Phospholipid scramblase 1 mediates type i interferon-induced protection against staphylococcal α-toxin

The opportunistic gram-positive pathogen Staphylococcus aureus is a leading cause of pneumonia and?sepsis. Staphylococcal α-toxin, a prototypical pore-forming toxin, is a major virulence factor of S.?aureus clinical isolates, and lung epithelial cells are highly sensitive to α-toxin's cytolytic activity. Type I interferon (IFN) signaling activated in response to S.?aureus increases pulmonary cell resistance to α-toxin, but the underlying mechanisms are uncharacterized. We show that IFNα protects human lung epithelial cells from α-toxin-induced intracellular ATP depletion and cell death by reducing extracellular ATP leakage. This effect depends on protein palmitoylation and induction of phospholipid scramblase 1 (PLSCR1). IFNα-induced PLSCR1 associates with the cytoskeleton after exposure to α-toxin, and cellular depletion of PLSCR1 negates IFN-induced protection from α-toxin. PLSCR1-deficient mice display enhanced sensitivity to inhaled α-toxin and an α-toxin-producing S.?aureus strain. These results uncover PLSCR1 activity as part of an innate protective mechanism to a bacterial pore-forming toxin.     

Isoalantolactone protects against Staphylococcus aureus pneumonia

Staphylococcus aureus is a versatile pathogen that can cause life-threatening infections. The growing emergence of methicillin-resistant S.?aureus strains and a decrease in the discovery of new antibiotics warrant the search for new therapeutic targets to combat infections. Staphylococcus aureus produces many extracellular virulence factors that contribute to its pathogenicity. Therefore, targeting bacterial virulence as an alternative strategy to the development of new antimicrobials has gained great interest. α-Toxin is a 33.2-kDa, water-soluble, pore-forming toxin that is secreted by most S.?aureus strains. α-Toxin is essential for the pathogenesis of pneumonia, as strains lacking α-toxin display a profound defect in virulence. In this report, we demonstrate that isoalantolactone (IAL), a naturally occurring compound found in Inula helenium (Compositae), has no anti-S.?aureus activity as per MIC evaluation in vitro. However, IAL can markedly inhibit the expression of α-toxin in S.?aureus at very low concentrations. Furthermore, the in vivo data indicate that treatment with IAL protects mice from S.?aureus pneumonia.     

Expression of δ-toxin by Staphylococcus aureus mediates escape from phago-endosomes of human epithelial and endothelial cells in the presence of β-toxin

Staphylococcus aureus is able to invade non-professional phagocytes by interaction of staphylococcal adhesins with extracellular proteins of mammalian cells and eventually resides in acidified phago-endosomes. Some staphylococcal strains have been shown to subsequently escape from this compartment. A functional agr quorum-sensing system is needed for phagosomal escape. However, the nature of this agr dependency as well as the toxins involved in disruption of the phagosomal membrane are unknown. Using a novel technique to detect vesicular escape of S. aureus, we identified staphylococcal virulence factors involved in phagosomal escape. Here we show that a synergistic activity of the cytolytic peptide, staphylococcal δ-toxin and the sphingomyelinase β-toxin enable the phagosomal escape of staphylococci in human epithelial as well as in endothelial cells. The agr dependency of this process can be directly explained by the location of the structural gene for δ-toxin within the agr effector RNAIII.     

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host-pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen.     

Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.     

Antimicrobial activity of community-associated methicillin-resistant Staphylococcus aureus is caused by phenol-soluble modulin derivatives

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are causing an ongoing pandemic of mostly skin and soft tissue infections. The success of CA-MRSA as pathogens is due to a combination of antibiotic resistance with high virulence. In addition, it has been speculated that CA-MRSA strains such as the epidemic U.S. clone USA300 have increased capacity to colonize human epithelia, owing to bacteriocin-based bacterial interference. We here analyzed the molecular basis of antimicrobial activity detected in S. aureus strains, including those of the USA300 lineage. In contrast to a previous hypothesis, we found that this activity is not due to expression of a lantibiotic-type bacteriocin, but proteolytically processed derivatives of the phenol-soluble modulin (PSM) peptides PSMα1 and PSMα2. Notably, processed PSMα1 and PSMα2 exhibited considerable activity against Streptococcus pyogenes, indicating a role of PSMs in the interference of S. aureus strains with the competing colonizing pathogen. Furthermore, by offering a competitive advantage during colonization of the human body, the characteristically high production of PSMs in USA300 and other CA-MRSA strains may thus contribute not only to virulence but also the exceptional capacity of those strains to sustainably spread in the population, which so far has remained poorly understood.     

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin

Clostridium sordellii lethal toxin and Clostridium novyi α-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (α-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi α-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin.     

Lactobacillus-mediated inhibition of clinical toxic shock syndrome Staphylococcus aureus strains and its relation to acid and peroxide production

The inhibitory activities of 39 strains representing 20 different species of Lactobacillus toward a menstrual toxic shock syndrome (TSS) Staphylococcus aureus archetype strain MN8 were investigated. Nearly every strain (38 of 39) produced an inhibitory effect under both aerobic and anaerobic conditions when assayed on agar medium. In addition, the MN8 inhibition was conserved against at least 10 other clinical TSS S. aureus isolates and, interestingly, required actively growing cultures of Lactobacillus (verified with a two-well co-culture system in broth medium). This general uniform inhibition could be ameliorated by organic buffer (PIPES) supplied in the growth medium and, with only one exception, MRS medium adjusted with non-organic acid (HCl) failed to support growth of TSS strains at or below pH 5.5. By comparison, the vast majority of lactobacilli in this study decreased culture pH to a range of 4-5. Hydrogen peroxide production by the lactobacilli was also assessed and verified by two different methodologies revealing a broad spectrum of phenotypes that, contrary to reports touting its effectiveness, did not seem to correspond with our inhibition studies. Furthermore, resistances to peroxide by MN8, other TSS strains, and a subset of lactobacilli used in this study were nearly identical whereas the S. aureus collection was slightly more sensitive to racemic lactic acid than the lactobacilli. Collectively, these data suggest that the underlying inhibition toward Staphylococcus is generally conserved in Lactobacillus sp. and is related to a common factor in this genus involving promotion of acidic conditions.     

Direct and synergistic hemolysis caused by Staphylococcus phenol-soluble modulins: implications for diagnosis and pathogenesis

Phenol-soluble modulins are secreted staphylococcal peptides with an amphipathic α-helical structure. Some PSMs are strongly cytolytic toward human neutrophils and represent major virulence determinants during Staphylococcus aureus skin and blood infection. However, capacities of PSMs to lyse human erythrocytes have not been investigated. Here, we demonstrate that many S. aureus and Staphylococcus epidermidis PSMs lyse human erythrocytes. Furthermore, synergism with S. aureus β-toxin considerably increased the hemolytic capacities of several PSMs. This synergism may be of key importance in PSM and β-toxin-producing S. aureus or in mixed-strain or -species infections with PSM and β-toxin producers. Of specific interest, several PSMs, in particular PSMα peptides, contributed to a considerable extent to synergistic hemolysis with β-toxin or when using the β-toxin-producing strain RN4220 in CAMP assays. Thus, CAMP-type assays should not be used to detect or quantify S. aureus δ-toxin production, but may be used for an overall assessment of Agr functionality. Our study suggests an additional role of PSMs in staphylococcal pathogenesis and demonstrates that the repertoire of staphylococcal hemolysins is not limited to S. aureus and is much larger and diverse than previously thought.     

Gram-positive bacterial superantigen outside-in signaling causes toxic shock syndrome

Staphylococcus aureus and Streptococcus pyogenes (group A streptococci) are Gram-positive pathogens capable of producing a variety of bacterial exotoxins known as superantigens. Superantigens interact with antigen-presenting cells (APCs) and T cells to induce T cell proliferation and massive cytokine production, which leads to fever, rash, capillary leak and subsequent hypotension, the major symptoms of toxic shock syndrome. Both S. aureus and group A streptococci colonize mucosal surfaces, including the anterior nares and vagina for S. aureus, and the oropharynx and less commonly the vagina for group A streptococci. However, due to their abilities to secrete a variety of virulence factors, the organisms can also cause illnesses from the mucosa. This review provides an updated discussion of the biochemical and structural features of one group of secreted virulence factors, the staphylococcal and group A streptococcal superantigens, and their abilities to cause toxic shock syndrome from a mucosal surface. The main focus of this review, however, is the abilities of superantigens to induce cytokines and chemokines from epithelial cells, which has been linked to a dodecapeptide region that is relatively conserved among all superantigens and is distinct from the binding sites required for interactions with APCs and T cells. This phenomenon, termed outside-in signaling, acts to recruit adaptive immune cells to the submucosa, where the superantigens can then interact with those cells to initiate the final cytokine cascades that lead to toxic shock syndrome.     

Capsaicin protects mice from community-associated methicillin-resistant Staphylococcus aureus pneumonia

Background

α-toxin is one of the major virulence factors secreted by most Staphylococcus aureus strains, which played a central role in the pathogenesis of S. aureus pneumonia. The aim of this study was to investigate the impact of capsaicin on the production of α-toxin by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA 300 and to further assess its performance in the treatment of CA-MRSA pneumonia in a mouse model.

Methodology/Principal Findings

The in vitro effects of capsaicin on α-toxin production by S. aureus USA 300 were determined using hemolysis, western blot, and real-time RT-PCR assays. The influence of capsaicin on the α-toxin-mediated injury of human alveolar epithelial cells was determined using viability and cytotoxicity assays. Mice were infected intranasally with S. aureus USA300; the in vivo protective effects of capsaicin against S. aureus pneumonia were assessed by monitoring the mortality, histopathological changes and cytokine levels. Low concentrations of capsaicin substantially decreased the production of α-toxin by S. aureus USA 300 without affecting the bacterial viability. The addition of capsaicin prevented α-toxin-mediated human alveolar cell (A549) injury in co-culture with S. aureus. Furthermore, the in vivo experiments indicated that capsaicin protected mice from CA-MRSA pneumonia caused by strain USA 300.

Conclusions/Significance

Capsaicin inhibits the production of α-toxin by CA-MRSA strain USA 300 in vitro and protects mice from CA-MRSA pneumonia in vivo. However, the results need further confirmation with other CA-MRSA lineages. This study supports the views of anti-virulence as a new antibacterial approach for chemotherapy.     

Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-toxin

Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.     

A novel core genome-encoded superantigen contributes to lethality of community-associated MRSA necrotizing pneumonia

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone.     

The dominant Australian community-acquired methicillin-resistant Staphylococcus aureus clone ST93-IV [2B] is highly virulent and genetically distinct

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.     

Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives.

The agr quorum-sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum-sensing system is a thiolactone-containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N-terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N-terminus and a thiolactone were absolute prerequisites for an agr-activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum-sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.