Protein adhesins as vaccine antigens for Group A Streptococcus.

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development.

The ability of Group A streptococcus to adhere to immortalized human skin versus throat cell lines does not reflect their predicted tissue tropism.

OBJECTIVES: To determine if emm pattern-inferred tissue tropisms of Group A streptococcus strains is reflected in their ability to adhere to immortalized human HaCat (keratinocyte) and Detroit 562 (pharyngeal) cell lines. METHODS: Human epithelial cell line monolayers were inoculated with Group A streptococcus, and the percentage of adhered bacteria after a 1-h incubation period was calculated. RESULTS: Of the 21/69 inferred-skin-tropic, and the 25/69 inferred-throat-tropic isolates no preferential adherence was observed to a particular cell line. The 23/69 strains classified as 'generalists', however, showed an overall greater ability to adhere to both cell lines. CONCLUSIONS: Predicted tissue-tropism based on emm patterns is not reflected by preferential adherence to a specific cell line, suggesting that early adhesion events may not be as important in establishing infection at a particular ecological niche than originally expected.

Pili in Gram-negative and Gram-positive bacteria - structure, assembly and their role in disease.

Many bacterial species possess long filamentous structures known as pili or fimbriae extending from their surfaces. Despite the diversity in pilus structure and biogenesis, pili in Gram-negative bacteria are typically formed by non-covalent homopolymerization of major pilus subunit proteins (pilins), which generates the pilus shaft. Additional pilins may be added to the fiber and often function as host cell adhesins. Some pili are also involved in biofilm formation, phage transduction, DNA uptake and a special form of bacterial cell movement, known as 'twitching motility'. In contrast, the more recently discovered pili in Gram-positive bacteria are formed by covalent polymerization of pilin subunits in a process that requires a dedicated sortase enzyme. Minor pilins are added to the fiber and play a major role in host cell colonization.This review gives an overview of the structure, assembly and function of the best-characterized pili of both Gram-negative and Gram-positive bacteria.

Induction of interleukin-8 in human neutrophils after MHC class II cross-linking with superantigens.

Neutrophils have been shown to express major histocompatibility complex class II (MHC II) after stimulation. However, reports concerning the functional effect of MCH II expression are still lacking. In our hands, granulocyte-monocyte colony-stimulating factor (GM-CSF) alone and in combination with interferon (IFN)-gamma, but not IFN-gamma or interleukin (IL)-3, induced a significant level of expression of human leukocyte antigen DR on neutrophils. The addition of staphylococcal enterotoxin E to neutrophils resulted in a significant increase in IL-8 production only after prestimulation with GM-CSF alone or in combination with IFN-gamma but had no effect on neutrophils preincubated with IFN-gamma alone or IL-3. Staphylococcal enterotoxin A, another bivalent superantigen, also stimulated production of IL-8 by preincubated polymorphonuclear neutrophils, whereas staphylococcal enterotoxin A mutants that are not able to cross-link MHC II molecules failed to induce IL-8 production. Taken together, our results clearly demonstrate that after induction of MHC II, neutrophils are able to respond to MHC II-specific stimulation. These findings support the ideas that the induced MHC II complex is completely functional and that neutrophils may be able to present antigens.

Immunological and biochemical characterization of streptococcal pyrogenic exotoxins I and J (SPE-I and SPE-J) from Streptococcus pyogenes.

Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II beta-chain in a zinc-dependent mode but not to the generic MHC class II alpha-chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DRbeta-SPE-J-SPE-J-HLA-DRbeta. SPE-I preferably stimulates T cells bearing the Vbeta18.1 TCR, which is not targeted by any other known SAG: SPE-J almost exclusively stimulates Vbeta2.1 T cells, a Vbeta that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistinguishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C.

Pyrogenicity and cytokine-inducing properties of Streptococcus pyogenes superantigens: comparative study of streptococcal mitogenic exotoxin Z and pyrogenic exotoxin A.

Streptococcal mitogenic exotoxin Z (SMEZ), a superantigen derived from Streptococcus pyogenes, provoked expansion of human lymphocytes expressing the Vbeta 2, 4, 7 and 8 motifs of T-cell receptor. SMEZ was pyrogenic in rabbits and stimulated the expression of the T-cell activation markers CD69 and cutaneous lymphocyte-associated antigen. A variety of cytokines was released by human mononuclear leukocytes stimulated with SMEZ, which was 10-fold more active than streptococcal pyrogenic exotoxin A. Th2-derived cytokines were elicited only by superantigens and not by streptococcal cells.

Conservation and variation in superantigen structure and activity highlighted by the three-dimensional structures of two new superantigens from Streptococcus pyogenes.

Bacterial superantigens (SAgs) are a structurally related group of protein toxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They are implicated in a range of human pathologies associated with bacterial infection whose symptoms result from SAg-mediated stimulation of a large number (2-20%) of T-cells. At the molecular level, bacterial SAgs bind to major histocompatability class II (MHC-II) molecules and disrupt the normal interaction between MHC-II and T-cell receptors (TCRs). We have determined high-resolution crystal structures of two newly identified streptococcal superantigens, SPE-H and SMEZ-2. Both structures conform to the generic bacterial superantigen folding pattern, comprising an OB-fold N-terminal domain and a beta-grasp C-terminal domain. SPE-H and SMEZ-2 also display very similar zinc-binding sites on the outer concave surfaces of their C-terminal domains. Structural comparisons with other SAgs identify two structural sub-families. Sub-families are related by conserved core residues and demarcated by variable binding surfaces for MHC-II and TCR. SMEZ-2 is most closely related to the streptococcal SAg SPE-C, and together they constitute one structural sub-family. In contrast, SPE-H appears to be a hybrid whose N-terminal domain is most closely related to the SEB sub-family and whose C-terminal domain is most closely related to the SPE-C/SMEZ-2 sub-family. MHC-II binding for both SPE-H and SMEZ-2 is mediated by the zinc ion at their C-terminal face, whereas the generic N-terminal domain MHC-II binding site found on many SAgs appears not to be present. Structural comparisons provide evidence for variations in TCR binding between SPE-H, SMEZ-2 and other members of the SAg family; the extreme potency of SMEZ-2 (active at 10(-15) g ml-1 levels) is likely to be related to its TCR binding properties. The smez gene shows allelic variation that maps onto a considerable proportion of the protein surface. This allelic variation, coupled with the varied binding modes of SAgs to MHC-II and TCR, highlights the pressure on SAgs to avoid host immune defences.

The streptococcal superantigen SMEZ exhibits wide allelic variation, mosaic structure, and significant antigenic variation.

The frequencies of the newly identified streptococcal superantigen genes smez, spe-g, and spe-h were determined in a panel of 103 clinical isolates collected between 1976 and 1998 at various locations throughout New Zealand. smez and spe-g were found in every group A Streptococcus (GAS) isolate, suggesting a chromosomal location. The spe-h gene was found in only 24% of the GAS isolates and is probably located on a mobile DNA element. The smez gene displays extensive allelic variation and appears to be in linkage equilibrium with the M/emm type. 22 novel smez alleles were identified from 21 different M/emm types in addition to the already reported alleles smez and smez-2 with sequence identities between 94. 5 and 99.9%. Three alleles are nonfunctional due to a single base pair deletion. The remaining 21 alleles encode distinct SMEZ variants. The mosaic structure of the smez gene suggests that this polymorphism has arisen from homologous recombination events rather than random point mutation. The recently resolved SMEZ-2 crystal structure shows that the polymorphic residues are mainly surface exposed and scattered over the entire protein. The allelic variation did not affect either Vbeta specificity or potency, but did result in significant antigenic differences. Neutralizing antibody responses of individual human sera against different SMEZ variants varied significantly. 98% of sera completely neutralized SMEZ-1, but only 85% neutralized SMEZ-2, a very potent variant that has not yet been found in any New Zealand isolate. SMEZ-specific Vbeta8 activity was found in culture supernatants of 66% of the GAS isolates, indicating a potential base for the development of a SMEZ targeting vaccine.

Superantigens - powerful modifiers of the immune system.

Superantigens are powerful microbial toxins that activate the immune system by binding to class II major histocompatibility complex and T-cell receptor molecules. They cause a number of diseases characterized by fever and shock and are important virulence factors for two human commensal organisms, Staphylococcus aureus and Streptococcus pyogenes, as well as for some viruses. Their mode of action and variation around the common theme of over-stimulating T cells, provides a rich insight into the constant battle between microbes and the immune system.

Superantigens in human disease.

Superantigens have been implicated in a wide variety of human diseases. Yet, solid evidence for their role in pathogenesis is available only for Toxic Shock Syndrome and a few other conditions. This evidence is critically reviewed herein.

Identification and characterization of novel superantigens from Streptococcus pyogenes.

Three novel streptococcal superantigen genes (spe-g, spe-h, and spe-j) were identified from the Streptococcus pyogenes M1 genomic database at the University of Oklahoma. A fourth novel gene (smez-2) was isolated from the S. pyogenes strain 2035, based on sequence homology to the streptococcal mitogenic exotoxin z (smez) gene. SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ and streptococcal pyrogenic exotoxin (SPE)-C, whereas SPE-H is most similar to the staphylococcal toxins than to any other streptococcal toxin. Recombinant (r)SMEZ, rSMEZ-2, rSPE-G, and rSPE-H were mitogenic for human peripheral blood lymphocytes with half-maximal responses between 0.02 and 50 pg/ml (rSMEZ-2 and rSPE-H, respectively). SMEZ-2 is the most potent superantigen (SAg) discovered thus far. All toxins, except rSPE-G, were active on murine T cells, but with reduced potency. Binding to a human B-lymphoblastoid line was shown to be zinc dependent with high binding affinity of 15-65 nM. Evidence from modeled protein structures and competitive binding experiments suggest that high affinity binding of each toxin is to the major histocompatibility complex class II beta chain. Competition for binding between toxins was varied and revealed overlapping but discrete binding to subsets of class II molecules in the hierarchical order (SMEZ, SPE-C) > SMEZ-2 > SPE-H > SPE-G. The most common targets for the novel SAgs were human Vbeta2.1- and Vbeta4-expressing T cells. This might reflect a specific role for this subset of Vbetas in the immune defense of gram-positive bacteria.

Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence.

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA-) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA+ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5' end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 Mr cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 Mr and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.

Sequence analysis and characterization of the hmw gene cluster of Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp) cytadherence requires the proper anchoring of cytadhesin proteins in the mycoplasmal membrane at an attachment organelle through their interaction with a cytoskeleton-like network of accessory proteins that includes HMW1 and HMW3. Approximately 8.25 kb of Mp DNA was sequenced, beginning at the 3' end of the hmw3 gene and continuing through hmw1. Comparison of the resulting deduced amino acid (aa) sequence with N terminus and internal peptide aa sequences from purified HMW1 permitted definitive identification of hmw1. HMW1 was characterized with respect to structure, hydrophobicity, possible phosphoacceptor sites and expression of the Mp recombinant protein in Escherichia coli. In addition, HMW1 membrane topography was examined for antibody accessibility on the mycoplasmal surface. hmw3 and hmw1 flank four open reading frames (ORFs) spanning approximately 4.3 kb and in the same orientation as the hmw genes. The sequences of their deduced products were evaluated for likely structural features and comparison with protein data banks. Finally, the Mp rpsD analog was identified immediately downstream from hmw1.

The P200 protein of Mycoplasma pneumoniae shows common features with the cytadherence-associated proteins HMW1 and HMW3.

The gene coding for the P200 protein of the bacterium, Mycoplasma pneumoniae (Mp), was cloned and sequenced. The sequence-derived data and biochemical data indicated that P200 has several features in common with the well characterized cytadherence-associated proteins, HMW1 and HMW3. These features consist of abnormal migration in SDS-PAGE, a central acidic domain with a high Pro content, repeated peptide blocks within the Pro-rich domain and P200 partitioning similar to HMW1 and HMW3 in the insoluble fraction after extraction of Mp with the detergent Triton X-100.

The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH.

Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta- and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated beta-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain.

Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins.

Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location. This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M. pneumoniae genome and expressing them in Escherichia coli. Positive clones were identified using antibodies against specific fractions of M. pneumoniae. The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein data-bank searches. Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M. pneumoniae proteins by immunoscreening Western blots of SDS-acrylamide gels from M. pneumoniae cell extracts.