Characterisation of the PrtM maturase of Streptococcus equi; a proven virulence factor in strangles

Streptococcus equi subspecies equi (S. equi) is the pathogen responsible for the prevalent and highly contagious equine disease called strangles. Strangles has been reported worldwide as a cause of a high level of animal suffering and economic loss. S. equi is susceptible to many antibiotics in vitro, but relapse due to insufficient vascularity often renders such treatment ineffective. Getting effective and universally accepted vaccines against S. equi have been slow mainly because of safety concerns. It was previously reported that colonization of air interface organ cultures, after inoculation with a mutant strain (ΔprtM138-213) deficient in the putative maturase lipoprotein (PrtM, a homologue of the pneumococcal PpmA) was less than that seen in cultures which were infected with wild-type S. equi strain 4047 (Hamilton et al, 2006), indicating that PrtM is a major virulence factor in strangles. It has also been demonstrated by in vitro and in vivo studies that many streptococcal adhesins, for example, serve as colonization or virulence factors and this makes them attractive targets for therapeutic and preventive strategies against streptococcal infections (Nobbs et al., 2009). S. equi adhesins or other colonization factors may be substrates for PrtM. Understanding PrtM is key to designing drugs or vaccines against the equine S. equi infection and strangles. In this research, advanced biomolecular techniques were systematically applied to investigate and characterise PrtM, and to evaluate its potential as a therapeutic or vaccine target. Bioinformatics, microbiological, biochemical and molecular biology techniques were used in screening the S. equi WT 4047 and Mutant (prtM138-213) strains to evaluate the immunogenicity and conservation of PrtM. Proteomics techniques: two-dimensional gel electrophoresis and mass spectrometry were employed in evaluating the cell-associated and secreted protein extracts of both the S. equi WT 4047 and mutant (prtM138-213) strains. In this study, genetic engineering technology involving targeted domain knock-out and/or knock-in, was employed in producing the central domain recombinant protein and mutant (prtM138-213) revertants. Following cloning, over-expression and purification of the full length and central domain, biochemical data on the PrtM protein (kcat/KM for S. equi 4047 PrtM full length recombinant protein = 5.84 x 106 /M/s) were derived via enzyme(peptydylprolyl isomerase - PPIase) assay; and crystallography was applied in an attempt to derive structural data on the PrtM protein. Advanced biomolecular techniques (including Western blots and Proteomics) were employed in screening the complemented mutants. It has been proven from this research that the PrtM of S. equi 4047 is involved in adaptation to NaCl stress and in regulating sensitivity to antibiotics; PrtM may have roles in speeding up the synthesis of hyaluronic acid and in the folding or remodeling of HPr Kinase. The parvulin-type structure of PrtM elucidated by bioinformatics analysis, the cross reactions of the WT and mutant with a number of antisera, the observation that PrtM may be a multisubstrate foldase due to the detectable and significant differences in the proteomes of the WT, mutant and complemented mutants, the dimeric protein formed by the full length recombinant protein of S. equi 4047 WT, and the PPIase-Chaperonine activities of PrtM, all observed from this study, validate PrtM of S. equi 4047 as a viable and novel therapeutic target which pharmaceutical industries should extensively evaluate for the prevention and treatment of S. equi infection and strangles

Founder Effect: Breeding a Dog for the Elderly Gentleman Reveals an Animal Model of a Human Genetic Disorder

Animal models of genetic disorders that have risen due to selective breeding can be used as a valuable model to teach the basic concepts of population genetics. The Clumber Spaniel is a breed of dog created in the mid-1700s by the 4th Duc du Noailles. He selectively bred this dog for the elderly gentleman. This sleepy-looking breed survives today, though 1% suffer from severe exercise intolerance due to an autosomal-recessive founder mutation in the pyruvate dehydrogenase phosphatase 1 (PDP1) gene. PDP1 deficiency was long suspected to be a human metabolic disorder and described at the molecular level in 2005 by Robinson and coworkers. The Robinson group later identified a founder mutation within the PDP1 gene of the Clumber spaniel. This case clearly illustrates how a detrimental mutant allele in a small population, when selecting for phenotype, can persist in the progeny of that group. In this review, we discuss the origin of the “Founder Effect” theory and present an example of how a bottleneck that occurred during the selective breeding of the Clumber spaniel over 250 years ago led to the current genetic status of the breed. Today, genotyping can help reduce the incidence of PDP1 in the Clumber breed

Characterisation of SEQ0694 (PrsA/PrtM) of Streptococcus equi as a functional peptidyl-prolyl isomerase affecting multiple secreted protein substrates

YesPeptidyl-prolyl isomerase (PPIase) lipoproteins have been shown to influence the virulence of a number of Gram-positive bacterial human and animal pathogens, most likely through facilitating the folding of cell envelope and secreted virulence factors. Here, we used a proteomic approach to demonstrate that the Streptococcus equi PPIase SEQ0694 alters the production of multiple secreted proteins, including at least two putative virulence factors (FNE and IdeE2). We demonstrate also that, despite some unusual sequence features, recombinant SEQ0694 and its central parvulin domain are functional PPIases. These data add to our knowledge of the mechanisms by which lipoprotein PPIases contribute to the virulence of streptococcal pathogens