Analysis of cytadherence-deficient, GapA-negative Mycoplasma gallisepticum strain R.

Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapA translational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapA in R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream of gapA in the same operon and was designated crmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in both Mycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed that crmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-type gapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.

Molecular and biochemical analysis of a 105 kDa Mycoplasma gallisepticum cytadhesin (GapA).

The identification of a gene (gapA) from Mycoplasma gallisepticum with homology to the P1 cytadherence gene of Mycoplasma pneumoniae is reported. The gapA gene is a 2895 bp ORF encoding a protein with a molecular mass of 105 kDa. Nucleotide sequence analysis of the gapA gene revealed 45% homology to the M. pneumoniae P1 gene, 46% homology to the Mycoplasma genitalium MgPa gene and 47% homology to the Mycoplasma pirum P1-like protein gene. It has a 64 mol % A+T content compared to 46, 60 and 72 mol % respectively for the P1, MgPa and the P1-like protein genes. As with the P1 and MgPa genes, gapA is a central gene in a multi-gene operon, but unlike the P1 and MgPa genes, there is only a single copy of gapA in the genome. GapA is a trypsin-sensitive surface-exposed protein. Chicken tracheal-ring inhibition-of-attachment assays, using anti-GapA Fab fragments, resulted in 64% inhibition of attachment. These results indicated that GapA plays a role in cytadherence of M. gallisepticum to host cells.