investigation examines the influence of alpha-toxin (Hla) during USA300 infection of human leukocytes. Survival of an USA300 isogenic deletion mutant of hla (USA300Deltahla) 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, USA300Deltahla, and USA300Deltahla transformed with a plasmid over-expressing Hla (USA300Deltahla 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 USA300Deltahla 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, USA300Deltahla, USA300Deltahla Comp, and USA300DeltasaeR/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.

Microglia are important innate immune effectors against invading CNS pathogens, such as Staphylococcus aureus (S. aureus), a common etiological agent of brain abscesses typified by widespread inflammation and necrosis. The NLRP3 inflammasome is a protein complex involved in IL-1beta and IL-18 processing following exposure to both pathogen- and danger-associated molecular patterns. Although previous studies from our laboratory have established that IL-1beta is a major cytokine product of S. aureus-activated microglia and is pivotal for eliciting protective anti-bacterial immunity during brain abscess development, the molecular machinery responsible for cytokine release remains to be determined. Therefore, the functional role of the NLRP3 inflammasome and its adaptor protein apoptosis-associated speck-like protein (ASC) in eliciting IL-1beta and IL-18 release was examined in primary microglia. Interestingly, we found that IL-1beta, but not IL-18 production, was significantly attenuated in both NLRP3 and ASC knockout (KO) microglia following exposure to live S. aureus. NLRP3 inflammasome activation was partially dependent on autocrine/paracrine ATP release and alpha- and gamma-hemolysins produced by live bacteria. A cathepsin B inhibitor attenuated IL-beta release from NLRP3 and ASC KO microglia, demonstrating the existence of alternative inflammasome-independent mechanisms for IL-1beta processing. In contrast, microglial IL-18 secretion occurred independently of cathepsin B and inflammasome action. Collectively, these results demonstrate that microglial IL-1beta processing is regulated by multiple pathways and diverges from mechanisms utilized for IL-18 cleavage. Understanding the molecular events that regulate IL-1beta production is important for modulating this potent proinflammatory cytokine during CNS disease

Staphylococcus aureus overproduces a subset of immunomodulatory proteins known as the staphylococcal superantigen-like proteins (Ssls) under conditions of pore-mediated membrane stress. In this study we demonstrate that overproduction of Ssls during membrane stress is due to the impaired activation of the two-component module of the quorum-sensing accessory gene regulator (Agr) system. Agr-dependent repression of ssl expression is indirect and mediated by the transcription factor repressor of toxins (Rot). Surprisingly, we observed that Rot directly interacts with and activates the ssl promoters. The role of Agr and Rot as regulators of ssl expression was observed across several clinically relevant strains, suggesting that overproduction of immunomodulatory proteins benefits agr-defective strains. In support of this notion, we demonstrate that Ssls contribute to the residual virulence of S. aureus lacking agr in a murine model of systemic infection. Altogether, these results suggest that S. aureus compensates for the inactivation of Agr by producing immunomodulatory exoproteins that could protect the bacterium from host-mediated clearance

Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin-resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis

Iron is required for bacterial proliferation, and Staphylococcus aureus steals this metal from host hemoglobin during invasive infections. This process involves hemoglobin binding to the cell wall of S. aureus, heme extraction, passage through the cell envelope, and degradation to release free iron. Herein, we demonstrate an enhanced ability of S. aureus to bind hemoglobin derived from humans as compared to other mammals. Increased specificity for human hemoglobin (hHb) translates into an improved ability to acquire iron and is entirely dependent on the staphylococcal hemoglobin receptor IsdB. This feature affects host-pathogen interaction as demonstrated by the increased susceptibility of hHb-expressing mice to systemic staphylococcal infection. Interestingly, enhanced utilization of human hemoglobin is not a uniform property of all bacterial pathogens. These results suggest a step in the evolution of S. aureus to better colonize the human host and establish hHb-expressing mice as a model of S. aureus pathogenesis.

The tremendous success of Staphylococcus aureus as a pathogen is due to the controlled expression of a diverse array of virulence factors. The effects of host environments on the expression of virulence factors and the mechanisms by which S. aureus adapts to colonize distinct host tissues are largely unknown. Vertebrates have evolved to sequester nutrient iron from invading bacteria, and iron availability is a signal that alerts pathogenic microorganisms when they enter the hostile host environment. Consistent with this, we report here that S. aureus senses alterations in the iron status via the ferric uptake regulator (Fur) and alters the abundance of a large number of virulence factors. These Fur-mediated changes protect S. aureus against killing by neutrophils, and Fur is required for full staphylococcal virulence in a murine model of infection. A potential mechanistic explanation for the impact of Fur on virulence is provided by the observation that Fur coordinates the reciprocal expression of cytolysins and a subset of immunomodulatory proteins. More specifically, S. aureus lacking fur exhibits decreased expression of immunomodulatory proteins and increased expression of cytolysins. These findings reveal that Fur is involved in initiating a regulatory program that organizes the expression of virulence factors during the pathogenesis of S. aureus pneumonia

The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating DeltahrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host

Helicobacter pylori genomes contain about 30 hop genes that encode outer membrane proteins. Helicobacter pylori hopQ alleles exhibit a high level of genetic diversity, and two families of hopQ alleles have been described. Type I hopQ alleles are found more commonly in cag-positive H. pylori strains from patients with peptic ulcer disease than in cag-negative strains from patients without ulcer disease. In this study, we mutated hopQ in four H. pylori strains that each contained a type I hopQ allele, and then analyzed interactions of the wild-type and hopQ mutant strains with AGS cells. In comparison with the wild-type strains, two of the hopQ mutant strains exhibited increased adherence to AGS cells and two hopQ mutants did not exhibit any detectable differences in adherence. Higher levels of tyrosine-phosphorylated CagA were detected when AGS cells were cocultured with a hyperadherent hopQ mutant strain than when cocultured with the corresponding wild-type strain. These data indicate that in some strains of H. pylori, the HopQ protein can attenuate bacterial adherence to gastric epithelial cells

Helicobacter pylori VacA is a secreted pore-forming toxin that is comprised of two domains, designated p33 and p55. The p55 domain has an important role in the binding of VacA to eukaryotic cell surfaces. A total of 111 residues at the amino terminus of p55 (residues 312 to 422) are essential for the intracellular activity of VacA, which suggests that this region may constitute a subdomain with an activity distinct from cell binding. To investigate the properties of this subdomain, a small deletion mutation (targeting aspartic acid 346 and glycine 347) was introduced into the H. pylori chromosomal vacA gene. Similar to wild-type VacA, the VacA Delta346-347 mutant protein was proteolytically processed, secreted, and bound to eukaryotic cells. However, VacA Delta346-347 did not cause cell vacuolation or membrane depolarization, and it was impaired in the ability to assemble into large water-soluble oligomeric structures. Interestingly, VacA Delta346-347 was able to physically interact with wild-type VacA to form mixed oligomeric complexes, and VacA Delta346-347 inhibited wild-type vacuolating activity in a dominant-negative manner. These data indicate that the assembly of functional oligomeric VacA complexes is dependent on specific sequences, including amino acids 346 and 347, within the p55 amino-terminal subdomain

During systemic infection, Staphylococcus aureus acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme, S. aureus up-regulates the expression of the heme-regulated transporter, HrtAB. Strains lacking hrtAB exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that hrtAB encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn(2+)/Mg(2+)-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of S. aureus Delta hrtA, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of hrtA mutants exposed to heme