Structure/function analyses of Mycoplasma pneumoniae cytadherence proteins HMW1 and HMW3

Abstract

Mycoplasma pneumoniae is a pathogen of the human respiratory tract. M. pneumoniae adheres to respiratory epithelial cells via a sophisticated structure, the attachment organelle. The attachment organelle is composed of cytadherence-accessory proteins including HMW1, HMW2, HMW3 and P65. P1, the primary adhesin, and P30 a candidate adhesin are prominently displayed on this structure. Biochemical analysis has demonstrated that many cytadherence-accessory proteins are components of a Triton X-100 insoluble complex. Recent studies have illuminated a circuit of interactions among these proteins. Mutations in cytadherence-accessory proteins result in a reduced or nonadherent phenotype. Evaluation of additional effects of such mutations has advanced our understanding of the contribution of each cytadherence-accessory protein to a functional attachment organelle. I describe here the isolation of the first HMW3 mutant. This transposon insertion derived mutant had an altered cellular morphology, at times with an aberrant electron-dense core, reduced levels of P65, and failed to localize P1 and P65 properly. Cytadherence mutant M6, lacks HMW1 and produces a truncated P30 protein (Layh-Schmitt et al., 1995). I describe an aberrant morphology and altered P1 distribution in this mutant. Additionally, I showed that the C-terminus of HMW1 is required for these activities. I used mutant M6 to demonstrate a reciprocal dependency between HMW1 and HMW2, whereby the entire C-terminus of HMW1 was needed for HMW2 stabilization. The C-terminus of HMW1 was previously implicated in the rapid turnover of HMW1, in the absence of HMW2 (Hahn et al., 1996; Popham et al., 1997). Potential proteolytic target sequences were identified within this section of HMW1 and I constructed truncation/deletion mutants based on these sequences. I assessed the stability of each mutant. Amino acids 906-926 destabilized HMW1, while amino acids 927-1018, especially 978-1018 stabilized HMW1, particularly in the presence of HMW2. I also assessed the contribution of regions of the C-terminus of HMW1 to morphology revealing that the entire C-terminus of HMW1 influences morphology. Finally, I observed that fusion of the C-terminus of HMW1 to mouse dihydrofolate reductase destabilized DHFR in Escherichia coli in a manner dependent on ClpP and Lon.