Biomedical subjects
Publications and source records attributed to G S Chhatwal.
Fibronectin-binding protein I (SfbI) of Streptococcus pyogenes binds to mouse immunoglobulin G (IgG) but not to IgA or IgM in a nonimmune fashion. The fibronectin-binding domains of SfbI were responsible for this activity, which was targeted to the IgG F(ab')(2) fragment. SfbI also binds to B cells but not to CD4(+) or CD8(+) lymphocytes.
Fibronectin binding proteins play an important role in the adherence and invasion of group A streptococci (GAS). Genotypically distinct GAS isolates were screened for the presence and expression of two streptococcal fibronectin binding protein genes, sfbI and sfbII. Of the tested strains, 64 and 36% were shown to harbor and express the sfbI and sfbII genes, respectively. All sfbII-positive strains tested were also positive for sfbI, but only 28% of the sfbII-negative strains were positive for sfbI. High levels of immunoglobulin G antibodies to both SfbI and SfbII were found in sera from 80 subjects with defined streptococcal infections.
Serum opacity factor (SOF) is produced by group A streptococci belonging to certain M types. SOF cleaves the apolipoprotein component of the high density lipoprotein fraction of serum rendering it insoluble which in turn leads to serum opacity. SfbII protein, a fibronectin binding surface protein cloned from group A streptococci, was obtained from a strain of M75. Here we show that this protein has a second functional domain responsible for SOF activity. The fibronectin binding region was located in the C-terminal end of the protein. Deletion analysis showed that the remainder of the protein was required for SOF activity. Sequence analysis of SfbII, when compared with the published sequence of SOF22, showed 99% identity with a difference of only four amino acids. In spite of this high homology, SOF from M75 was type-specific and antibody evoked specifically inhibited only SOF produced by M75. Antibodies found in human serum following natural infection also inhibited the SOF of SfbII in a type-specific manner. The results showed that the SfbII protein from M75 is SOF with a high serotype-specific enzyme activity.
Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.
The genus Streptococcus consists of large number of species many of which are pathogenic to humans and animals. Although streptococci have long been considered as extracellular pathogens, they are capable of causing serious invasive infections such as necrotizing fasciitis and meningitis. Streptococcal invasion, therefore, has been a focus of many studies in recent years. Streptococci are efficiently internalized by nonprofessional phagocytes and the current research interest has shifted to determine the role of this invasion in the natural infection process. Moreover, characterization of bacterial and eukaryotic components involved in the uptake process might be useful in developing new strategies for combating streptococcal infections.
Despite the significant impact on human health of Streptococcus pyogenes, an efficacious vaccine has not yet been developed. Here, the potential as a vaccine candidate of a major streptococcal adhesin, the fibronectin-binding protein SfbI, was evaluated. Intranasal immunization of mice with either SfbI alone or coupled to cholera toxin B subunit (CTB) triggered efficient SfbI-specific humoral (mainly IgG) and lung mucosal (14% of total IgA) responses. CTB-immunized control mice were not protected against challenge with S. pyogenes (90%-100% lethality), whereas SfbI-vaccinated animals showed 80% and 90% protection against homologous and heterologous challenge, respectively. Multiple areas of consolidation with diffused cellular infiltrates (macrophages and neutrophils) were observed in lungs from control mice; the histologic structure was preserved in SfbI-vaccinated animals, which occasionally presented focal infiltrates confined to the perivascular, peribronchial, and subpleural areas. These results suggest that SfbI is a promising candidate for inclusion in acellular vaccines against S. pyogenes.
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To identify novel putative staphylococcal adhesins, lithium chloride extraction (an established method for selective surface molecule solubilization) was employed. N-terminal sequencing and functional assays identified a 42-kDa fibronectin-binding protein from Staphylococcus epidermidis as ornithine carbamoyltransferase (OCTase). However, OCTase was not recognizable extracellularly, and this fact together with the fact that LiCl induced DNA release and a decrease in viability suggests that LiCl extraction may not be the method of choice for selective surface molecule extraction from staphylococci.
Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10(-18) M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 x 10(-8) and 2.74 x 10(-7) M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the S. pneumoniae proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci. S. pneumoniae may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.
A common problem in human vaccinology is the limited availability of efficient and non-toxic adjuvants capable of promoting mucosal responses. The potential usefulness of fibronectin-binding protein I (Sfbl) of Streptococcus pyogenes as immunological adjuvant was assessed using ovalbumin (OVA) as a model antigen. Mice were immunized by intranasal route, either with soluble OVA or OVA covalently coupled to Sfbl. Immunization with OVA-Sfbl resulted in the elicitation of about 100-fold higher titers of anti-OVA serum IgG than using OVA alone. The anti-OVA IgG subclass pattern was dominated in both groups of mice by IgG1, followed by IgG2b, IgG2a, and IgG3. Immunization with OVA-Sfbl also resulted in the elicitation of OVA-specific IgA in lung washes (24% of the total IgA), which was absent in mice immunized with OVA alone. Spleen cells from OVA-Sfbl-immunized mice also gave a much stronger proliferative response to restimulation with soluble OVA in vitro. Phenotypic analysis of proliferating cells showed an enrichment in CD4+ T cells, producing a pattern of cytokines (IL-4, IL-5, IL-6 and IL-10) characteristic of Th2-type cells. In contrast to immunization with soluble OVA alone, OVA-Sfbl induced the generation of CD8+ OVA-specific cytotoxic cells. These results demonstrate that Sfbl represents a promising mucosal adjuvant able to substantially improve cellular, humoral and mucosal responses when coupled to an antigen administered by intranasal route.
The invasiveness of 96 group A streptococci (GAS) isolates (56 from throat or skin and 40 from blood) were analyzed. GAS invasion strongly correlated with the source of the isolates, whereas no correlation was observed with the Vir type. Isolates from throat or skin exhibited the highest invasion efficiency (57% were between 0.1% and 10%). In contrast, 77.5% of the blood isolates were noninvasive (efficiency <0.01%) and only 7.5% exhibited rates comparable to those of throat or skin isolates (>0.1%). Immunofluorescence studies of 34 selected isolates showed that attachment and invasion are strain-related. Although isolates with high invasiveness usually exhibit high attachment, isolates that showed high attachment and no invasion or poor attachment and efficient internalization were identified. The ability of GAS to invade and survive within eukaryotic cells may provide bacteria a sure niche, in which they are protected against host defense mechanisms or antimicrobial agents favoring their local persistence.
We have previously shown that fibronectin-binding protein I (SfbI) of Streptococcus pyogenes can act as an adjuvant for mucosal-delivered antigens (Medina, E., Talay, S. R., Chhatwal, G. S. and Guzmán, C. A. 1998. Eur. J. Immunol. 28:1069). To characterize the underlying mechanism of the adjuvancity, we investigated the in vitro stimulating activity of SfbI. The SfbI protein promoted a dose-dependent proliferation of mouse spleen cells. Studies performed using cellular subpopulations showed that proliferation involved B cells and was T cell- and macrophage-independent. SfbI also induced lg production by B cells in a T cell-independent manner. The kinetics of lg isotype accumulation in supernatant fluids and the analysis of Ig-secreting cells suggested that SfbI stimulates B cells expressing different Ig isotypes rather than promoting the isotype switching of single subpopulations. Experiments performed with recombinant proteins encompassing different functional domains of SfbI showed that the fibronectin-binding repeats were responsible for B cell activation. The sera from mice immunized by the intranasal route with SfbI did not react with either double-stranded DNA, cardiolipin or collagen. Interestingly, stimulation with Sfbl also resulted in the up-regulation of MHC class 11 molecules expression by B cells and macrophages. The elucidation of the underlying molecular events to the immunomodulatory effect exerted by SfbI will facilitate the exploitation of the potential of this molecule for the generation of mucosal vaccines.
Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA'-'lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of beta-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37 degrees C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.
The specific binding of vitronectin to Streptococcus pyogenes is believed to play an important role in the infection process by mediating adherence of the bacteria to host cells. The domain of vitronectin involved in the interaction with S. pyogenes is unknown. In the present study, we constructed a vitronectin random epitope phage display library, which was used to pan against intact cells of S. pyogenes. Several phage-displayed vitronectin peptides containing a hydrophobic pentapeptide motif within the hemopexin-type repeats were found to bind to streptococci. These data were supported by competition experiments, in which a representative 23-amino acid synthetic vitronectin peptide comprising part of a hemopexin-type repeat inhibited binding of the bacteria to vitronectin, while a control peptide with identical amino acid composition but a scrambled sequence had no effect. Moreover, cells of S. pyogenes were shown to bind to the synthetic peptide as well as to immobilized hemopexin, whose structural homology to the hemopexin-type repeats in the vitronectin molecule has long been underlined. Soluble vitronectin could inhibit streptococcal binding to immobilized hemopexin. These results provide first evidence for a biological role of hemopexin itself and respective repeats in vitronectin in bacterial binding, suggesting that during an infection process these or other hemopexin-type repeat-containing proteins could be potential targets for bacterial attachment and subsequent colonization.
Vitronectin is a multifunctional serum protein which provides a unique regulatory link between cell adhesion, humoral defense mechanism and the hemostatic system, and the heparin-binding properties of vitronectin are thought to have participated in various functional aspects. In addition to the carboxy-terminal glycosaminoglycan-binding motif, we report on two novel heparin-binding domains which were identified using phage display technique. One heparin-binding domain is located between amino acids Asp82 and Cys137 at the end of the connector region, while the other is in the second hemopexin-type repeat, between amino acids Lys175 and Asp219 of the vitronectin molecule. Our findings may shed new light to the activities of vitronectin and its binding to cells, which could not be explained solely on the basis of the known heparin-binding domain.
The invasion of group B streptococci (GBS) in HEp-2 epithelial cells was analyzed by electron microscopy and a quantitative antibiotic survival assay. Invasion of GBS involved intimate attachment of streptococcal chains, engulfment of the adherent bacteria by cellular protrusions, entry of the bacteria in a 'polar' fashion and formation of membrane-bound vacuoles in which most of the intracellular streptococci resided. At later stages of infection bacteria were also found free in the cytoplasm. Efficient uptake of streptococci by HEp-2 cells occurred within 20 min and live intracellular bacteria were detectable up to 48 h post-infection. Invasion of GBS required activation of the eukaryotic actin microfilament system involving, at least partially, protein kinase signal transduction pathways. Invasion was inhibited in a dose-dependent manner by decreasing extracellular Ca2+ levels as well as by substances known to interfere with eukaryotic calcium regulatory systems. These results suggest that GBS invade HEp-2 cells by triggering calcium-dependent phagocytosis-like internalization mechanisms and persist intracellularly both in vacuoles and free in the cytoplasm.
Bordetella bronchiseptica has the ability to invade and survive intracellularly. This potential to survive for extended periods within eukaryotic cells might play an important role in the pathogenesis of the infections caused by this microorganism. The bacterial factors involved in this process, however, have not yet been determined. In this study we have identified an acid phosphatase produced by B. bronchiseptica, but not by other Bordetella spp. The expression of this enzyme was demonstrated to be strictly regulated by temperature (optimal expression at 30 degrees C) and seems to be partially repressed by the product of the bvg locus. The enzyme was localized in the cytoplasmic fraction, the optimal activity was observed at pH 5.5, and the apparent molecular mass obtained by zymogram was 40 kDa. To further investigate the pathogenic role of this enzyme, mutants lacking acid phosphatase activity were obtained from both bvg-positive and bvg-negative parental strains using minitransposons. The growth pattern of these recombinant clones in vitro was similar to the parent strains, however, the tested clones exhibited a significant reduction (P < or = 0.05) in their intracellular survival ability. This newly described acid phosphatase from B. bronchiseptica seems to play a role in intracellular survival, and therefore represents a novel pathogenicity factor.
The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 x 10(-9) M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA- or SC-binding domain is located in the N-terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA-binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.