Genetic and serological analysis of lipoprotein LppA in Mycoplasma mycoides subsp. mycoides LC and Mycoplasma mycoides subsp, capri

Abstract

The genes encoding the 62-kDa lipoproteins from the Mycoplasma mycoides subsp, mycoides large-colony type (LC) strain Y-goat and the M. mycoides subsp, capri strain PG3 were cloned and analyzed by sequencing. These two lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes have been named lppA[MmymyLC] and lppA[Mmyca], respectively. The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas. Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M. mycoides subsp, mycoides small-colony type and P67 of the Mycoplasma species bovine group 7. Based on lppA gene sequences of M. mycoides subsp, mycoides LC and M. mycoides subsp, capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplified lppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoides subsp, mycoides LC and M. mycoides subsp, capri but does not distinguish between these two closely related subspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in all M. mycoides subsp, mycoides LC and M. mycoides subsp, capri type strains and field strains tested but not with the other members of the M. mycoides cluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.