Analysis of protein-protein interactions linked to the formation of a bacterial cytoskeleton in Mycoplasma pneumoniae

Mycoplasma pneumoniae has a cytoskeleton-like structure. Based on genetic evidence, it was proposed that the 1818 amino acids long protein HMW2 plays a central role in both formation of the cytoskeleton-like structure and adherence to its host cell (cytadherence). As gene products of the hmw2 gene (MPN310), two proteins were identified, the full-length protein HMW2 with a molar mass of 216 kDa and a smaller one (HMW2-s) with only 25 kDa. HMW2-s was considered to be the processing product of HMW2, but it could be shown by determining the N-terminus of HMW2-s and by expression studies with an artifical hmw2-s gene in M. pneumoniae that HMW2-s was synthesized by a new internal start within the hmw2 gene but in the same raster as HMW2. This internal expression unit also ensures the transcription of the two genes, MPN311 and MPN312, located immediately downstream. To characterize HMW2, it was expressed in Escherichia coli under various condition and with alternative E. coli strains, but, it was poorly expressed and degraded rapidly independent of the applied conditions. Therefore, it was impossible to isolate enough soluble full-length protein to do biochemical and structural analyses. The proposed function of HMW2 requires its interaction with other proteins of M. pneumoniae. Pilot experiments with the two-hybrid system suggested several candidates. By applying the “pairwise tests”, an internal fragment of HMW2 was found to interact with the C-terminal fragment of HMW1 (MPN447) and with the MPN297 encoded 17-kDa protein. The latter one has not yet been implicated in cytoskeleton formation. In addition, the interaction between the main adhesin P1 with HMW1 and the gene product of MPN297 was established linking indirectly HMW2 to the P1 adhesion complex consisting of at least three proteins: P1, P40 and P90. Further evidence for the interaction between HMW2 and the P1 adhesin (complex) derived from comparative protein analyses of M. pneumoniae WT and the mutant M. pneumoniae A3 (hmw2−). Western blot analyses showed that in M. pneumoniae A3 the turnover rate of the proteins of the P1 complex was significantly higher. This was interpreted as a consequence of the missing binding partner, because without HMW2, the P1 complex can not be formed and inserted properly in the membrane, making those proteins useless for the bacterium. First attempts to isolate protein complexes containing HMW2, to which a TAP tag was fused, were succesful. Twelve genes/ proteins were identified from the purified complexes: MPN015, MPN140 (ORF4 gene product), MPN141 (P1), MPN142 (P90), MPN160, MPN297, MPN392 (PdhB), MPN426 (P115), MPN430 (GAPDH), MPN447 (HMW1), MPN573 (GroEL), MPN665 (EF-Tu). These results confirm the two-hybrid analyses of proteins interacting with HMW2. Eight of them (P1, P90, PdhB, GAPDH, HMW1, GroEL, EF-tu and the gene product of MPN297) were also found in the Triton X-100 insoluble fraction, which contains almost all of the known cytoskeletal proteins including HMW2. Furthermore, the interaction of HMW2 with EF-Tu (elongation factor Tu) and PdhB (pyruvate dehydrogenase E1-beta subunit), of which a subfraction was reported to be surface exposed, provide evidence, that HMW2 might also have an important function in organizing other proteins than cytoskeletal proteins. Finally, a new antiserum against the N-terminal part of HMW2 was generated, which improved the immunocytochemistry and allowed to co-localize HMW2 with the rod structure (co-operation with Dr. Hegermann), which is one of the predominant structures seen in thin sections of M. pneumoniae.