The bacterium Mycoplasma pneumoniae is a human pathogen, chronically infecting both the respiratory tract and extrapulmonary sites, causing high rates of morbidity and considerable expense. Antibiotic resistance is in- creasing rapidly and no vaccines are available to combat this prevalent infectious agent. Adherence to host cells, which is critical for the virulence of M. pneumoniae, is primarily mediated by the immunodominant but anti- genically variable P1 adhesin protein, which must be clustered at a membrane protrusion called the attachment organelle (AO) to function. Although preliminary data indicate that its intracellular C-terminal region is required for localization, neither the mechanism by which P1 is localized to the AO nor the interactions its C-terminal region has with other AO proteins are known. Continuing not to know how P1 is localized to and maintained within the AO would prevent a complete understanding of M. pneumoniae virulence and impede development of therapeutics aimed at this process. The long-term goal of our research is to understand construction and function of the mycoplasma AO to develop it as a novel therapeutic target. The overall objective of this applica- tion is to identify the mechanism by which the primary adhesin of M. pneumoniae, P1, reaches and interacts with the AO, enabling adherence to host cells. The underlying working hypothesis being addressed is that P1 locali- zation is conferred by interactions between the positively charged sequence in the intracellular C-terminal region of P1 and negatively charged proteins on the AO interior. The rationale underlying the proposed research is that understanding the molecular basis for AO assembly and function is expected to lead to development of critically important therapeutic agents that prevent or reduce the severity of infection with M. pneumoniae, potentially by impairing AO function or engineering non-variable proteins to cluster at the AO, increasing their immunogenic- ity and resulting in successful vaccines. The central hypothesis of this proposal will be objectively tested, thereby attaining the goal of this application, by pursuing the specific aims of determining the AO localization signal within the C-terminal regions of the P1 adhesin protein and screening for binding partners, including AO struc- tural proteins. Identification of the AO targeting regions will be achieved by testing their ability to drive a chi- meric reporter protein to the AO, a classic, powerful, and appropriate cell biology approach, followed by testing of mutants in potential key amino acids within targeting regions. Testing for interactions with AO structural proteins will be done by both a bacterial two-hybrid approach and probing for interactions between a recombi- nant fusion protein and proteins from M. pneumoniae cells. This contribution will be significant because it is expected to direct future research efforts toward development of therapeutic intervention strategies targeting AO-mediated processes during M. pneumoniae infection and disease. The proposed research is innovative be- cause it represents a substantive departure from the status quo by focusing on a critical stage of AO development that has never been examined, namely the mechanism by which P1 is recruited to and maintained within the AO.
The proposed research is relevant to public health because the development of alternative therapeutic agents targeting the interaction between Mycoplasma pneumoniae and the patient requires deeper understanding of the molecular mechanisms used by the M. pneumoniae attachment organelle, the specialized but poorly understood structure required for interactions with host cells. The work of this proposal is expected to direct future research on developing treatments for M. pneumoniae-infected patients targeting the attachment organelle, which will lead to reduction in infection and alleviation of symptoms from increasingly antibiotic- resistant infections, improvement of the quality of life of patients, and substantial reduction in costs from health care and hospitalization. The project is directly relevant to NIH?s mission in that part of the NIH?s stated mission is to ?foster fundamental creative discoveries, innovative research strategies, and their applications as a basis for ultimately protecting and improving health.?
