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Hurd AF Garcia-Lara J Rauter Y Cartron M Mohamed R Foster SJ 《FEMS microbiology letters》2012,329(1):93-100
The iron-regulated surface determinant proteins (Isd) of Staphylococcus aureus are expressed during iron limitation and have been proposed to be involved in the scavenging of iron from heme. In this study, the genes encoding the surface proteins IsdA, IsdB, and IsdH were inactivated in order to determine their combined role. The triple mutant was found to have no defect in growth under any conditions of iron limitation tested. Also using a mouse septic arthritis model of S.?aureus systemic disease, no significant difference in bacterial load was observed for the triple mutant, compared with its otherwise isogenic parent. 相似文献
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The ability to gain access to iron is pivotal for bacterial pathogens during infection. Although much is known about iron acquisition systems in Gram-negative bacteria, comparatively little is known about how Gram-positive pathogens access iron from host iron sources. A previous study showed that, in the Gram-positive human pathogen Staphylococcus aureus, a cell surface-associated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme (Gap, or Tpn) is capable of binding human transferrin, representing a potential means by which this bacterium is able to access iron in vivo. We have investigated this property of S. aureus further and shown that, in S. aureus RN6390, GAPDH is expressed on the S. aureus cell surface independent of exogenous iron concentrations, and that overexpressed and purified Gap, although retaining GAPDH activity, has no affinity for human transferrin. Moreover, although a S. aureus gap mutant was devoid of surface-associated and cytoplasmic GAPDH activity, it retained the ability to bind human transferrin, equivalent to wild type. We concluded from these results that the Gap protein is not involved in S. aureus binding to human transferrin. We identified the transferrin-binding protein as a novel cell wall-anchored protein, designated StbA for staphylococcal transferrin-binding protein A, which shared no significant similarities with any other bacterial transferrin-binding proteins. StbA contained a C-terminal cell wall-anchoring motif (LPKTG), and expression of StbA in the cell wall was strictly controlled by exogenous iron concentrations. The stbA gene is found within a 7 kb region in the S. aureus chromosome that contains a total of six iron-regulated genes. Immediately downstream from stbA is an iron-regulated gene whose product was predicted to be another cell wall-anchored protein with no significant similarity to proteins with characterized functions. Transcribed in the opposite direction from stbA is a four-gene operon whose expression is also regulated by iron. While the deduced products of the first two genes lack similarity to known proteins, the last two genes encode, respectively, putative lipoprotein and permease components of an ABC transporter that shares significant similarities with several iron(III) ABC transporters in a variety of bacteria. 相似文献
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Jeremy M. Yarwood John K. McCormick Michael L. Paustian Vivek Kapur Patrick M. Schlievert 《Journal of bacteriology》2002,184(4):1095-1101
Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (agr locus) as well as multiple virulence-associated genes in Staphylococcus aureus. Gene expression was examined during growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of agr and any staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of agr and virulence factors was also observed. These results suggest that agr activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity. 相似文献
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Heilbronner S Holden MT van Tonder A Geoghegan JA Foster TJ Parkhill J Bentley SD 《FEMS microbiology letters》2011,322(1):60-67
Staphylococcus lugdunensis is an opportunistic pathogen related to Staphylococcus aureus and Staphylococcus epidermidis. The genome sequence of S. lugdunensis strain N920143 has been compared with other staphylococci, and genes were identified that could promote survival of S. lugdunensis on human skin and pathogenesis of infections. Staphylococcus lugdunensis lacks virulence factors that characterize S. aureus and harbours a smaller number of genes encoding surface proteins. It is the only staphylococcal species other than S. aureus that possesses a locus encoding iron-regulated surface determinant (Isd) proteins involved in iron acquisition from haemoglobin. 相似文献
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Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus
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The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including Staphylococcus aureus, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an S. aureus genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the cbr locus of the plant pathogen Erwinia chrysanthemi. The first gene in the locus, which we have designated sir for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in Escherichia coli. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the S. aureus culture medium. sir-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents. 相似文献
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Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor. 相似文献
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Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes 总被引:4,自引:1,他引:3
C. Greene D. McDevitt P. Francois P.E. Vaudaux D.P. Lew T.J. Poster 《Molecular microbiology》1995,17(6):1143-1152
Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB . A double fnbA fnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbA fnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo . 相似文献
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Molecular cloning and genetic analysis of the determinant for gamma-lysin, a two-component toxin of Staphylococcus aureus 总被引:2,自引:0,他引:2
The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus. 相似文献
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Ulcerative keratitis is among the leading ocular bacterial infections, and Streptococcus aureus accounts for approximately 25% of cases in some surveys. Although S. aureus expresses numerous virulence factors, many of which are under the control of staphylococcal global regulatory genes, their pathophysiologic roles in keratitis are largely unknown. Similarly, the nature of the host response during S. aureus keratitis is unclear. Following a review of previously published research on the pathophysiology of S. aureus ocular infection, we present the results of a study designed to assess the host-parasite relationship between S. aureus and human corneal epithelial cells (HCECs) in vitro. In this model system, a wild-type S. aureus strain and its isogenic mutants harboring mutations in agr and sar global regulatory genes or fibronectin-binding proteins A and B (fnbAB) were tested for their ability to bind and invade confluent HCEC monolayers. The contribution of host cell factors was assessed by preincubating HCECs with various inhibitory agents. These studies demonstrated that S. aureus not only adhered to the surface of HCECs but was also internalized, as has been previously observed in other nonocular cell lines. Adherence and invasion of HCECs was saturable at 1 h of incubation in the presence of approximately 10(7) CFU per HCEC monolayer (multiplicity of infection approximately 10). A mutant defective in both agr and sar global regulators was not significantly different in invasive capacity compared to its isogenic wild-type parent strain. In contrast, mutations in fibronectin-binding proteins A and B (fnbAB) reduced the invasiveness of S. aureus by 99% compared to the wild-type strain. Pretreatment of HCECs with colchicine had little effect on S. aureus invasion. In sharp contrast, cytochalasin D and genistein were each capable of inhibiting invasion by >99%. In summary, the results of this study point to fibronectin-binding protein as a key S. aureus surface adhesin facilitating invasion of HCECs in vitro. Furthermore, these results suggest an active mechanism for S. aureus internalization by HCECs, likely involving actin polymerization and tyrosine kinase activity. Additional studies are warranted to determine the applicability of these findings in vivo, and to facilitate the rational design of therapeutic agents aimed at blocking the establishment and progression of S. aureus keratitis. 相似文献
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Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen. 相似文献
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As an important facet of host-pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad-spectrum ligand-binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron-regulated proteins of S. aureus and in other Gram-positive organisms. The isdA gene is only expressed in iron-limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron-limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy. 相似文献