Characterization of a novel fibronectin-binding surface protein in group A streptococci

Streptococcus pyogenes interacts with host fibronectin via distinct surface components. One of these components is the Sfbl protein (streptococcal fibronectin-binding protein, now specified as class I), an adhesin that represents a protein family with characteristic features. Here we present the complete structure of a novel fibronectin-binding protein of S. pyogenes , designated SfbII, which is distinct from the previously described Sfbl proteins. The sfbII gene originated from a λ EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113kDa protein exhibiting features of membrane-anchored surface proteins of Gram-positive cocci. The expression of biologically active fusion proteins allowed the determination of the location of the fibronectin-binding domain within the C-terminal part of the protein. It consisted of two and a half repeats which share common motifs with fibronectin-binding repeats of other streptococcal and staphylococcal proteins. Purified recombinant fusion protein containing this domain competitively inhibited the binding of fibronectin to the parental S. pyogenes strain. Furthermore, polyclonal antibodies against the binding domain specifically blocked the Sfbll receptor site on the streptococcal surface. No cross-reactivity could be detected between anti-Sfbll antibodies and the sfbl gene product, and vice versa, indicating that the two proteins do not share common immunogenic epitopes. Southern hybridization experiments performed with specific sfbll gene probes revealed the presence of the sfbll gene in more than 55% of 93 streptococcal isolates tested. The majority of the strains also harboured the sfbl gene, and 86% carried at least one of the two sfb genes.     

Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.     

Molecular cloning and expression of the coagulase gene of Staphylococcus aureus 8325-4

The gene coding for coagulase (coa) was cloned from Staphylococcus aureus 8325-4 in a lambda replacement vector in Escherichia coli. Coagulase (plasma-clotting) activity was measured in lambda coa lysates and an immunoreactive protein of 60 kDa was detected by Western immunoblotting with anti-coagulase serum. This protein comigrated with the major immunoreactive protein in supernatants of S. aureus 8325-4. The coa gene was subcloned in pUC vectors. One recombinant expressed a 60 kDa immunoreactive protein and plasma-clotting activity. A putative beta-galactosidase-coagulase fusion protein and truncated peptides were expressed by variants formed by subcloning. These results are consistent with previously published biochemical data that the prothrombin-binding domain of coagulase is located in the N-terminus of the protein. The cloned coa gene was transferred into S. aureus on a shuttle plasmid. Expression of coagulase was higher in a strain with a mutation in the agr locus, which controls the level of several exoproteins in S. aureus, suggesting that agr normally regulates coagulase expression negatively.     

The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells

Invasive serotype M3 Streptococcus pyogenes are among the most frequently isolated organisms from patients suffering from invasive streptococcal disease and have the potential to invade primary human endothelial cells (EC) via a rapid and efficient mechanism. FbaB protein, the fibronectin-binding protein expressed by M3 S. pyogenes, was herein identified as a potent invasin for EC. By combining heterologous gene expression with allelic replacement, we demonstrate that FbaB is essential and sufficient to trigger EC invasion via a Rac1-dependent phagocytosis-like uptake. FbaB-mediated uptake follows the classical endocytic pathway with lysosomal destination. FbaB is demonstrated to be a streptococcal invasin exhibiting EC tropism. FbaB thus initiates a process that may contribute to the deep tissue tropism and spread of invasive S. pyogenes isolates into the vascular EC lining.     

Molecular cloning of the S-layer protein gene of Campylobacter rectus ATCC 33238

Abstract A genomic DNA library of Campylobacter rectus (Wolinella recta) ATCC 33238 was constructed using bacteriophage lambda EMBL3 as a vector. One clone expressing the S-layer protein was identified immunologically with the antiserum to the S-layer protein of C. rectus ATCC 33238. Western immunoblotting using monoclonal antibodies directed against the S-layer protein showed a single blot of recombinant protein at 150 kDa, suggesting that this clone contained the entire coding region of the S-layer protein gene. Additionally, the S-layer protein gene was subcloned into plasmid vector pUC18. Southern hybridization revealed that the S-layer protein gene was present on the chromosome of C. rectus as a single-copy gene, and that there were minor heterogeneities among the S-layer protein genes of clinical isolates. Moreover, a spontaneous stable mutant strain, which has no S-layer expression, also conserved the full-length gene of the S-layer protein.     

Cloning and expression of an adhesin antigen of Streptococcus sanguis G9B in Escherichia coli

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a lambda gt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-beta-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to saliva-coated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.     

牛分枝杆菌MPB83基因的原核表达及免疫活性分析

利用PCR技术,以牛型分枝杆菌(M.bovis)Vallee菌株的全基因组DNA为模板,扩增出了一条600bp的MPB83基因片段,将其克隆至pMD18T载体中,经核苷酸序列测定确证后,KpnI/EcoRI双酶切,然后亚克隆到原核表达载体pET30a的相同酶切位点,构建表达质粒pETMPB83,将鉴定的重组质粒转化大肠杆菌BL21(DE3),IPTG诱导后SDSPAGE检测表达情况,重组质粒pETMPB83在30kDa处有一特异表达带,与预计大小相符。经Ni柱纯化后,Westernblot检测纯化蛋白具有免疫活性,用纯化的该蛋白进行动物(兔)接种制备抗血清,用Westernblot和ELISA检测该抗血清的效价和特异性,结果表明特异性较好。     

Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes

Fibronectin-binding surface proteins are found in many bacterial species. Most strains of Streptococcus pyogenes, a major human pathogen, express the fibronectin-binding protein F1, which promotes bacterial adherence to and entry into human cells. In this study, the role of fibronectin in S. pyogenes virulence was investigated by introducing the protein F1 gene in an S. pyogenes strain lacking this gene. Furthermore, transgenic mice lacking plasma fibronectin were used to examine the relative contribution of plasma and cellular fibronectin to S. pyogenes virulence. Unexpectedly, protein F1-expressing bacteria were less virulent to normal mice, and virulence was partly restored when these bacteria were used to infect mice lacking plasma fibronectin. Dissemination to the spleen of infected mice was less efficient for fibronectin-binding bacteria. These bacteria also disseminated more efficiently in mice lacking plasma fibronectin, demonstrating that plasma fibronectin bound to the bacterial surface downregulates S. pyogenes virulence by limiting bacterial spread. From an evolutionary point of view, these results suggest that reducing virulence by binding fibronectin adds selective advantages to the bacterium.     

Two distinct pathways for the invasion of Streptococcus pyogenes in non-phagocytic cells

Adherence to and invasion of epithelial cells represent important pathogenic mechanisms of Streptococcus pyogenes . A fibronectin-binding surface protein of S. pyogenes , SfbI protein, has been implicated in both adherence and invasion processes. Invasion of SfbI-containing strains has been suspected to be responsible for the failure of antibiotics treatment to eradicate S. pyogenes . In this study, we tested the adherence and invasion properties of two well-characterized clinical isolates: A40, which expresses SfbI; and A8, which is SfbI negative and is unable to bind fibronectin. In strain A40, SfbI was the main factor required for attachment and invasion by using fibronectin as a bridging molecule and the α₅β₁ integrin as cellular receptor. The uptake process was characterized by the generation of large membrane invaginations at the bacteria–cell interface without evidence of actin recruitment or cellular injury. A40 cells were located in phagosomes and, only 24 h after infection, a consistent part of the bacterial population reached the cytoplasm. In contrast, uptake of strain A8 required major rearrangements of cytoskeletal proteins underneath attached bacteria. In A8, a proteinaceous moiety was involved, which does not interact with α₅β₁ or need any known bridging molecule. Bacterial attachment stimulated elongation and massive recruitment of neighbouring microvilli, which fused to surround streptococcal chains. They led to the generation of large pseudopod-like structures, which engulfed bacteria that were rapidly released and replicated in the cytoplasm. The identification of two completely different uptake pathways reported here provided further evidence regarding the diversity of S. pyogenes isolates and might contribute towards understanding the pathogenesis and persistence of S. pyogenes .     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

Ketolide resistance in Streptococcus pyogenes correlates with the degree of rRNA dimethylation by Erm

Macrolide and ketolide antibiotics inhibit protein synthesis on the bacterial ribosome. Resistance to these antibiotics is conferred by dimethylation at 23S rRNA nucleotide A2058 within the ribosomal binding site. This form of resistance is encoded by erm dimethyltransferase genes, and is found in many pathogenic bacteria. Clinical isolates of Streptococcus pneumoniae with constitutive erm(B) and Streptococcus pyogenes with constitutive erm(A) subtype (TR) are resistant to macrolides, but remain susceptible to ketolides such as telithromycin. Paradoxically, some strains of S. pyogenes that possess an identical erm(B) gene are clinically resistant to ketolides as well as macrolides. Here we explore the molecular basis for the differences in these streptococcal strains using mass spectrometry to determine the methylation status of their rRNAs. We find a correlation between the levels of A2058-dimethylation and ketolide resistance, and dimethylation is greatest in S. pyogenes strains expressing erm(B). In constitutive erm strains that are ketolide-sensitive, appreciable proportions of the rRNA remain monomethylated. Incubation of these strains with subinhibitory amounts of the macrolide erythromycin increases the proportion of dimethylated A2058 (in a manner comparable with inducible erm strains) and reduces ketolide susceptibility. The designation 'constitutive' should thus be applied with some reservation for most streptococcal erm strains. One strain worthy of the constitutive designation is S. pyogenes isolate KuoR21, which has lost part of the regulatory region upstream of erm(B). In S. pyogenes KuoR21, nucleotide A2058 is fully dimethylated under all growth conditions, and this strain displays the highest resistance to telithromycin (MIC > 64 microg ml-1).     

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus-infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and titre of anti-S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.     

Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide- resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E. coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA . Furthermore, intracellular accumulation of [¹⁴C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.     

Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS–PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli , and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.     

Immunoblotting of a Staphylococcus aureus DNA library as a tool for isolating potential antigenic proteins

Abstract A Staphylococcus aureus DNA library was created in the lambda vector EMBL4 and expressed in Escherichia coli strain Y1090. Plaques were screened using serum from a S. aureus -infected patient. A number of positive clones that expressed proteins recognised by serum antibodies were further analysed using Western blots of total lysates from cultures infected with the lambda clones. Differences were found between patient and control sera in both the specificity and tite of anti- S. aureus antibodies. Expressed staphylococcal proteins appeared to be stable and were easily distinnguishable from E. coli proteins in the Western blot. This method may have applications in specifically selecting clones encoding antigens associated with infection and may be of potential use clinically.     

Broadening the insecticidal spectrum of Lepidoptera-specific Bacillus thuringiensis strains by chromosomal integration of cry3A

A TnpI-TnpIA-mediated and thermosensitive recombination system was developed to construct genetically modified Bacillus thuringiensis strains encoding a crystal protein particularly active against Coleopteran species. Based on B. thuringiensis transposon Tn4430, an integrative vector, pBMB-R14E, was constructed, by which the cry3A delta-endotoxin gene highly toxic to Lepidoptera was delivered into a wildtype B. thuringiensis subsp. kurstaki strain YBT1520. The cry3A gene was integrated into the chromosome of the host strain. Then the integrative vector was eliminated by moving recombinant cultures to 46 degrees C. Two recombinant B. thuringiensis strains, BMB1520-S and BMB1520-T, were obtained. In recombinant strains, the cry3A gene was stably expressed in measurable amounts and did not reduce the expression of endogenous crystal protein genes. Bioassay results showed that BMB1520-S and BMB1520-T, in addition to the activity against lepidopteran Plutella xylostella third-instar larvae present in the parental strains, exhibited a high level of activity against coleopteran Rhyllodecta vulgatissima third-instar larvae, absent from the parental strains.     

Streptococcus pyogenes recruits collagen via surface-bound fibronectin: a novel colonization and immune evasion mechanism

This study aimed to characterize matrix assembly mechanisms on the surface of the human pathogen Streptococcus pyogenes. Among 125 S. pyogenes isolates, 61% were able to recruit collagen type IV via surface-bound fibronectin. Streptococcus gordonii expressing the fibronectin-binding repeat domain of S. pyogenes SfbI protein was equally potent in recruiting collagen, indicating that this domain was sufficient to promote fibronectin-mediated collagen recruitment. Electron microscopic analysis of streptococci revealed that fibronectin-mediated collagen recruitment led to matrix deposition on and between streptococcal cells, which induced the formation of large bacterial aggregates. Furthermore, collagen-recruiting streptococci were able to colonize collagen fibres and were protected from adhering to human polymorphonuclear cells in the presence of opsonizing antibodies. Fibronectin-mediated collagen recruitment thus represents a novel aggregation, colonization and immune evasion mechanism of S. pyogenes.     

Identification and characterization of a novel fibronectin-binding protein gene from Streptococcus equi subspecies zooepidemicus strain VTU211

This work describes the cloning and sequencing of genes encoding fibronectin-binding proteins from Streptococcus equi subspecies zooepidemicus strain VTU211. A gene encoding a cell-wall protein FNZ was amplified and sequenced. In the same bacterial strain, a second gene termed fnz2 was now discovered, encoding another fibronectin-binding protein (FNZ2). The complete amino acid sequence encoded by fnz2 was deduced and compared to that deduced from fnz. The sequence comparison of the fnz and fnz2 predicted that fibronectin-binding activity is localizing a domain in the C terminal part of FNZ2, since this domain is composed of three repeats, which contain a motif similar to what has earlier been found in other fibronectin-binding proteins in streptococci. Three parts of fnz2 [fnz2(1-8), fnz2(2-4), and fnz2(4-3)] were amplified using polymerase chain reaction and ligated into an expression vector, and recombinant FNZ2 proteins were produced in Escherichia coli. Fibronectin bound to the FNZ2(1-8) [amino acids 212-396] and FNZ2(2-4) (amino acids 36-448) but not to the FNZ2(4-3) (amino acids 36-191) in a Western ligand blot, showing that repeat domain of FNZ2 protein was sufficient for binding of fibronectin. Purified FNZ2(2-4) protein was also shown to display collagen-binding activity to collagen-coated microtiter wells. These results show that recombinant FNZ2 has fibronectin- and collagen-binding activities.     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。