The surface of virtually all bacterial pathogens is coated with polysaccharides (exopolysaccharides, EPS) that can protect bacteria from host defenses and modulate the ability of the bacteria to adhere to various surfaces including host cells and tissue and other bacteria. The field of mycoplasmology has largely ignored polysaccharides although electron micrographs suggest that many species produce a capsule and some species form biofilms that should contain EPS in the extracellular matrix. Mycoplasmas cause chronic respiratory, genital and arthritic diseases in many animals including man. The lack of a cell wall might seem to make mycoplasmas highly susceptible to killing by host defenses, but these organisms are often difficult to eradicate from the host even with the help of antibiotic therapy. Polysaccharides may be a contributing factor to the chronicity of mycoplasmal infections. Our long-range goals are to unravel the pathogenic mechanisms of mycoplasmas, with the murine pathogen Mycoplasma pulmonis serving as a model organism. One factor that contributes to the avoidance of host defenses is the mycoplasma's Vsa (variable surface antigen) proteins that protect the cells from lysis by complement and modulate adherence to solid surfaces and thus biofilm formation. Our studies on biofilms led to the recent realization that polysaccharides are produced despite the fact that the complete genome sequence of M. pulmonis has few genes annotated as having a potential role in EPS synthesis. Nevertheless, M. pulmonis produces at least two EPS molecules (EPS-I and EPS-II).
Aim 1 of this proposal is to determine the structure of EPS-I and II. Knowledge of the structures will be instrumental in understanding the mechanism of their synthesis and the mechanics of how they interact with mycoplasmal proteins and host molecules.
Aim 2 is to study the pathway of EPS synthesis through the isolation and study of mutants that do not produce EPS-I or II. There are several reasons for suspecting that the mechanism for synthesis may be fundamentally novel. Some data suggest that nucleotide sugars are not needed as substrates for EPS synthesis in this system. Several of the genes that are required for synthesis of EPS-I or EPS-II have already been identified. These genes code for proteins that are annotated as ABC transporters. However, EPS-I is synthesized when an operon containing two of these genes are cloned into other species of mycoplasma. Thus, these genes may code for novel glycosyltransferases that are transporters only in the sense that they export the growing polysaccharide chain during synthesis. The synthesis machinery in this system will likely be important and found in other mycoplasmas and possibly walled bacteria.
Aim 3 is to examine the role of each EPS in pathogenesis. Mutants that do not produce EPS-I or EPS-II will be compared to wild-type mycoplasmas for the ability to avoid killing by complement, cytadhere, avoid phagocytosis, and cause disease in mice. These studies will lead to the development of new approaches for the control of mycoplasmal infections, such as the targeting of the polysaccharides or the machinery for their synthesis.
Probably all bacterial pathogens produce one or more polysaccharides that are important for full virulence and can be the targets for vaccines. Using the murine pathogen Mycoplasma pulmonis as a model organism for studying host-pathogen interactions, fundamentally novel machinery for polysaccharide synthesis has been discovered. Through the study of the structure of the polysaccharides produced, the novel pathway for their synthesis, and the role of these polysaccharides in pathogenesis, significant strides will be made towards understanding pathogenic mechanisms, leading to the development of better measures to control infections.
|Simmons, Warren L; Daubenspeck, James M; Osborne, John D et al. (2013) Type 1 and type 2 strains of Mycoplasma pneumoniae form different biofilms. Microbiology 159:737-47|
|Jordan, David S; Daubenspeck, James M; Laube, Audra H et al. (2013) O-linked protein glycosylation in Mycoplasma. Mol Microbiol 90:1046-53|
|Jordan, David S; Daubenspeck, James M; Dybvig, Kevin (2013) Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide. Mol Microbiol 89:918-28|
|Bolland, Jeffrey R; Dybvig, Kevin (2012) Mycoplasma pulmonis Vsa proteins and polysaccharide modulate adherence to pulmonary epithelial cells. FEMS Microbiol Lett 331:25-30|
|Dybvig, Kevin; Lao, Ping; Jordan, David S et al. (2010) Fewer essential genes in mycoplasmas than previous studies suggest. FEMS Microbiol Lett 311:51-5|
|Daubenspeck, James M; Bolland, Jeffrey R; Luo, Wenyi et al. (2009) Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis. Mol Microbiol 72:1235-45|
|French, Christopher T; Lao, Ping; Loraine, Ann E et al. (2008) Large-scale transposon mutagenesis of Mycoplasma pulmonis. Mol Microbiol 69:67-76|
|Dybvig, Kevin; Cao, Z; French, C Todd et al. (2007) Evidence for type III restriction and modification systems in Mycoplasma pulmonis. J Bacteriol 189:2197-202|