Incorporation of carbon from decomposing litter of two pioneer plant species into microbial communities of the detritusphere

For several Staphylococci, such as Staphylococcus aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis, invasion of eukaryotic cells has been described and this mechanism has been considered an important part of the infection process. The fibrinogen-binding protein (Fbl) of Staphylococcus lugdunensis, a homolog of the clumping factor A of S. aureus, has been described as fibrinogen-binding adhesin and might promote invasion of cells. We therefore characterized several clinical strains of S. lugdunensis in terms of whole cell fibrinogen and fibronectin binding and correlated these results with the invasion of epithelial and endothelial cells by S. lugdunensis. We described for the first time invasion of cells by S. lugdunensis. As invasion of cells by S. lugdunensis was only partly inhibited by cytochalasin D in contrast to a complete inhibition of invasion of cells by S. aureus, further invasion mechanisms are likely to be present in S. lugdunensis. In addition, the Fbl of S. lugdunensis is not involved in the invasion of cells as ruled out by an isogenic fbl mutant.     

A von Willebrand factor-binding protein from Staphylococcus lugdunensis

In the present study, a phage display library covering the genome of Staphylococcus lugdunensis, was affinity-selected against von Willebrand factor (vWf). This led to the identification of a gene, vwbl, encoding a putative cell surface protein of 2060 amino acids, denoted vWbl. The deduced protein has an overall organisation typical of staphylococcal cell surface proteins, with an N-terminal signal peptide, and a C-terminal cell wall sorting signal. The vWf-binding part is located in repetitive domains and antibodies against vWbl or vWf can inhibit the binding. Southern blot analysis showed that vwbl was present in the 12 S. lugdunensis strains tested.     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Cadmium-resistance plasmid in Staphylococcus lugdunensis

Plasmids that confer resistance to cadmium (MIC > or = 125 microM), were found in 18 out of 30 independent Staphylococcus lugdunensis strains from clinical specimens. Variants that were cured of their plasmid were cadmium sensitive. Restriction endonuclease sites of a 3.2-kb cadmium-resistance plasmid of S. lugdunensis, designated pLUG10, were similar to those of pOX6, a S. aureus cadmium-resistance plasmid containing the cadB gene. Southern-blot hybridisation was performed with a probe intragenic to cadB. Hybridisation was found with the cadB probe in the cadmium-resistant S. lugdunensis isolates to the 2.9-, 3.2- and 3.7-kb plasmids. These findings suggest that cadmium-resistance in some S. lugdunensis strains is due to a gene sharing homology with the cadB gene of S. aureus.     

Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the approximately 2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the approximately 2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.     

The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus

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.     

Silkworm apolipophorin protein inhibits Staphylococcus aureus virulence

Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding α-hemolysin, hlb encoding β-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin.     

Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus

Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C-terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of peptidoglycan cross-bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.     

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes

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 .     

Cloning of an agr homologue of Staphylococcus saprophyticus

An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.     

An embarrassment of sortases - a richness of substrates?

A range of surface proteins is anchored to the cell walls of Gram-positive pathogens such as Staphylococcus aureus by the transpeptidase sortase. Until now, sortase-like proteins and their substrates appeared to be limited mainly to such pathogens. However, by searching for sortase homologues among complete and incomplete genome sequences, we have found them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean. There is usually more than one sortase-like protein encoded in each Gram-positive genome, and the genes encoding the sortase-like proteins are often clustered with genes encoding their likely substrates.     

金黄葡萄球菌fnbB基因的克隆及在大肠杆菌中的表达

金黄色葡萄球菌（Staphylococcus aureus）是引起奶牛乳房炎主要致病菌之一,主要通过其菌体表面的黏附素侵入寄主细胞引起疾病,为奶牛业造成巨大损失。金黄色葡萄球菌表面蛋白纤连蛋白结合蛋白(fibronectin-binding protein,FnBP)是其关键的黏附因子,在研制抗金黄色葡萄球菌的新型疫苗中占有重要地位.本文根据GenBank中纤连蛋白结合蛋白B基因（fnbB）序列设计特异性引物,以金黄葡萄球菌基因组DNA为模板,进行PCR扩增,获得3 458 bp 的DNA片段。使用T-A克隆技术,将PCR产物克隆至pGEM T easy Vector中,成功构建出了克隆质粒pGEM-fnbB。以 BamHI和XhoI 双酶切pGEM-fnbB和pET28a(+),并将纯化的基因fnbB 亚克隆至pET28a(+)中,构建出原核表达质粒pET28a-fnbB,并将其转化至E.coli BL21(DE3)感受态细胞中,经1 mmol/L的IPTG诱导和SDS-PAGE分析,在约165 ku 处出现了与预期目的蛋白相一致的外源蛋白带,Western blot分析结果表明该蛋白具有金黄葡萄球菌的抗原性。金黄葡萄球菌pET28a-fnbB成功表达为金黄葡萄球菌引起的奶牛乳房炎的诊断和研究新型疫苗奠定基础。     

Characterization of Streptococcus suis Genes Encoding Proteins Homologous to Sortase of Gram-Positive Bacteria

Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptidoglycan by a specific enzyme called sortase. Using the type strain of Streptococcus suis, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated srtA, was linked to gyrA, as were the sortase and sortase-like genes of other streptococci. Three genes, designated srtB, srtC, and srtD, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated srtE, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of Streptococcus gordonii and Staphylococcus aureus, except that SrtA of S. suis had 65% identity with that of S. gordonii. Isogenic mutants deficient for srtA, srtBCD, or srtE were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with N-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the srtA mutant compared with that of the parent strain, and this phenotype was completely complemented by srtA cloned from S. suis. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the srtA mutant appeared in the profiles of the srtBCD and srtE mutants. These results provide evidence that the cell wall sorting system involving srtA is also present in S. suis.     

Analysis of the beta-lactamase plasmid of borderline methicillin-susceptible Staphylococcus aureus: focus on bla complex genes and cadmium resistance determinants cadD and cadX

Borderline methicillin-susceptible Staphylococcus aureus strains are a rather homogeneous group, characterized by MICs of penicillinase-resistant penicillins (PRPs) at or just below the susceptibility breakpoint. Other features unique to this group include the presence of a pBW15-like beta-lactamase plasmid, the association with phage complex 94/96, and the production of a PRP-hydrolyzing beta-lactamase activity in addition to the classical penicillinase activity. The four HindIII fragments of pBORa53, a pBW15-like plasmid from the well-studied borderline S. aureus strain a53, were cloned in Escherichia coli, sequenced and analyzed. The plasmid (17,334 bp in size) contains 14 open reading frames (ORFs) and a complete copy of transposon Tn552, which harbors the three genes of the bla complex (blaZ, blaR1, and blaI) necessary for penicillinase production. Among the other 11 ORFs identified, two were homologous to cadmium resistance determinants of Staphylococcus lugdunensis and to the cadD and cadX genes recently detected in S. aureus. Consistent with this, strain a53 was found to be cadmium resistant. From a collection of 30 S. aureus isolates with borderline PRP MIC levels, 27 matched strain a53 in the positive amplification reactions with all of the four primer pairs targeting the cadD-cadX region, the presence of the 17.3-kb plasmid, and the level of cadmium resistance. The well-established S. aureus laboratory strain ATCC 29213 was also found to express cadD-cadX-mediated cadmium resistance. pBORa53 could be re-isolated from transformants obtained by transferring it into a PRP-susceptible recipient. However, while the transformants demonstrated levels of cadmium and penicillin resistance similar to those of strain a53, they remained fully susceptible to PRPs.