Analysis of P97 and P102 paralogs of Mycoplasma hyopneumoniae
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Mycoplasma hyopneumoniae, the causative agent of enzootic pneumonia in pigs, is an economically significant disease in the swine industry. Mycoplasmas cause a chronic infection characterized by high morbidity but low mortality. The disease has been implicated as an important agent in the porcine respiratory disease complex, which results in slower growth and decreased feed efficiency. M. hyopneumoniae colonizes the porcine respiratory tract by adherence to the cilia of ciliated epithelial cells. Recent studies have identified P97 as the major protein responsible for this adherence. The gene for P97 is located within a two gene operon that also includes the gene for P102. The function of P102 is unknown, but it is also thought to play an important role in adherence. The recent sequencing and annotation of the M. hyopneumoniae genome has identified several P97 and P102 paralogous genes, comprising two significant gene families. The goal of this study was to determine if these paralogs are expressed and if they could contribute to the adherence process. To accomplish this, we examined the paralogous gene expression patterns at the transcriptional and translational levels in high and low adherent phenotypic variants of M. hyopneumoniae strain 232, as well as in vivo grown organisms harvested from bronchoalveolar lavage fluids from mycoplasma-infected pigs. Transcriptional analysis of each paralog open reading frame was performed using reverse transcriptase-polymerase chain reaction assays. These results indicated the transcription of the paralogous genes between these isolates were variable and allowed us to identify several genes whose transcription might be responsive to changing environmental conditions. For translational analysis, monospecific, polyclonal antisera was produced in rabbits using a genetic vaccination approach against each of the paralogous family members. The design of the study allowed us to utilize unique protein sequences present on the carboxy terminus of each paralog as a gene-specific tag. Immunoblots against lysed, whole cell M. hyopneumoniae strain 232 antigen indicated that we were able to produce antibodies specific for paralogous gene products and demonstrated their translation into protein products.