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Abstract
Mycoplasmas are cell wall-less prokaryotes considered to be among the simplest known microorganisms capable of self-replication. Several mycoplasma species are distinguished by the presence of a complex assemblage of proteins comprising a differentiated terminal organelle containing a Triton X-100 insoluble electron-dense core. This terminal organelle is an extension of the mycoplasma cell and is thought to function in cell division, host cell interactions, and gliding motility. Through mutant analysis, several proteins have been found to be required for cytadherence and are components of the terminal organelle including HMW1-HMW3, P1, B, C, and P30. Protein P30 consists of a predicted signal sequence, cytoplasmic domain, membrane-spanning region, and extracellular proline-rich C-terminal domain. Protein P65 localizes to the terminal organelle, yet its role in cytadherence is unknown as no mutant lacking only P65 has been reported. Upon analysis of previously isolated cytadherence mutants and P30 structure-function constructs created here and expressed in a P30 null mutant, we found that HMW1- HMW3, and P30 are required for P65 stability and in the case of P30, stabilization was mediated by the surface-exposed and not cytoplasmic domain. In contrast, P65 localization only required HMW1-HMW3 and not P30. We examined gliding motility of M. pneumoniae using two assays we developed and found that cytadherence mutants absent of electron-dense cores (HMW1- HMW2) were incapable of motility. Of the strains possessing electron-dense cores an intermediate level of gliding was seen in the absence of HMW3, and in strains having certain defects in the surface-exposed domain of P30. Despite the presence of electron-dense cores, no motility was seen in the absence of proteins B and C and in certain P30 mutants. P30 stability was most affected by deletion of a highly conserved region in the surface-exposed domain of P30 and although hemadsorption was the most sensitive phenotype to any alteration in the P30 sequence, a P30 revertant strain was isolated which exhibited wild-type levels of attachment to hamster respiratory epithelium yet only demonstrated an intermediate motility phenotype. Thus, this strain might be useful to determine if gliding motility is a virulence determinant for M. pneumoniae pathogenesis.