Nuclear factor kappa B-dependent persistence of Salmonella Typhi and Paratyphi in human macrophages.

Salmonella serovars Typhi and Paratyphi cause a prolonged illness known as enteric fever, whereas other serovars cause acute gastroenteritis. Mechanisms responsible for the divergent clinical manifestations of nontyphoidal and enteric fever Salmonella infections have remained elusive. Here, we show that S. Typhi and S. Paratyphi A can persist within human macrophages, whereas S. Typhimurium rapidly induces apoptotic macrophage cell death that is dependent on Salmonella pathogenicity island 2 (SPI2). S. Typhi and S. Paratyphi A lack 12 specific SPI2 effectors with pro-apoptotic functions, including nine that target nuclear factor κB (NF-κB). Pharmacologic inhibition of NF-κB or heterologous expression of the SPI2 effectors GogA or GtgA restores apoptosis of S. Typhi-infected macrophages. In addition, the absence of the SPI2 effector SarA results in deficient signal transducer and activator of transcription 1 (STAT1) activation and interleukin 12 production, leading to impaired TH1 responses in macrophages and humanized mice. The absence of specific nontyphoidal SPI2 effectors may allow S. Typhi and S. Paratyphi A to cause chronic infections. IMPORTANCE: Salmonella enterica is a common cause of gastrointestinal infections worldwide. The serovars Salmonella Typhi and Salmonella Paratyphi A cause a distinctive systemic illness called enteric fever, whose pathogenesis is incompletely understood. Here, we show that enteric fever Salmonella serovars lack 12 specific virulence factors possessed by nontyphoidal Salmonella serovars, which allow the enteric fever serovars to persist within human macrophages. We propose that this fundamental difference in the interaction of Salmonella with human macrophages is responsible for the chronicity of typhoid and paratyphoid fever, suggesting that targeting the nuclear factor κB (NF-κB) complex responsible for macrophage survival could facilitate the clearance of persistent bacterial infections.

Scratching the Surface Takes a Toll: Immune Recognition of Viral Proteins by Surface Toll-like Receptors.

Early innate viral recognition by the host is critical for the rapid response and subsequent clearance of an infection. Innate immune cells patrol sites of infection to detect and respond to invading microorganisms including viruses. Surface Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) that can be activated by viruses even before the host cell becomes infected. However, the early activation of surface TLRs by viruses can lead to viral clearance by the host or promote pathogenesis. Thus, a plethora of research has attempted to identify specific viral ligands that bind to surface TLRs and mediate progression of viral infection. Herein, we will discuss the past two decades of research that have identified specific viral proteins recognized by cell surface-associated TLRs, how these viral proteins and host surface TLR interactions affect the host inflammatory response and outcome of infection, and address why controversy remains regarding host surface TLR recognition of viral proteins.

The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction.

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 (USA300ΔsaeP and USA300ΔsaeQ). S. aureus survival was increased following phagocytosis of USA300ΔsaeP compared to USA300 by neutrophils. Additionally, secreted extracellular proteins produced by USA300ΔsaeP 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 USA300ΔsaeP 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 (USA300ΔsaePQ) showed a significant increase in survival following neutrophil phagocytosis that was comparable to the USA300ΔsaeP 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.

Quantifying the Cytotoxicity of Staphyloccus aureus Against Human Polymorphonuclear Leukocytes.

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Staphylococcus aureus SaeR/S-Regulated Factors Decrease Monocyte-Derived Tumor Necrosis Factor-α to Reduce Neutrophil Bactericidal Activity.

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-α) to subsequently influence human neutrophil priming. Methods: Using flow cytometry and TNF-α specific enzyme-linked immunosorbent assays we identify the primary cellular source of TNF-α 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-α 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-α concentrations. Conclusions: Our findings identify an immune evasion strategy used by S. aureus to impede neutrophil priming and subsequent bactericidal activity.

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.

Immune evasion by a staphylococcal inhibitor of myeloperoxidase.

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

Epic Immune Battles of History: Neutrophils vs. Staphylococcus aureus.

Neutrophils are the most abundant leukocytes in human blood and the first line of defense after bacteria have breached the epithelial barriers. After migration to a site of infection, neutrophils engage and expose invading microorganisms to antimicrobial peptides and proteins, as well as reactive oxygen species, as part of their bactericidal arsenal. Ideally, neutrophils ingest bacteria to prevent damage to surrounding cells and tissues, kill invading microorganisms with antimicrobial mechanisms, undergo programmed cell death to minimize inflammation, and are cleared away by macrophages. Staphylococcus aureus (S. aureus) is a prevalent Gram-positive bacterium that is a common commensal and causes a wide range of diseases from skin infections to endocarditis. Since its discovery, S. aureus has been a formidable neutrophil foe that has challenged the efficacy of this professional assassin. Indeed, proper clearance of S. aureus by neutrophils is essential to positive infection outcome, and S. aureus has developed mechanisms to evade neutrophil killing. Herein, we will review mechanisms used by S. aureus to modulate and evade neutrophil bactericidal mechanisms including priming, activation, chemotaxis, production of reactive oxygen species, and resolution of infection. We will also highlight how S. aureus uses sensory/regulatory systems to tailor production of virulence factors specifically to the triggering signal, e.g., neutrophils and defensins. To conclude, we will provide an overview of therapeutic approaches that may potentially enhance neutrophil antimicrobial functions.

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

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.

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.