Mechanisms of Mycoplasmal Disease Pathogenesis

描述(申请人提供)：几乎所有细菌病原体的表面都覆盖有多糖(胞外多糖，EPS)，可以保护细菌免受宿主防御，并调节细菌附着在包括宿主细胞和组织以及其他细菌在内的各种表面的能力。尽管电子显微照片显示许多物种产生一个囊膜，一些物种形成的生物膜应该在细胞外基质中含有EPS，但支原体研究领域在很大程度上忽略了多糖。支原体在包括人类在内的许多动物中引起慢性呼吸道、生殖器和关节炎疾病。缺乏细胞壁似乎使支原体非常容易被宿主防御系统杀死，但即使在抗生素治疗的帮助下，这些微生物通常也很难从宿主中根除。多糖可能是支原体感染慢性化的一个因素。我们的长期目标是揭开支原体的致病机制，以小鼠的病原体肺支原体作为模式生物。一个有助于避免宿主防御的因素是支原体的VSA(可变表面抗原)蛋白，它通过补体保护细胞免受裂解，并调节对固体表面的黏附，从而形成生物膜。我们对生物膜的研究导致了最近意识到，尽管肺支原体的完整基因组序列中几乎没有被注释为在EPS合成中具有潜在作用的基因，但仍能产生多糖。然而，肺支原体至少产生两个EPS分子(EPS-I和EPS-II)。这项建议的目的1是确定EPS-I和EPS-II的结构。了解这些结构将有助于理解它们的合成机制以及它们如何与支原体蛋白和宿主分子相互作用的机制。目的2是通过分离和研究不产生EPS-I或II的突变体来研究EPS合成的途径。有几个原因可以怀疑合成的机制可能是根本上的新的。一些数据表明，在这个系统中，不需要核苷酸糖作为EPS合成的底物。合成EPS-I或EPS-II所需的几个基因已经被鉴定。这些基因编码的蛋白质被注释为ABC转运蛋白。然而，当包含其中两个基因的操纵子被克隆到其他种类的支原体中时，EPS-I就被合成了。因此，这些基因可能编码新的糖基转移酶，这些糖基转移酶只是在合成过程中输出不断增长的多糖链的意义上的转运蛋白。这个系统中的合成机制可能很重要，可以在其他支原体和可能的壁状细菌中找到。目的3是研究每个EPS在发病机制中的作用。不产生EPS-I或EPS-II的突变株将与野生型支原体进行比较，以避免被补体杀死，细胞黏附，避免吞噬，并在小鼠中引起疾病。这些研究将导致开发控制支原体感染的新方法，例如靶向多糖或其合成机制。

DESCRIPTION (provided by applicant): 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.