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ABSTRACT: Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's disease. Scanning electron microscopy and colonization levels of the M. ap mutant indicated that the pstA gene significantly contributes to the ability of M. ap to form biofilms. Digital measurements taken during electron microscopy identified a unique morphology for the DeltapstA mutant, which consisted of significantly shorter bacilli than the wild type. Analysis of the lipid profiles of the mycobacterial strains identified a novel lipopeptide that was present in the cell wall extracts of wild-type M. ap, but missing from the DeltapstA mutant. Interestingly, the calf infection model suggested that pstA contributes to intestinal invasion of M. ap. Furthermore, immunoblot analysis of peptides encoded by pstA identified a specific and significant level of immunogenicity. Taken together, our analysis revealed a novel cell wall component that could contribute to biofilm formation and to the virulence and immunogenicity of M. ap. Molecular tools to better control M. ap infections could be developed utilizing the presented findings.
Microbial Pathogenesis 05/2009; 46(4):222-30. · 1.94 Impact Factor
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ABSTRACT: The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the P-selectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Pre-incubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse.
Veterinary Immunology and Immunopathology 05/2007; 116(3-4):115-30. · 2.08 Impact Factor
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ABSTRACT: We previously reported that the number of acid-fast bacilli within Mycobacterium paratuberculosis-infected bovine monocytes increased steadily during an 8-day incubation period in vitro, despite a decrease in the number of viable bacilli as estimated by a radiometric method. In this study, we used differential live/dead staining of bacilli from infected monocytes to show that the percentage of viable bacilli decreased during an 8-day incubation period. We observed poor phagosome-lysosome fusion in monocytes that had ingested viable M. paratuberculosis (30% phagosome-lysosome fusion), while monocytes that ingested heat killed M. paratuberculosis exhibited 94% phagosome-lysosome fusion at 24h after infection. Treatment with the selective Ca(2+)/CaM and PI3 kinase inhibitors (i.e. KN62 and Wortmannin) in combination increased the survival of M. paratuberculosis in bovine monocytes without significantly altering phagosome-lysosome fusion. Scanning electron microscopy suggested that M. paratuberculosis-infected monocytes were less differentiated (smaller and less spreading) than uninfected monocytes at 4 and 8 days of infection. Overall, these data suggest that both multiplication and killing of intracellular M. paratuberculosis occur concomitantly in bovine monocytes. Monocytes in turn may be adversely affected by the bacilli, their products, or factors released from infected monocytes.
Microbial Pathogenesis 43(2-3):106-13. · 1.94 Impact Factor
