α-cyperone alleviates lung cell injury caused by Staphylococcus aureus via attenuation of α-hemolysin expression

In this study, we aimed to evaluate the effect of α- cyperone on S. aureus. We used a hemolysin test to examine the hemolytic activity in supernatants of S. aureus cultured with increasing concentrations of α- cyperone. In addition, we evaluated the production of α- hemolysin (Hla) by Western blotting. Real-time RT-PCR was performed to test the expression of hla (the gene encoding Hla) and agr (accessory gene regulator). Furthermore, we investigated the protective effect of α- cyperone on Hla-induced injury of A549 lung cells by live/ dead and cytotoxicity assays. We showed that in the presence of subinhibitory concentrations of α-cyperone, Hla production was markedly inhibited. Moreover, α- cyperone protected lung cells from Hla-induced injury. These findings indicate that α-cyperone is a promising inhibitor of Hla production by S. aureus and protects lung cells from this bacterium. Thus, α-cyperone may provide the basis for a new strategy to combat S. aureus pneumonia.     

Population studies of methicillin-resistant and -sensitive Staphylococcus aureus strains reveal a lack of variability in the agrD gene, encoding a staphylococcal autoinducer peptide

The virulence of Staphylococcus aureus is controlled by the accessory gene regulator (agr) system, including an extracellular inducer encoded by agrD. Variable agr PCR restriction fragment length polymorphism (RFLP) patterns of unique S. aureus strains (n = 192) were determined for a region comprising agrD and parts of the neighboring agrC and agrB genes. Twelve unique RFLP patterns were identified among S. aureus strains in general; these patterns were further specified by sequencing. All sequences could be catalogued in the three current agr groups. A major proportion of the S. aureus strains belong to agr group 1, whereas only 6% of the methicillin-susceptible S. aureus strains and 5% of the methicillin-resistant S. aureus strains belong to agr groups 2 and 3, respectively. The homology between groups varied from 75 to 80%, and within groups it varied from 96 to 100%. Different levels of sequence variability were observed in the different agr genes. agr-related bacterial interference among colonizing S. aureus strains in the noses of persistent and intermittent human carriers was studied. S. aureus strains belonging to different agr groups were encountered in the same individual. This may suggest that the activity of the agrD gene product does not define colonization dynamics, which is further substantiated by the rarity of agr group 2 and 3 strains.     

Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

Induction of multiple matrix metalloproteinases in human dermal and synovial fibroblasts by Staphylococcus aureus: implications in the pathogenesis of septic arthritis and other soft tissue infections

Infections of body tissue by Staphylococcus aureus are quickly followed by degradation of connective tissue. Patients with rheumatoid arthritis are more prone to S. aureus-mediated septic arthritis. Various types of collagen form the major structural matrix of different connective tissues of the body. These different collagens are degraded by specific matrix metalloproteinases (MMPs) produced by fibroblasts, other connective tissue cells, and inflammatory cells that are induced by interleukin-1 (IL-1) and tumor necrosis factor (TNF). To determine the host's contribution in the joint destruction of S. aureus-mediated septic arthritis, we analyzed the MMP expression profile in human dermal and synovial fibroblasts upon exposure to culture supernatant and whole cell lysates of S. aureus. Human dermal and synovial fibroblasts treated with cell lysate and filtered culture supernatants had significantly enhanced expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, and MMP-11 compared with the untreated controls (p < 0.05). In the S. aureus culture supernatant, the MMP induction activity was identified to be within the molecular-weight range of 30 to >50 kDa. The MMP expression profile was similar in fibroblasts exposed to a combination of IL-1/TNF. mRNA levels of several genes of the mitogen-activated protein kinase (MAPK) signal transduction pathway were significantly elevated in fibroblasts treated with S. aureus cell lysate and culture supernatant. Also, tyrosine phosphorylation was significantly higher in fibroblasts treated with S. aureus components. Tyrosine phosphorylation and MAPK gene expression patterns were similar in fibroblasts treated with a combination of IL-1/TNF and S. aureus. Mutants lacking staphylococcal accessory regulator (Sar) and accessory gene regulator (Agr), which cause significantly less severe septic arthritis in murine models, were able to induce expression of several MMP mRNA comparable with that of their isogenic parent strain but induced notably higher levels of tissue inhibitors of metalloproteinases (TIMPs). To our knowledge, this is the first report of induction of multiple MMP/TIMP expression from human dermal and synovial fibroblasts upon S. aureus treatment. We propose that host-derived MMPs contribute to the progressive joint destruction observed in S. aureus-mediated septic arthritis.     

RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus

Cell-density-dependent gene regulation by quorum-sensing systems has a crucial function in bacterial physiology and pathogenesis. We demonstrate here that the Staphylococcus aureus agr quorum-sensing regulon is divided into (1) control of metabolism and PSM cytolysin genes, which occurs independently of the small regulatory RNA RNAIII, and (2) RNAIII-dependent control of additional virulence genes. Remarkably, PSM expression was regulated by direct binding of the AgrA response regulator. Our findings suggest that quorum-sensing regulation of PSMs was established before wide-ranging control of virulence was added to the agr regulon, which likely occurred by development of the RNAIII-encoding region around the gene encoding the PSM delta-toxin. Moreover, the agr regulon in the community-associated methicillin-resistant S. aureus MW2 considerably differed from that previously determined using laboratory strains. By establishing a two-level model of quorum-sensing target gene regulation in S. aureus, our study gives important insight into the evolution of virulence control in this leading human pathogen.     

Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.     

Beyond conventional antibiotics for the future treatment of methicillin-resistant Staphylococcus aureus infections: two novel alternatives

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S.?aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S.?aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S?aureus. These are generally small molecules that inhibit virulence targets in S.?aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.     

Signaling and DNA-binding activities of the Staphylococcus aureus HssR-HssS two-component system required for heme sensing

For the important human pathogen Staphylococcus aureus, host heme is a vital source of nutrient iron during infection. Paradoxically, heme is also toxic at high concentrations and is capable of killing S. aureus. To maintain cellular heme homeostasis, S. aureus employs the coordinated actions of the heme sensing two-component system (HssRS) and the heme regulated transporter efflux pump (HrtAB). HssRS-dependent expression of HrtAB results in the alleviation of heme toxicity and tempered staphylococcal virulence. Although genetic experiments have defined the role of HssRS in the heme-dependent activation of hrtAB, the mechanism of this activation is not known. Furthermore, the global effect of HssRS on S. aureus gene expression has not been evaluated. Herein, we combine multivariable difference gel electrophoresis with mass spectrometry to identify the heme-induced cytoplasmic HssRS regulon. These experiments establish hrtAB as the major target of activation by HssRS in S. aureus. In addition, we show that signaling between the sensor histidine kinase HssS and the response regulator HssR is necessary for growth of S. aureus in high concentrations of heme. Finally, we show that a direct repeat DNA sequence within the hrtAB promoter is required for heme-induced, HssR-dependent expression driven by this promoter and that phosphorylated HssR binds to this direct repeat upon exposure of S. aureus to high concentrations of heme. Taken together, these data establish the mechanism for HssRS-dependent expression of HrtAB and, in turn, provide a functional understanding for how S. aureus avoids heme-mediated toxicity.     

金黄色葡萄球菌附属基因调节系统研究进展

金黄色葡萄球菌的微生物病原特性非常复杂并且不断地产生抗生素抗性,目前迫切需要增加对金黄色葡萄球菌的了解。它所引起的疾病与大量毒力因子相关,这些毒力因子的表达是受多个基因调控,其中agr（accessory gene regulator,附属基因调节）是最主要的一个。由于agr系统与人类的多种疾病有关,研究的较为深入,现已成为一个理解群体感应激活和抑制机制的模型系统。agr系统以及其它菌的群体感应系统已经引起了越来越多研究者的注意,本文对agr系统的研究现状、研究过程中发现的问题及其潜在应用价值作了深入地探讨。     

Late-stage polyribitol phosphate wall teichoic acid biosynthesis in Staphylococcus aureus

Wall teichoic acids are cell wall polymers that maintain the integrity of the cellular envelope and contribute to the virulence of Staphylococcus aureus. Despite the central role of wall teichoic acid in S. aureus virulence, details concerning the biosynthetic pathway of the predominant wall teichoic acid polymer are lacking, and workers have relied on a presumed similarity to the putative polyribitol phosphate wall teichoic acid pathway in Bacillus subtilis. Using high-resolution polyacrylamide gel electrophoresis for analysis of wall teichoic acid extracted from gene deletion mutants, a revised assembly pathway for the late-stage ribitol phosphate-utilizing enzymes is proposed. Complementation studies show that a putative ribitol phosphate polymerase, TarL, catalyzes both the addition of the priming ribitol phosphate onto the linkage unit and the subsequent polymerization of the polyribitol chain. It is known that the putative ribitol primase, TarK, is also a bifunctional enzyme that catalyzes both ribitol phosphate priming and polymerization. TarK directs the synthesis of a second, electrophoretically distinct polyribitol-containing teichoic acid that we designate K-WTA. The biosynthesis of K-WTA in S. aureus strain NCTC8325 is repressed by the accessory gene regulator (agr) system. The demonstration of regulated wall teichoic acid biosynthesis has implications for cell envelope remodeling in relation to S. aureus adhesion and pathogenesis.     

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-α), interferon gamma (IFN-γ), 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-γ 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.