The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus.
ABSTRACT: Methicillin-resistant Staphylococcus aureus is problematic both in hospitals and in the community. Currently, we have limited understanding of mechanisms of innate immune evasion used by S. aureus. To that end, we created an isogenic deletion mutant in strain MW2 (USA400) of the saeR/S 2-component gene regulatory system and studied its role in mouse models of pathogenesis and during human neutrophil interaction. In this study, we demonstrate that saeR/S plays a distinct role in S. aureus pathogenesis and is vital for virulence of MW2 in a mouse model of sepsis. Moreover, deletion of saeR/S significantly impaired survival of MW2 in human blood and after neutrophil phagocytosis. Microarray analysis revealed that SaeR/S of MW2 influences expression of a wide variety of genes with diverse biological functions. These data provide new insight into how virulence is regulated in S. aureus and associates a specific staphylococcal gene-regulatory system with invasive staphylococcal disease.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is problematic both in hospitals and the community. Currently, we have limited understanding of mechanisms of innate immune evasion used by S. aureus. To that end, we created an isogenic deletion mutant in strain MW2 (USA400) of the saeR/S two-component gene regulatory system and studied its role in mouse models of pathogenesis and during human neutrophil interaction. In this study, we demonstrate saeR/S plays a distinct role in S. aureus pathogenesis and is vital for virulence of MW2 in a mouse model of sepsis. Moreover, deletion of saeR/S significantly impaired survival of MW2 in human blood and after neutrophil phagocytosis. Microarray analysis of genes influenced by saeR/S demonstrated SaeR/S of MW2 influences a wide variety of genes with diverse biological functions. These data shed new insight into how virulence is regulated in S. aureus and associates a specific staphylococcal gene-regulatory system with invasive staphylococcal disease. Wild type control vs mutant at two different growth phases
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is problematic both in hospitals and the community. Currently, we have limited understanding of mechanisms of innate immune evasion used by S. aureus. To that end, we created an isogenic deletion mutant in strain MW2 (USA400) of the saeR/S two-component gene regulatory system and studied its role in mouse models of pathogenesis and during human neutrophil interaction. In this study, we demonstrate saeR/S plays a distinct role in S. aureus pathogenesis and is vital for virulence of MW2 in a mouse model of sepsis. Moreover, deletion of saeR/S significantly impaired survival of MW2 in human blood and after neutrophil phagocytosis. Microarray analysis of genes influenced by saeR/S demonstrated SaeR/S of MW2 influences a wide variety of genes with diverse biological functions. These data shed new insight into how virulence is regulated in S. aureus and associates a specific staphylococcal gene-regulatory system with invasive staphylococcal disease. Overall design: Wild type control vs mutant at two different growth phases
Project description: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-?), interferon gamma (IFN-?), 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-? production 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.
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:Staphylococcus aureus is a common Gram-positive bacteria that is a major cause of human morbidity and mortality. The SaeR/S two-component sensory system of S. aureus is important for virulence gene transcription and pathogenesis. However, the influence of SaeR phosphorylation on virulence gene transcription is not clear. To determine the importance of potential SaeR phosphorylation sites for S. aureus virulence, we generated genomic alanine substitutions at conserved aspartic acid residues in the receiver domain of the SaeR response regulator in clinically significant S. aureus pulsed-field gel electrophoresis (PFGE) type USA300. Transcriptional analysis demonstrated a dramatic reduction in the transcript abundance of various toxins, adhesins, and immunomodulatory proteins for SaeR with an aspartic acid to alanine substitution at residue 51. These findings corresponded to a significant decrease in cytotoxicity against human erythrocytes and polymorphonuclear leukocytes, the ability to block human myeloperoxidase activity, and pathogenesis during murine soft-tissue infection. Analysis of SaeR sequences from over 8,000 draft S. aureus genomes revealed that aspartic acid residue 51 is 100% conserved. Collectively, these results demonstrate that aspartic acid residue 51 of SaeR is essential for S. aureus virulence and underscore a conserved target for novel antimicrobial strategies that treat infection caused by this pathogen.
Project description:This investigation examines the role of the SaeR/S 2-component system in USA300, a prominent circulating clone of community-associated methicillin-resistant Staphylococcus aureus. Using a saeR/S isogenic deletion mutant of USA300 (USA300DeltasaeR/S) in murine models of sepsis and soft-tissue infection revealed that this sensory system is critical to pathogenesis of USA300 during both superficial and invasive infection. Oligonucleotide microarray and real-time reverse-transcriptase polymerase chain reaction identified numerous extracellular virulence genes that are down-regulated in USA300DeltasaeR/S. Unexpectedly, an up-regulation of mecA and mecR1 corresponded to increased methicillin resistance in USA300DeltasaeR/S. 5'-RACE analysis defined transcript start sites for sbi, efb, mecA, lukS-PV, hlb, SAUSA300_1975, and hla, to underscore a conserved consensus sequence within promoter regions of genes under strong SaeR/S transcriptional regulation. Electrophoretic mobility shift assay experiments illustrated direct binding of SaeR(His) to promoter regions containing the conserved consensus sequence. Collectively, the findings of this investigation demonstrate that SaeR/S directly interacts with virulence gene promoters to significantly influence USA300 pathogenesis.
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:Staphylococcus aureus is a predominant cause of fatal pneumonia following influenza A virus (IAV) infection. Herein we investigate the influence of antecedent IAV infection on S. aureus virulence gene expression. Using a murine model, comparing the USA300 and USA300ΔsaeR/S strains, we demonstrate that S. aureus pathogenesis following IAV infection is SaeR/S dependent. Furthermore, we show that IAV modulates the lung environment to rapidly up-regulate S. aureus virulence factors containing the SaeR-binding domain. Data demonstrate that the pathogen response to IAV infection impacts host outcome and provides evidence that the ability of S. aureus to sense and respond to the lung environment determines severity of pneumonia.
Project description:The saeRS two-component regulatory system regulates transcription of multiple virulence factors in Staphylococcus aureus. In the present study, we demonstrated that the saePQRS region in Staphylococcus epidermidis is transcriptionally regulated in a temporal manner and is arranged in a manner similar to that previously described for S. aureus. Studies using a mouse foreign body infection model demonstrated that the virulence of strain 1457 and the virulence of a mutant, strain 1457 saeR, were statistically equivalent. However, histological analyses suggested that the polymorphonuclear neutrophil response at 2 days postinfection was significantly greater in 1457-infected mice than in 1457 saeR-infected mice, demonstrating that SaeR influences the early, acute phases of infection. Microarray analysis demonstrated that a saeR mutation affected the transcription of 65 genes (37 genes were upregulated and 28 genes were downregulated); in particular, 8 genes that facilitate growth under anaerobic conditions were downregulated in 1457 saeR. Analysis of growth under anaerobic conditions demonstrated that 1457 saeR had a decreased growth rate compared to 1457. Further metabolic experiments demonstrated that 1457 saeR had a reduced capacity to utilize nitrate as a terminal electron acceptor and exhibited increased production of lactic acid in comparison to 1457. These data suggest that in S. epidermidis SaeR functions to regulate the transition between aerobic growth and anaerobic growth. In addition, when grown anaerobically, 1457 saeR appeared to compensate for the redox imbalance created by the lack of electron transport-mediated oxidation of NADH to NAD+ by increasing lactate dehydrogenase activity and the subsequent oxidation of NADH.
Project description: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.