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The survival, replication, and virulence of mycoplasmas depend on their ability to capture and import host-derived nutrients using poorly characterized membrane proteins. Previous studies on the important bovine pathogen Mycoplasma 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 contains glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. We mutagenized the gene to facilitate expression of a series of regions spanning the gene in Escherichia coli. Using monospecific antibodies against these recombinant proteins, we showed that MilA was proteolytically processed into 226-kDa and 50-kDa fragments that were both partitioned into the detergent phase by Triton X-114 phase fractionation. Trypsin treatment of intact cells showed that P226 was surface exposed. In vitro, the recombinant regions of MilA bound to 1-anilinonaphthalene-8-sulfonic acid and to a variety of lipids. The MilA fragments were also shown to bind heparin. Antibody against the carboxyl-terminal fragment inhibited the growth of M. bovis in vitro. This carboxyl end also bound and hydrolyzed ATP, suggestive of a potential role as an autotransporter. Our studies have demonstrated that DUF445 has lipid binding activity and that MilA is a multifunctional protein that may play multiple roles in the pathogenesis of infection with M. bovis.

Mycoplasma bovis cause serious infection in ruminants leading to huge economic losses. Lipoproteins are key components of mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interaction with the host and inducing the host immune response to infection. Genomic data arising from M. bovis show that many of the genes are not assigned any functions, owing in part to their low sequence similarity to those of other bacteria making it difficult to extrapolate gene functions. This study identifies a surface localised LRR lipoprotein encoded by mbfN of M. bovis PG45. Homologues of MbfN were detected as a 48-kDa peptide on Western blot of all strains of M. bovis and the predicted 70 kDa full-length polypeptide in some strains included in this study. Sequence analysis of the gene revealed the absence of a region encoding the carboxyl terminal 147 amino acids in some strains when compared with PG45, which could account for the variation detected on immunoblot. In silico analysis of MbfN suggest that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, showing the contribution of MbfN as an adhesin. IMPORTANCE: Experimental validation of putative functions of a particular gene in M. bovis will advance our understanding of the basic biology of this economically important pathogen, which is crucial in developing prevention strategies. This study demonstrates the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis and the contribution of this protein in adhesion of M. bovis suggest that it could play a role in virulence.

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