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ItemMolecular and functional characterisation of membrane proteins of Mycoplasma bovisAdamu, James Yazah ( 2017)Mycoplasma bovis is a pathogen of considerable economic importance that has been associated with pneumonia, arthritis, mastitis and otitis in cattle, reducing productivity and causing or predisposing to mortality. The control of M. bovis infections has typically relied on antimicrobial therapy, which is expensive and has variable success. Understanding the basic biology of this pathogen and the mechanisms it uses for colonisation and survival could lead to development of cheaper and more effective control strategies. Although the complete genomic sequences of four strains of M. bovis have been available for some time, the use of these data to increase our understanding of the pathogenesis of M. bovis has been limited and the annotations of a large proportion of the genes in these sequences remain hypothetical. This thesis focuses on exploring the function of membrane–associated proteins of M. bovis, as they are likely to be most directly involved in interactions with the host. The studies utilised a combination of bioinformatic analysis, three-dimensional ab initio structural modeling and exploration of biochemical functions of purified recombinant proteins expressed from genes that were previously uncharacterised to attribute a range of roles to several of these proteins. The genes encoding these membrane proteins were first codon optimised by mutating the TGA codons to TGG to allow full-length expression of the proteins in E. coli. These proteins were expressed in E. coli JM109 and the soluble GST-tagged recombinant proteins were then purified by affinity chromatography and their immunogenicity established by Western immunoblotting. Monospecific antisera were raised against each recombinant protein and the growth inhibitory effects of the antisera were tested on running drop cultures of M. bovis. All the genes targeted were successfully expressed and purified, with the exception of the product of MBOVPG45_0318. The recombinant product of MBOVPG45_0353, MbLolA, MbFg-bA, MbFn-bA and MilA were recognised by antibodies in sera from calves experimentally infected with M. bovis, while antisera raised in rats against the recombinant product of MBOVPG45_0353, MbLolA and MilA-EF could inhibit the growth of M. bovis cells in vitro. Previous studies on M. bovis demonstrated that the amino terminal end of an immunogenic 226-kDa (P226) protein, encoded by milA (the full length product of which has a predicted molecular weight of 303 kDa) had lipase activity. The predicted sequence of MilA has glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. Recombinant regions of MilA bound to 1-anilinonaphthalene-8-sulfonic acid and a variety of lipids, with the amino-terminus binding more tightly. MilA was also able to bind ATP, suggesting a potential autotransporter function. Other ATP binding proteins identified in this study included the products of MBOVPG45_0305, MBOVPG45_0353 and MBOVPG45_0779, which may play important roles in cellular processes. The pathogenesis of disease caused by infection with mycoplasmas depends in part on interactions between their cellular components and the host’s extracellular matrix, which aid their adherence to host cells and thus colonisation and the subsequent development of disease. The results of this study identified novel proteins of M. bovis that bound heparin (the product of MBOVPG45_0232, MbFn-bA, MbLolA and MilA) and fibronectin (the product of MBOVPG45_0232, MbFn-bA, MbFg-bA and MbLolA). A novel fibrinogen binding protein of M. bovis (MbFg-bA) was also identified and the binding was shown to be regulated by divalent cations, with magnesium ions increasing binding, while calcium and manganese ions inhibited binding. MilA and MbFn-bA were shown to be surface exposed and accessible to the proteolytic effect of trypsin. MilA was shown to be post-translationally processed into 226-kDa and 50-kDa polypeptides, while MbFn-bA was detected as a 48-kDa polypeptide in all strains and also as the predicted full length 70-kDa polypeptide in some strains. Sequencing of mbFn-bA in several strains of M. bovis detected the deletion of the region encoding the carboxyl terminal 147 amino acids in the mbFn-bA of the PG45 type strain, which could account for the variation detected in immunoblots of MbFn-bA in these strains. The significance of M. bovis infection has become increasingly apparent in recent times and the increasing resistance of this bacterium to antimicrobials necessitates a search for better methods of control. Serological diagnosis is an alternative strategy for the control of M. bovis infection and offers the opportunity to separate infected from uninfected cattle. Current diagnostic assays are suboptimal necessitating the search for antigens that are specific for M. bovis and desirable for the development of such diagnostic assays. The studies described in this thesis have identified additional immunogenic membrane proteins of M. bovis, including the product of MBOVPG45_0353, MbFg-bA, MbFn-bA and MbLolA, that may warrant further analyses as possible diagnostic antigens. In conclusion, the results of these studies have increased our understanding of the functions of previously hypothetical membrane proteins of M. bovis. In some instances individual proteins were shown to have a number of functions and thus can be predicted to play multiple roles in the pathogenesis of disease caused by M. bovis. These results complement genetic studies that have identified genes required for colonisation and persistence in animals infected with M. bovis.