Microbiology & Immunology - Theses
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ItemA genetic analysis of the virulence determinants of Moraxella bovis( 2000)Limited information is available regarding the process by which Moraxella bovis induces the condition known as infectious bovine keratoconjunctivitis or IDK in cattle. Previous work has indicated that there are two proteins (type IV pili and a haemolysin) secreted by the bacterium that are essential for disease progression. Of these two proteins, the majority of work has been carried out on the type IV pili. Many of the genes required for type IV pili expression have been identified and characterised. To date the gene encoding the haemolysin secreted by M bovis has not been identified and therefore its role in IBK infection has not been fully investigated. In addition to these recognised virulence determinants, the bacterium secretes a number of other proteins that have the potential to play a role in the disease. This study describes the isolation and characterisation of a number of these known and putative virulence determinants produced by M bovis. Using a cosmid library generated using genomic DNA from the M. hovis strain Dalton 2d, an open reading frame (ORF) that encoded proteolytic activity was identified (mbp). The ORF was 3.4kb in size and had the capability of encoding a protein with a molecular weight of 120kDa. This protein was expressed using a Gram-positive expression system and this data, together with sequence analysis, suggested that Mbp belonged to the autotransporter secretion family. The generation of an isogenic protease mutant allowed for the preliminary analysis of the role Mbp may play in the pathogenesis of an M. hovis infection. This revealed that perhaps Mbp in some instances may have either a regulatory role for expression of the haemolysin and possibly type IV pili. A gene that encoded lipase activity (plb) was found to be an ORF of 1.8kb, encoding a protein of65.8kDa molecular weight. This protein was abundantly expressed in recombinant E coli. Further analysis of the enzymatic activity of PLB revealed that it had the specificity of a phospholipase B and this activity was found to be relatively heat stable. Partial sequence of an operon encoding the haemolytic activity observed for M. hovis was obtained and this suggested that the M bovis haemolysin belongs to the RTX toxin family. Complementation experiments using the a.-haemolysin from E. coli confirmed that the M. bovis haemolysin had RTX-like requirements for activation and secretion. The conservation of each of these possible virulence factors in all of the pathogenic M. bovis strains that were tested indicated that they may have the potential to be used in a vaccine preparation to protect against a heterologous M bovis infection.