Aspartic Acid Residue 51 of SaeR Is Essential for Staphylococcus aureus Virulence

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

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

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-a), interferon gamma (IFN-c), 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-cproduction 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

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 ROSwas assessed during exposure to the clinically relevant S. aureus USA300 (strain LAC) and its isogenic mutant LACDsaeR/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 newevasion strategy used by S. aureus to diminish a vital neutrophil antimicrobial mechanism

Delayed neutrophil recruitment allows nascent <i>Staphylococcus aureus </i>biofilm formation and immune evasion

Biofilms that form on implanted medical devices cause recalcitrant infections. The early events enabling contaminating bacteria to evade immune clearance, before a mature biofilm is established, are poorly understood. Live imaging in vitro demonstrated that Staphylococcus aureus sparsely inoculated on an abiotic surface can go undiscovered by human neutrophils, grow, and form aggregates. Small (~50 μm(2)) aggregates of attached bacteria resisted killing by human neutrophils, resulting in neutrophil lysis and bacterial persistence. In vivo, neutrophil recruitment to a peritoneal implant was spatially heterogenous, with some bacterial aggregates remaining undiscovered by neutrophils after 24 hours. Intravital imaging in mouse skin revealed that attached S. aureus aggregates grew and remained undiscovered by neutrophils for up to three hours. These results suggest a model in which delayed recruitment of neutrophils to an abiotic implant presents a critical window in which bacteria establish a nascent biofilm and acquire tolerance to neutrophil killing