The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction.
ABSTRACT: Staphylococcus aureus (S. aureus) causes a range of diseases ranging from superficial skin and soft-tissue infections to invasive and life-threatening conditions (Klevens et al., 2007; Kobayashi et al., 2015). S. aureus utilizes the Sae sensory system to adapt to neutrophil challenge. Although the roles of the SaeR response regulator and its cognate sensor kinase SaeS have been demonstrated to be critical for surviving neutrophil interaction and for causing infection, the roles for the accessory proteins SaeP and SaeQ remain incompletely defined. To characterize the functional role of these proteins during innate immune interaction, we generated isogenic deletion mutants lacking these accessory genes in USA300 (USA300?saeP and USA300?saeQ). S. aureus survival was increased following phagocytosis of USA300?saeP compared to USA300 by neutrophils. Additionally, secreted extracellular proteins produced by USA300?saeP cells caused significantly more plasma membrane damage to human neutrophils than extracellular proteins produced by USA300 cells. Deletion of saeQ resulted in a similar phenotype, but effects did not reach significance during neutrophil interaction. The enhanced cytotoxicity of USA300?saeP cells toward human neutrophils correlated with an increased expression of bi-component leukocidins known to target these immune cells. A saeP and saeQ double mutant (USA300?saePQ) showed a significant increase in survival following neutrophil phagocytosis that was comparable to the USA300?saeP single mutant and increased the virulence of USA300 during murine bacteremia. These data provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeP and saeQ together impact pathogenesis in vivo.
Project description:Background:The ability of Staphylococcus aureus to evade killing by human neutrophils significantly contributes to disease progression. In this study, we characterize an influential role for the S. aureus SaeR/S 2-component gene regulatory system in suppressing monocyte production of tumor necrosis factor alpha (TNF-?) to subsequently influence human neutrophil priming. Methods:Using flow cytometry and TNF-? specific enzyme-linked immunosorbent assays we identify the primary cellular source of TNF-? in human blood and in purified peripheral blood mononuclear cells (PBMCs) during interaction with USA300 and an isogenic saeR/S deletion mutant (USA300?saeR/S). Assays with conditioned media from USA300 and USA300?saeR/S exposed PBMCs were used to investigate priming on neutrophil bactericidal activity. Results:TNF-? production from monocytes was significantly reduced following challenge with USA300 compared to USA300?saeR/S. We observed that priming of neutrophils using conditioned medium from peripheral blood mononuclear cells stimulated with USA300?saeR/S significantly increased neutrophil bactericidal activity against USA300 relative to unprimed neutrophils and neutrophils primed with USA300 conditioned medium. The increased neutrophil bactericidal activity was associated with enhanced reactive oxygen species production that was significantly influenced by elevated TNF-? concentrations. Conclusions:Our findings identify an immune evasion strategy used by S. aureus to impede neutrophil priming and subsequent bactericidal activity.
Project description:Cigarette smoke (CS) predisposes exposed individuals to respiratory infections not only by suppressing immune response but also by enhancing the virulence of pathogenic bacteria. As per our observations, in methicillin-resistant Staphylococcus aureus strain USA300, CS extract (CSE) potentiates biofilm formation via the down-regulation of quorum-sensing regulon accessory gene regulator. Because accessory gene regulator is a global regulator of the staphylococcal virulome, in the present study we sought to identify the effects of CS exposure on staphylococcal gene expression using RNAseq. Comparative analysis of RNAseq profiles revealed the up-regulation of important virulence genes encoding surface adhesins (fibronectin- and fibrinogen-binding proteins A and B and clumping factor B) and proteins involved in immune evasion, such as staphylocoagulase, staphylococcal protein A, and nuclease. In concurrence with the RNAseq data, we observed: (1) significant up-regulation of the ability of CSE-exposed USA300 to evade phagocytosis by macrophages and neutrophils, a known function of staphylococcal protein A; and (2) twofold higher (P?<?0.001) number of CSE-exposed USA300 escaping neutrophil extracellular trap-mediated killing by neutrophils as a result of CS-mediated induction of nuclease. Importantly, in three different mouse strains, C57BL6/J, Balb/C, and A/J, we observed significantly higher pulmonary bacterial burden in animals infected with CSE-exposed USA300 as compared with medium-exposed control USA300. Taken together, these observations indicate that bioactive chemicals in CS induce hypervirulence by augmenting the ability of USA300 to evade bactericidal functions of leukocytes, such as phagocytosis and neutrophil extracellular trap-mediated killing.
Project description:Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are incompletely defined, but presumably include evasion of killing by human polymorphonuclear leukocytes (PMNs or neutrophils). To better understand this phenomenon, we investigated the basis of rapid PMN lysis after phagocytosis of USA300, a prominent CA-MRSA strain. Survival of USA300 clinical isolates after phagocytosis ultimately resulted in neutrophil lysis. PMNs containing ingested USA300 underwent morphological changes consistent with apoptosis, but lysed rapidly thereafter (within 6 h), whereas cells undergoing FAS-mediated apoptosis or phagocytosis-induced cell death remained intact. Phagosome membranes remained intact until the point of PMN destruction, suggesting lysis was not caused by escape of S. aureus from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated S. aureus and healthcare-associated MRSA strains revealed changes unique to community-associated S. aureus strains, such as upregulation of transcripts involved in regulation of calcium homeostasis. Collectively, the data suggest that neutrophil destruction after phagocytosis of USA300 is in part a form of programmed necrosis rather than direct lysis by S. aureus pore-forming toxins. We propose that the ability of CA-MRSA strains to induce programmed necrosis of neutrophils is a component of enhanced virulence.
Project description: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 LAC?saeR/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 LAC?saeR/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.
Project description:In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate the sensor kinase's phosphatase activity. Efficient activation of the phosphatase activity required the presence of both SaeP and SaeQ. When SaeP and SaeQ were ectopically expressed, the expression of coagulase, a sae target with low affinity for phosphorylated SaeR, was greatly reduced, while the expression of alpha-haemolysin, a sae target with high affinity for phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by the SaeRS TCS, these results suggest that the SaeRS TCS returns to the pre-activation state by a negative feedback mechanism.
Project description:In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulator is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a ternary complex with the sensor kinase SaeS and activate the sensor kinase’s phosphatase activity. Efficient activation of the phosphatase activity of SaeS required the presence of both SaeP and SaeQ. When SaeP and SaeQ were expressed, the expression of coagulase, a sae target with low affinity to phosphorylated SaeR, was greatly reduced, while the expression of alpha-hemolysin, a sae target with high affinity to phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by SaeRS TCS, these results suggest that the SaeRS TCS returns to pre-activation state by a negative feedback mechanism. To examine the global effect of SaePQ on sae target gene expression, we treated the wild type strain of USA300-P23 and its P1 promoter mutant with HNP-1 and analyzed the transcription of sae target genes by microarray assays. WT and P1 cells were compared against a each other at the same time point and against a T=0 reference.
Project description: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 ?-defensin. Furthermore, mouse neutrophils promoted transcription of saeR/S targets despite lacking ?-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 USA300?saeR/S and USA300?agr 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.
Project description:BACKGROUND:Previously we have shown that volatile anesthetic isoflurane attenuated neutrophil recruitment and phagocytosis in mouse sepsis and skin inflammation models. The objectives of this study were to test ex vivo function of neutrophils in patients who underwent cardiac catheterization under volatile anesthesia versus intravenous anesthesia (IA), and also to assess the effect of anesthesia on surgical site infections (SSIs) using mouse model to understand the clinical relevance of anesthesia-induced immunomodulation. METHODS:Whole blood from patients who underwent cardiac catheterization procedures either by volatile anesthesia or IA was collected and subjected to phagocytosis assay and a lipopolysaccharide-induced tumor necrosis factor-? assay. Mouse SSI with Staphylococcus aureus USA300 was created, and the effect of isoflurane and propofol exposure (short or long exposure) on bacterial loads was tested. RESULTS:Neutrophil phagocytosis was significantly attenuated after the induction of volatile anesthesia in patients, but not by IA. Monocyte phagocytosis was not affected by the anesthesia regimen. Bacterial loads following SSIs were significantly higher in mice receiving long, but not short, isoflurane exposure. Propofol exposure did not affect bacterial loads. DISCUSSION:Neutrophil phagocytosis can be affected by the type of anesthesia, and preclinical model of SSIs showed potential clinical relevance. The effects of anesthesia regimen on SSIs in patients needs to be studied extensively in the future.
Project description:Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LAC?hlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LAC?hlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LAC?hlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.
Project description:Recurrent Staphylococcus aureus skin and soft tissue infections (SSTIs) are common despite detectable antibody responses, leading to the belief that the immune response elicited by these infections is not protective. We recently reported that S. aureus USA300 SSTI elicits antibodies that protect against recurrent SSTI in BALB/c but not C57BL/6 mice, and in this study, we aimed to uncover the specificity of the protective antibodies. Using a proteomic approach, we found that S. aureus SSTI elicited broad polyclonal antibody responses in both BALB/c and C57BL/6 mice and identified 10 S. aureus antigens against which antibody levels were significantly higher in immune BALB/c serum. Four of the 10 antigens identified are regulated by the saeRS operon, suggesting a dominant role for saeRS in protection. Indeed, infection with USA300?sae failed to protect against secondary SSTI with USA300, despite eliciting a strong polyclonal antibody response against antigens whose expression is not regulated by saeRS. Moreover, the antibody repertoire after infection with USA300?sae lacked antibodies specific for 10 saeRS-regulated antigens, suggesting that all or a subset of these antigens are necessary to elicit protective immunity. Infection with USA300?hla elicited modest protection against secondary SSTI, and complementation of USA300?sae with hla restored protection but incompletely. Together, these findings support a role for both Hla and other saeRS-regulated antigens in eliciting protection and suggest that host differences in immune responses to saeRS-regulated antigens may determine whether S. aureus infection elicits protective or nonprotective immunity against recurrent infection.