Molecular pathogenesis of Mycoplasma bovis

© 2019 Filimon Mitiku HaileMycoplasma bovis is an important pathogen in cattle production systems worldwide and causes significant economic losses. It causes mastitis, pneumonia, otitis media, keratoconjunctivitis and polyarthritis in affected cattle. Antibiotics have limited efficacy in treating M. bovis infections so vaccines are considered the method most likely to achieve effective control of the disease it causes. As M. bovis lacks a cell wall, lipoproteins, which are exposed on the cell surface, play a key role in its survival in the host through their role in the acquisition of essential nutrients, in adherence to cell surfaces, in colonisation of the host and in evasion of the immune system. Mycoplasmas are the simplest free-living organisms, and are generally regarded as having evolved to retain only the essential machinery necessary for survival in their hosts. This study sought to understand the functions of selected membrane genes of M. bovis and their roles in adhesion, colonisation and immune evasion. The gross pathology of respiratory disease caused by M. bovis is characterised by the presence of areas of caseous necrosis and purulent exudates in the affected pulmonary tissues. Calves experimentally infected with M. bovis have an infiltration of neutrophils into the bronchioles. The studies described in this thesis showed that these neutrophilic infiltrates contained neutrophil extracellular traps (NETs) by detecting changes in the morphology of neutrophils in these sites, and free histones and neutrophil elastase, which are derived from the nuclear components of NETs. Further investigations of the induction of NETs by M. bovis were conducted in vitro using neutrophils isolated from the blood of healthy cattle. These studies demonstrated that M. bovis does induce the formation of NETs by neutrophils, but that the stability of these NETs was compromised by the product of the major membrane nuclease gene mnuA. The MnuA nuclease is the major nuclease of M. bovis detected in vitro. A fluorescence assay was developed to quantify NET formation when neutrophils were stimulated with M. bovis strain PG45 and mutants derived from it. The formation of NETs in bovine neutrophils was dependent on the presence of a relatively high concentration of M. bovis (a multiplicity of infection of 100), but NETs were degraded at very low concentrations of M. bovis, demonstrating the potency of the MnuA gene product in the degradation of NETS and its likely role in enabling M. bovis, and possibly other bacteria at the sites of infection, to escape from NETs. Ovine neutrophils were also shown to release NETs after stimulation with M. bovis and the MnuA nuclease also degraded these NETs. M. bovis-induced NETosis was found to result from stimulation of an alternative pathway to the classical NET induction pathway. It was dependent on neutrophil elastase and the ERK pathway, but was induced independently of the formation of reactive oxygen species (ROS), or the protein kinase C (PKC) and MAPK pathways. These studies suggest that the MnuA nuclease assists M. bovis in evading the innate immune system and in the establishing chronic infection. Mycoplasmas must adhere to epithelial cell surfaces to successfully colonise their host. To determine the role of selected genes of M. bovis in adhesion, an in vitro adhesion assay was developed using the Madin-Darby bovine kidney (MDBK) cell line. The adhesion of wildtype M. bovis strain PG45 was compared to mutants in which a transposon had been inserted into the open reading frames MBOVPG45_0416 or MBOVPG45_0565. The products of both of these genes have been found to have fibronectin binding activity in earlier studies. Both mutants had significantly reduced binding to MDBKs compared to the wildtype strain. In addition, adhesion of wildtype PG45 to MDBK cells was significantly inhibited by treatment of the M. bovis PG45 wildtype strain with polyclonal sera raised against the products of MBOVPG45_0565 or MBOVPG45_0416. Taken together, these findings are consistent with a significant role of these gene products in the adhesion of M. bovis to cell surfaces. In conclusion, the studies described in this thesis have demonstrated the likely role of three membrane genes of M. bovis in its survival in the host and have highlighted some of the mechanisms that contribute to the virulence of this pathogen. This in turn has enhanced our understanding of the pathogenesis of the disease caused by this organism and will assist in the design of vaccines and diagnostic tools that can be used to more effectively control disease caused by M. bovi