HMW1 Is Required for Stability and Localization of HMW2 to the Attachment Organelle of Mycoplasma pneumoniae

Abstract

The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Mycoplasma adherence to host respiratory epithelium (cytadherence) is mediated largely by the attachment organelle, a polar, membrane-bound, differentiated cell extension that is defined by an electron-dense core (7, 15). Extraction of M. pneumoniae cells with the nonionic detergent Triton X-100 (TX) yields a TX-insoluble fraction that includes this core structure and consists largely of cytoskeletal proteins such as HMW1, HMW2, HMW3, and P65 (Table 1) (6,11,13,20,30,36). These proteins contribute to the architecture of the attachment organelle, including the localization of the major adhesin protein P1 to this structure, but little is known regarding their specific roles or the dynamics of their assembly (19).HMW1 is a peripheral membrane protein on the mycoplasma cell surface but lacks typical secretion signal or transmembrane sequences or significant homology to any proteins found beyond the mycoplasmas (2, 3, 9). HMW1 has been localized to the attachment organelle (30, 32) as well as the trailing filament (32), depending upon the experimental approach. Cytadherence mutant M6 (Table 1) lacks HMW1 and produces the truncated cytadherence-associated protein P30 due to an internal, in-frame deletion (22). This mutant has a striking morphology, lacking the tapered filamentous appearance characteristic of wild-type cells, and fails to localize P1 to the attachment organelle (13). Recombinant wild-type HMW1, but not a truncated derivative lacking the C-terminal 112 amino acids, restores a near-normal morphology and P1 clustering to mutant M6 (13), indicating that HMW1 contributes to the architecture of the attachment organelle and that the C-terminal domain of HMW1 is essential for normal function.HMW2 is a large protein predicted to form dimeric coiled coils, as well as trimeric coiled coils with P28, also a product of the hmw2 gene (5, 21). HMW2 is a component of the attachment organelle and app...

Functional Analysis of the Mycoplasma genitalium MG312 Protein Reveals a Specific Requirement of the MG312 N-Terminal Domain for Gliding Motility

“…The loss of MG312 is also correlated with the loss of the terminal organelle and reduced levels of MG218, MG317, and MG217 proteins, as has been previously reported for M. pneumoniae (10,45). In addition, our results also show reduced levels of P140, P110, and MG386 proteins.…”

“…However, it remains unclear whether the contribution of MG312 to gliding motility is a direct consequence of the loss of the terminal organelle and/or whether MG312 may also play a specific and independent role in M. genitalium cell locomotion. The C terminus of HMW1 has been previously linked with functions related to cytadherence and cell morphology (45). We have addressed the question of whether the functions related to adhesion and locomotion may be located in separate MG312 protein domains, as suggested by the presence of an EAGR box in the MG312 N terminus (Fig.…”

“…Our results demonstrate that, in the absence of the MG312 protein, the M. genitalium cells adhere poorly to surfaces and they are nonmotile. In addition, we have found that the MG312 protein has two separate functional domains: a C-terminal domain related to cytadherence and cell morphology, as previously described for M. pneumoniae (45), and an N-terminal domain not directly related to surface attachment and terminal organelle development but clearly involved in M. genitalium cell locomotion. The presence in this region of a conserved ATP binding site, known as the P-loop or Walker A box (44), suggests that this structural protein could also play an active function in the gliding mechanism.…”

“…Characterization of an M. pneumoniae cytadherence-negative mutant lacking HMW1 and with defects in the P30 adhesin gene (22) showed that HMW1 is required for normal cell morphology and cytadherence (10). More-detailed analyses revealed that the HMW1 C-terminal domain is needed for HMW2 stabilization, proper cell morphology, P1 trafficking to the tip structure (45), and HMW1 turnover in the absence of HMW2 (27).…”

Synthesis, Stability, and Function of Cytadhesin P1 and Accessory Protein B/C Complex of Mycoplasma pneumoniae

“…This nonreciprocal requirement for stability is similar to that seen in M. pneumoniae with HMW3 and the two proteins HMW1 and HMW2. In that case, HMW3 is unstable if HMW1 or HMW2 levels are reduced (23,31), but HMW1 and HMW2 are present at wild-type levels in the absence of HMW3 (32).…”