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Biomedical subjects

G S Chhatwal

Publications and source records attributed to G S Chhatwal.

At least 19 recordsLinked to original sources
PubMed2006

Genes for the majority of group a streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease.

BACKGROUND: The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. METHODS: In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. RESULTS: Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. CONCLUSIONS: Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/or host factors.

Humans↗
PubMed2006

Trigger for group A streptococcal M1T1 invasive disease.

The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant delta speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous (s.c.) infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a subpopulation of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

Animals↗
PubMed2004

Intranasal vaccination with streptococcal fibronectin binding protein Sfb1 fails to prevent growth and dissemination of Streptococcus pyogenes in a murine skin infection model.

Fibronectin binding protein F1 (Sfb1) of Streptococcus pyogenes (group A streptococcus [GAS]) is a well-characterized adhesin that has been shown to induce protection in mice against a lethal intranasal GAS challenge after intranasal immunization with cholera toxin B subunit (CTB) as adjuvant. With a murine skin infection model, we have shown that Sfb1/CTB vaccination neither elicits opsonizing antibodies nor prevents systemic bacterial growth and dissemination to internal organs after a subcutaneous GAS challenge. These results indicate that an Sfb1-based vaccine should be complemented with additional protective antigens in order to be used in areas such as the tropical north of Australia, where the skin is the primary route of entry for invasive streptococcal diseases.

Adhesins, Bacterial↗
PubMed2004

Immune responses of a liposome/ISCOM vaccine adjuvant against streptococcal fibronectin binding protein 1 (Sfb1) in mice.

BACKGROUND & OBJECTIVES: The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4. METHODS: Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes. RESULTS: Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P<0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P<0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%). INTERPRETATION & CONCLUSION: These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

Adhesins, Bacterial↗
PubMed2002

Hydrogen peroxide-mediated killing of Caenorhabditis elegans by Streptococcus pyogenes.

Caenorhabditis elegans is currently introduced as a new, facile, and cheap model organism to study the pathogenesis of gram-negative bacteria such as Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium. The mechanisms of killing involve either diffusible exotoxins or infection-like processes. Recently, it was shown that also some gram-positive bacteria kill C. elegans, although the precise mechanisms of killing remained open. We examined C. elegans as a pathogenesis model for the gram-positive bacterium Streptococcus pyogenes, a major human pathogen capable of causing a wide spectrum of diseases. We demonstrate that S. pyogenes kills C. elegans, both on solid and in liquid medium. Unlike P. aeruginosa and S. enterica serovar Typhimurium, the killing by S. pyogenes is solely mediated by hydrogen peroxide. Killing required live streptococci; the killing capacity depends on the amount of hydrogen peroxide produced, and killing can be inhibited by catalase. Major exotoxins of S. pyogenes are not involved in the killing process as confirmed by using specific toxin inhibitors and knockout mutants. Moreover, no accumulation of S. pyogenes in C. elegans is observed, which excludes the involvement of infection-like processes. Preliminary results show that S. pneumoniae can also kill C. elegans by hydrogen peroxide production. Hydrogen peroxide-mediated killing might represent a common mechanism by which gram-positive, catalase-negative pathogens kill C. elegans.

Animals↗
PubMed2001

Genetic control of susceptibility to group A streptococcal infection in mice.

The influence of genetic background on the ability to control infection with group A streptococci was investigated in different inbred strains of mice. Whereas BALB/c, C57BL/10, and DBA/2 mice were the most resistant strains, with lower bacteria loads and higher survival times, C3H/HeN and CBA/J mice exhibited substantially higher bacterial growth and 100% mortality. Differences in susceptibility were not dependent on the inoculum size. Resistance was influenced by sex, with males being much more susceptible than females. B cell- and T cell-deficient mice from the resistant background were as resistant to infection as were immunocompetent mice, which suggests that the effector mechanisms are independent of adaptive immunity. These results demonstrate for the first time the influence of genetic background and sex on susceptibility to infection with Streptococcus pyogenes in mice. The use of this mouse model of group A streptococcal infection will allow for a better definition of parameters involved in the outcome of the disease.

Animals↗
PubMed2001

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.

Bacterial Adhesion↗
PubMed2001

alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface.

Binding of human plasminogen to Streptococcus pneumoniae and its subsequent activation promotes penetration of bacteria through reconstituted basement membranes. In this study, we have characterized a novel pneumococcal surface protein with a molecular mass of 47 kDa, designated Eno, which specifically binds human plasmin(ogen), exhibits alpha-enolase activity and is necessary for viability. Using enzyme assays, we have confirmed the alpha-enolase activity of both pneumococcal surface-displayed Eno and purified recombinant Eno protein. Immunoelectron microscopy indicated the presence of Eno in the cytoplasm as well as on the surface of encapsulated and unencapsulated pneumococci. Plasminogen-binding activity was demonstrated with whole pneumococcal cells and purified Eno protein. Binding of activated plasminogen was also shown for Eno; however, the affinity for plasmin is significantly reduced compared with plasminogen. Results from competitive inhibition assays indicate that binding is mediated through the lysine binding sites in plasmin(ogen). Carboxypeptidase B treatment and amino acid substitutions of the C-terminal lysyl residues of Eno indicated that the C-terminal lysine is pivotal for plasmin(ogen)-binding activity. Eno is ubiquitously distributed among pneumococcal serotypes, and binding experiments suggested the reassociation of secreted Eno to the bacterial cell surface. The reassociation was also confirmed by immunoelectron microscopy. The results suggest a mechanism of plasminogen activation for human pathogens that might contribute to their virulence potential in invasive infectious processes.

Amino Acid Sequence↗
PubMed2001

Characterization of the domain of fibronectin-binding protein I of Streptococcus pyogenes responsible for elicitation of a protective immune response.

Fibronectin-binding protein I (SfbI) represents a major adhesin of Streptococcus pyogenes. Mice were intranasally immunized with recombinant proteins spanning different portions of SfbI to identify the minimal fragment able to elicit a protective response against a lethal challenge with S. pyogenes. The strongest cellular responses and the highest levels of antigen-specific secretory immunoglobulin A (IgA) were detected in mice immunized with the fibronectin-binding region of SfbI. In contrast, animals vaccinated with a polypeptide spanning the aromatic and proline-rich regions showed the highest titers and fastest IgG response in serum. Vaccination with either SfbI without a membrane anchor and signal peptide or a polypeptide encompassing its fibronectin-binding regions resulted in efficient protection against heterologous challenge (60% and 80%, respectively), whereas the use of a polypeptide lacking this region conferred marginal protection (10%) with respect to the control group (0%). These results demonstrate that the fibronectin-binding region of SfbI is a promising candidate antigen for developing anti-S. pyogenes vaccines.

Adhesins, Bacterial↗
PubMed2001

Identification and characterization of a novel secreted immunoglobulin binding protein from group A streptococcus.

Immunoglobulin binding proteins are one of several pathogenicity factors which have been associated with invasive disease caused by group A streptococci. The surface-bound M and M-like proteins of Streptococcus pyogenes are the most characterized of these immunoglobulin binding proteins, and in most cases they bind only a single antibody class. Here we report the identification of a novel non-M-type secreted protein, designated SibA (for secreted immunoglobulin binding protein from group A streptococcus), which binds all immunoglobulin G (IgG) subclasses, the Fc and Fab fragments, and also IgA and IgM. SibA has no significant sequence homology to any M-related proteins, is not found in the vir regulon, and contains none of the characteristic M-protein regions, such as the A or C repeats. Like M proteins, however, SibA does have relatively high levels of alanine, lysine, glutamic acid, leucine, and glycine. SibA and M proteins also share an alpha-helical N-terminal secondary structure which has been previously implicated in immunoglobulin binding in M proteins. Evidence presented here indicates that this is also the case for SibA. SibA also has regions of local similarity with other coiled-coil proteins such as Listeria monocytogenes P45 autolysin, human myosin heavy chain, macrogolgin, and Schistoma mansoni paramyosin, some of which are of potential significance since cross-reactive antibodies between myosin proteins and M proteins have been implicated in the development of the autoimmune sequelae of streptococcal disease.

Amino Acid Sequence↗
PubMed2001

Identification and molecular analysis of PcsB, a protein required for cell wall separation of group B streptococcus.

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated pcsB, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (M(r), 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from Listeria monocytogenes. Northern blot analysis revealed a monocistronic transcriptional organization for pcsB in GBS. Insertional inactivation of pcsB in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.

Anti-Bacterial Agents↗
PubMed2000

A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3.

Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.

ADP Ribose Transferases↗
PubMed2000

Pharyngeal carriage of group C and group G streptococci and acute rheumatic fever in an Aboriginal population.

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) represent an autoimmune response to group A streptococcal pharyngitis. In the Aboriginal population of the Northern Territory of Australia, some of the highest rates of ARF in the world have been reported, although throat carriage rates of group A streptococcus in this population are extremely low and symptomatic group A streptococcal pharyngitis is uncommon; by contrast, carriage rates of group C and G streptococci are high. Therefore, we questioned the involvement of these groups in ARF and showed that they have the potential to elicit an autoimmune response that may trigger ARF.

Adult↗
PubMed2000

Protein A is the von Willebrand factor binding protein on Staphylococcus aureus.

Endovascular infection is a highly critical complication of invasive Staphylococcus aureus disease. For colonization, staphylococci must first adhere to adhesive endovascular foci. Von Willebrand factor (vWF) is a large, multimeric glycoprotein mediating platelet adhesion at sites of endothelial damage. Earlier it was demonstrated that vWF binds to and promotes the surface adhesion of S. aureus, prompting this effort to identify the vWF adhesin. In Western ligand assays of S. aureus lysates, staphylococcal protein A (SPA) was recognized by purified vWF. Surface plasmon resonance demonstrated the binding of soluble vWF to immobilized recombinant protein A with a K(d) of 1.49 x 10(-8) mol/L. Using flow cytometry, the binding of fluorescein isothiocyanate-labeled vWF to S. aureus was found to be saturable and inhibitable by unlabeled vWF, antiprotein-A antibodies, or IgG. Isogenic Deltaspa::Tc(r) mutants were constructed by the insertion of a tetracycline resistance cassette into spa using allelic replacement, and it exhibited decreased binding of soluble vWF and decreased adhesion to vWF-adsorbed surfaces. The interaction was restored on complementation of the mutants with spa-containing plasmid pSPA7235. In conclusion, protein A confers interaction of S. aureus with soluble and immobilized vWF in a newly discovered function characterizing protein A as a novel member of the staphylococcal surface protein adhesin superfamily and suggesting its potential role in the pathogenesis of endovascular staphylococcal disease.

Bacterial Adhesion↗
PubMed2000

Species-specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif.

SpsA, a pneumococcal surface protein belonging to the family of choline-binding proteins, interacts specifically with secretory immunglobulin A (SIgA) via the secretory component (SC). SIgA and free SC from mouse, rat, rabbit and guinea-pig failed to interact with SpsA indicating species-specific binding to human SIgA and SC. SpsA is the only pneumococcal receptor molecule for SIgA and SC as confirmed by complete loss of SIgA and SC binding to a spsA mutant. Analysis of recombinant SpsA fusion proteins showed that the binding domain is located in the N-terminal region of SpsA. By the use of different truncated N-terminal SpsA fusion proteins, the minimum binding domain was shown to be composed of 112 amino acids (residues 172-283). The sequence of this 112-amino-acids domain was used to spot synthesize 34 overlapping peptides, consisting of 15 amino acids each, with an offset of three amino acids on a cellulose membrane. One of the peptides reacted specifically with both SIgA and SC. By using a second membrane with immobilized synthetic peptides of decreasing length containing parts of the identified 15-amino-acid motif a hexapeptide, YRNYPT was identified as the binding motif for SC and SIgA. SpsA proteins with a size smaller than the assay-positive domain of 112 amino acids were able to inhibit the interaction of SIgA and pneumococci provided they contained the binding motif. The results indicated that the hexapeptide YRNYPT located in SpsA of pneumococcal strain type 1 (ATCC 33400) between amino acids 198 and 203 is involved in SIgA and SC binding. Because synthetic peptides containing only parts of the hexapeptide also assayed positive, these results further suggest that at least the amino acids YPT of the identified hexapeptide are critical for binding to SC and SIgA. Amino acid substitutions in the identified putative binding motif abolished SC-/SIgA-binding activity of the mutated SpsA protein, confirming the functional activity of this hexapeptide and the critical role of the amino acids YPT in SC and SIgA binding. Identification of this motif, which is highly conserved in SpsA protein among different serotypes, might contribute towards a new peptide based vaccine strategy.

Amino Acid Sequence↗
PubMed2000

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, Sfbl protein, has been implicated in both adherence and invasion processes. Invasion of Sfbl-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 Sfbl; and A8, which is Sfbl negative and is unable to bind fibronectin. In strain A40, Sfbl was the main factor required for attachment and invasion by using fibronectin as a bridging molecule and the alpha5beta1 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 alpha5beta1 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.

Actins↗
PubMed2000

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells.

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii, we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria-fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.

Adhesins, Bacterial↗