Search

Streptococcus pyogenes (group A Streptococcus (GAS)) causes ∼700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in serotype geographical distribution, and the production of antibodies crossreactive with human tissue that may lead to autoimmune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors. Here, we applied a proteomic analysis of serotype M1T1 GAS cell wall extracts for the purpose of vaccine development. This approach catalogued several anchorless proteins and identified two protective vaccine candidates, arginine deiminase and trigger factor. These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting =99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes. Our study demonstrates anchorless cell surface antigens as promising vaccine candidates for the prevention of GAS disease. © Springer-Verlag 2012.

A peptide (J14) containing conformationally restricted epitopes from the M protein of group A streptococcus (GAS) is capable of eliciting protective immune response against GAS infection. However, the protective response may be lost possibly due to its weak secondary-structure when the antigen is fused with other antigens in a recombinant polyepitope vaccine construct. We previously showed that JJo, a conformationally stabilized derivative of dimeric J14, overcomes this problem. We now show that anti JJo antibodies react with diverse GAS isolates found in the Indian sub-continent and that these antibodies are opsonic for GAS. The GAS strains used in this study were isolated from throat and skin swabs from Mumbai, Chennai and Vellore. Sera from mice immunized with recombinant JJo peptide were tested by ELISA, immunofluorescence, flow-cytometry, indirect bactericidal assay and mouse challenge assays to determine specific immunogenicity, opsonic functions and protection against an Indian isolate. We propose that JJo is a robust antigen suitable for inclusion in recombinant multi-epitope vaccines which are potentially affordable option for the pediatric population of developing countries.

The group AStreptococcus(GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. TheprtF2gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes ofprtF2which are mutually exclusive. The two genotypes have been identified previously aspfbpandfbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated thepfbptype and thefbaBtype. Phylogenetic analysis of sevenpfbptypes, 14fbaBtypes, and 11prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that thepfbptype had a recent origin compared to thefbaBtype. A comparison of multiple DNA sequences of thepfbpandfbaBtypes revealed a mosaic pattern for the amino-terminal region of thepfbptypes. ThefbaBtype is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of thepfbpgenotype withsof(84.2%), while thefbaBgenotype was found in a majority of the GAS strains negative forsof(90.6%), indicating that these twoprtF2subtypes may be under different selective pressures.

Shiga toxin-producing Escherichia coli (STEC) strains possessing genes for enterohemolysin ( ehxA ) and/or intimin ( eae ), referred to here as complex STEC (cSTEC), are more commonly recovered from the feces of humans with hemolytic uremic syndrome and hemorrhagic colitis than STEC strains that do not possess these accessory virulence genes. Ruminants, particularly cattle and sheep, are recognized reservoirs of STEC populations that may contaminate foods destined for human consumption. We isolated cSTEC strains from the feces of longitudinally sampled pasture-fed sheep, lot-fed sheep maintained on diets comprising various combinations of silage and grain, and sheep simultaneously grazing pastures with cattle to explore the diversity of cSTEC serotypes capable of colonizing healthy sheep. A total of 67 cSTEC serotypes were isolated, of which 21 (31.3%), mainly isolated from lambs, have not been reported. Of the total isolations, 58 (86.6%) were different from cSTEC serotypes isolated from a recent study of longitudinally sampled healthy Australian cattle (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68: 6439-6445, 2002). Our data suggest that cSTEC serotypes O5:H − , O75:H8, O91:H − , O123:H − , and O128:H2 are well adapted to colonizing the ovine gastrointestinal tract, since they were the most prevalent serotypes isolated from both pasture-fed and lot-fed sheep. Collectively, our data show that Australian sheep are colonized by diverse cSTEC serotypes that are rarely isolated from healthy Australian cattle.

Unlike Shiga toxin 2 ( stx 2 ) genes, most nucleotide sequences of Shiga toxin 1 ( stx 1 ) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae , and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx 1 among STEC isolates derived from human or animal sources. However stx 1OX3 , originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx 1 subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx 1c . In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx 1c from other stx 1 subtypes. The PCR-RFLP assay was used to study 378 stx 1 -containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene ( ehxA , eaeA , and/or stx 2 ); the combination stx 1 , stx 2 , and ehxA was the most common (175 of 355 [49.3%]), and 90 of 355 (25.4%) isolates possessed eaeA . One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx 1c alone and belonged to 41 serotypes. Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H − , O128:H2, and O123:H − . Fifty-two of 207 isolates (25.1%) possessed an stx 1 subtype; 27 (51.9%) of these belonged to serotype O91:H − . Nineteen of 207 isolates (9.2%) contained both stx 1c and stx 1 subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx 1 subtype, 6 isolates possessed stx 1c , and the remaining isolate possessed both stx 1c and stx 1 subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx 1c (serotypes O5:H − , O75:H8/H40, O81:H26, O88:H25, O104:H − /H7, O123:H − /H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep. Serotypes containing stx 1 recovered from cattle rarely were the same as those isolated from sheep. Although an stx 1c subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx 1c . Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H − isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H − (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep.

Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx 2 subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx 2 subtype in the ovine isolates was shown to be stx 2d-Ount (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H − /H8/H40, O91:H − , O123:H − , O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H − possessed a stx 2d-O111/OX3a subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx 2d ( stx 2d-Ount/O111/OX3a ) subtype. These studies suggest that a specific stx 2 subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.

Gram-positive streptococci are non-motile, chain-forming bacteria commonly found in the normal oral and bowel flora of warm-blooded animals. Over the past decade, a proteomic approach combining 2-DE and MS has been used to systematically map the cellular, surface-associated and secreted proteins of human pathogenic streptococcal species. The public availability of complete streptococcal genomic sequences and the amalgamation of proteomic, genomic and bioinformatic technologies have recently facilitated the identification of novel streptococcal vaccine candidate antigens and therapeutic agents. The objective of this review is to examine the constituents of the streptococcal cell wall and secreted proteome, the mechanisms of transport of surface and secreted proteins, and describe the current methodologies employed for the identification of novel surface-displayed proteins and potential vaccine antigens.

A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis.

The intimin gene eae , located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli . EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (α1, α2, β, γ, κ, ε, η, ι, λ, θ, and ζ) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int-μ, Int-ν, and Int-ξ. The PCR-RFLP assay was used to screen 213 eae -positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx -negative ( stx − ) and ehxA -positive ( ehxA + ) isolates, and 42 were stx − and ehxA -negative isolates. Int-β, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-ζ (39 of 213 [18.3%] isolates; 11 serotypes), Int-θ (25 of 213 [11.7%] isolates; 15 serotypes), Int-γ (19 of 213 [8.9%] isolates; 9 serotypes), and Int-ε (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes α1, α2, κ, λ, ξ, μ, ν, and ι were infrequently identified; and Int-η was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (α, β-ξ, γ, κ, ε-η-ν, ι-μ, λ, θ, and ζ). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.