Mycoplasma penetrans is a bacterium associated with immunocompromised patients, and has been implicated as a potential cofactor in AIDS progression. Although exactly how it harms host cells is unclear, like many other pathogenic mycoplasmas it attaches to host cells by a polarized appendage, the attachment organelle (AO), which is also the leading end of the cell during gliding motility. Interference with AO-mediated processes, including its assembly, adherence, and motility, is a potential target for the development of therapeutic agents that could relieve infected patients, which include a substantial number of HIV-positive individuals. Furthermore, understanding how mycoplasmas, which have served as models for minimalistic cells, generate cell polarity, will be generally informative to cell biologists attempting to understand generation of cell polarity in more complex organisms, ranging from other prokaryotes to human cells. Comparison of the M. penetrans genome with more distantly related mycoplasma species like Mycoplasma pneumoniae and Mycoplasma mobile reveals the absence of homologs of AO proteins of these species. The M. penetrans AO has distinct ultrastructural features relative to these other species, including those of its underlying cytoskeletal elements. Furthermore, unlike M. mobile, M. penetrans motility does not directly depend on ATP hydrolysis. All these data indicate that the M. penetrans AO is only superficially convergent with analogous structures in other species, and therefore understanding the molecular basis for M. penetrans AO assembly and function cannot be inferred from information from these species. On the other hand, Mycoplasma iowae, a close relative of M. penetrans, has an ultrastructurally similar AO, and its genome contains homologs of proteins we have preliminarily identified as M. penetrans AO proteins, including a series of cytoskeletal proteins and a candidate adhesin, P42. Although biochemical and cell biological studies have begun to reveal the components and mechanisms associated with the M. penetrans AO, and we will continue to employ them, the absence of a genetic system in this organism has significantly slowed progress toward solving these problems. However, our preliminary results indicate that unlike M. penetrans, M. iowae is capable of being transformed with Tn4001- derived transposons, with tetracycline resistance available as a selectable marker. In this proposal we describe experiments aimed at developing M. iowae as a genetic system to model the M. penetrans AO, studying the localization of a green fluorescent protein fusion to a preliminarily identified AO component as well as generating a null mutant in the gene for that protein. We will also investigate the role of P42 in M. penetran adherence and motility through antibody inhibition studies. Finally, we will use biochemical fractionation and mass spectrometry to identify other proteins of the M. penetrans AO. Finally, this AREA proposal renewal will also accomplish the training of undergraduate and graduate students at Miami University.

Public Health Relevance

Mycoplasma penetrans is a bacterium that infects people whose immune systems are chronically challenged, including AIDS patients. To understand how this organism infects and potentially harms people, we will study the proteins that make up the structure it uses to attach itself to the cells of the victims. We will use standard techniques that have led to some valuable preliminary information about these proteins, and we will also use another bacterium with the same structure to study this problem using genetics, because genetic techniques do not work in M. penetrans.

Agency
National Institute of Health (NIH)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15AI073994-03
Application #
8768738
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Taylor, Christopher E,
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Miami University Oxford
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
City
Oxford
State
OH
Country
United States
Zip Code
45056
Pritchard, Rachel E; Prassinos, Alexandre J; Osborne, John D et al. (2014) Reduction of hydrogen peroxide accumulation and toxicity by a catalase from Mycoplasma iowae. PLoS One 9:e105188
Balish, Mitchell F (2014) Mycoplasma pneumoniae, an underutilized model for bacterial cell biology. J Bacteriol 196:3675-82
Jurkovic, Dominika A; Hughes, Michael R; Balish, Mitchell F (2013) Analysis of energy sources for Mycoplasma penetrans gliding motility. FEMS Microbiol Lett 338:39-45
Relich, Ryan F; Balish, Mitchell F (2011) Insights into the function of Mycoplasma pneumoniae protein P30 from orthologous gene replacement. Microbiology 157:2862-70
Relich, Ryan F; Friedberg, Aaron J; Balish, Mitchell F (2009) Novel cellular organization in a gliding mycoplasma, Mycoplasma insons. J Bacteriol 191:5312-4