PavB is a surface‐exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections

The genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dose‐dependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with ΔpavB pneumococci showed significantly increased survival times compared with wild‐type bacteria. Importantly, the pavB‐mutant showed a delay in transmigration to the lungs as observed in real‐time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co‐infection experiments the wild‐type out‐competed the pavB‐mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface‐exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.     

The adhesin structures involved in the adherence of group B streptococci to human vaginal cells

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.     

A novel bacterial resistance mechanism against human group IIA-secreted phospholipase A2: role of Streptococcus pyogenes sortase A

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA(2)-IIA. Analysis of a set of clinical isolates demonstrated that an ~500-fold higher concentration of sPLA(2)-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA(2)-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA(2)-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (~30-fold) to sPLA(2)-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA(2)-IIA. Importantly, using transgenic mice expressing human sPLA(2)-IIA, we showed that the sortase A-mediated sPLA(2)-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA(2)-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA(2)-IIA and we showed that sPLA(2)-IIA contributes to the host defense against S. pyogenes infection.     

DsbA: a protein-folding catalyst contributing to bacterial virulence

DsbA, a periplasmic thiol:disulphide oxidoreductase, catalyses the folding of various factors, among which are virulence determinants or the components of type III secretory machinery. It is also necessary for intracellular survival and cell-to-cell spread of the intracellular pathogen Shigella flexneri.     

RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.     

CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen

Clostridium difficile is the leading cause of antibiotic‐associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (C ollagen b inding p rotein A ), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG‐like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent K_d in the order of 10^?9 M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild‐type C. difficile 630 strain was indistinguishable to the cbpA knock‐out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM‐producing cells. The binding activity of L. lactis–CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface‐exposed adhesin contributing to the C. difficile interaction with the host.     

Peptide mapping of a novel discontinuous epitope of the major surface adhesin from Streptococcus mutans

Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170-218 and 956-969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation.     

HtpS, a novel immunogenic cell surface-exposed protein of Streptococcus suis, confers protection in mice

Streptococcal histidine triad protein was identified recently as a cell surface-associated protein family. Five members of this family (PhtA, PhtB, PhtD, PhtE and HtpA), derived from Streptococcus pneumoniae and Streptococcus pyogenes, have been shown as antigens that confer protection to the host on infection. In this report, a gene sequence highly homologous to htpA and phtD (designated htpS, the histidine triad protein of Streptococcus suis) was identified from S. suis 2 Chinese strain 05ZYH33. Our data revealed that htpS is extremely conserved in S. suis 2 and widely distributed in 83% (29/35) of 35 S. suis serotypes. It was also demonstrated by Western blot and flow cytometry that HtpS is a cell surface-associated protein that was expressed during S. suis 2 infection. An antibody against HtpS could increase the deposition of human complement 3 on S. suis 2 and also enhance the clearance of S. suis 2 in whole blood. In addition, mice could be immunized against S. suis 2 infection and were well protected after immunization with recombinant HtpS.     

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.     

The effects of growth in human serum on an acapsular group B Streptococcus mutant

Abstract A Group B Streptococcus Type III (GBS) mutant which, when grown in Todd Hewitt broth (THB), does not produce any detectable capsule, produced a clearly visible polysaccharide capsule when grown in human serum. We isolated cytoplasmic membranes from GBS and separated the component membrane proteins by polyacrylamide gel electrophoresis. A significant change in membrane composition was found during growth in human serum. Several unique proteins were produced on serum growth and there was both up- and down-regulation of other proteins. We measured the intracellular levels of sialic acid for a variety of GBS serotype III isolates. Interestingly, while there was little difference between the intracellular sialic levels of most isolates, the sialic acid level of COH31-15 grown in THB was over 100% higher than that of any other isolate. When grown in serum this pool was reduced to a level similar to that in other strains. The concentration of bacterial cell sialic acid was directly correlated with the sialic acid content of the serum. Exogenous sialic acid content, in concert with other serum factors, plays a role in determining the capsular size in GBS.     

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

Sufu (Suppressor of Fused), a two‐domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu‐like proteins have previously been identified based on sequence similarity to the N‐terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu‐like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu‐like protein. The structure revealed a striking similarity to the N‐terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ～15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu‐like proteins that are present in ～200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.     

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A₂ kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A₂ kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Galα1–4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of Gaα1–3(Fucα1–2)Galβ-HexNac-Galα1–4Galβ-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant glood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which migh explain the low expression of type-4 chain blood group heptaglycosylceramides in human blood group B kidneys.