Linked Virulence and Fitness of Mycoplasma alligatoris

描述(申请人提供)：支原体病通常是轻微的，但鳄支原体会引起易感宿主的急性致死性原发感染。这项工作的目标是解释这种毒力的机制和进化起源。基因组研究表明，鳄鱼分枝杆菌利用唾液酸酶(NAN1)和透明质酸酶(NAGH)从宿主细胞糖链中产生糖酵解的燃料。它的兄弟种鳄支原体具有NagH，但不具有NagL，因此单靠对宿主细胞外基质的破坏不能解释鳄鱼支原体的特殊毒力。虽然NaN和NAG系统可以在营养获取方面做出贡献，但我们的假设是，毒力是这些糖苷酶协同作用的结果，感染共同定位，加强宿主CD44转导的细胞凋亡、坏死和炎症，与观察到的暴发性疾病一致。推论是，像NaN这样的适应性增强操纵子在水平转移到像鳄鱼分枝杆菌这样预先适应了NAG的基因组后，可以产生毒力，NAG是病原体出现的重要范例。这项建议集中在自然简化的小基因组病原体和宿主中的NaN和NAG糖苷酶及其相互作用，这代表了微生物病原体和脊椎动物免疫共同进化史上的一个重要阶段。我们的目标是：1)通过转座子介导的关键中间酶基因的敲除和互补，展示NaN和NAG系统对鳄鱼微囊藻生长速度和营养通量所反映的适合性的优势；2)通过使用体外原代成纤维细胞感染模型，通过灭活和互补NaN1和NAG H来确定反映在Fas(CD95)、NO和干扰素表达中的糖苷酶的细胞病变效应；以及3)注释鳄鱼微囊藻基因组，并建立水平转移NaN基因对鳄鱼微囊藻适合性和毒力的影响的模型。这些结果有望为扬子鳄适合度和毒力因子之间的联系提供直接证据，并支持通过营养适合性选择有利于系统的水平转移，作为引发某些细菌毒力的进化机制。相关性：除了解释鳄鱼分枝杆菌的显著致病性外，这项工作还将调查细菌在感染过程中CD44信号是如何调节的，这可能会为许多侵袭性病原体提供新的治疗方式。评估基因组与透明质酸酶等适度优势基因的预适应对水平转移NaN等系统后严重毒力沉淀的重要性，可能会导致病原体的出现和进化的预测模型。

DESCRIPTION (provided by applicant): Mycoplasmosis is usually subtle, but Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. The goal of this work is to explain the mechanisms and evolutionary origins of that virulence. A genome survey indicated that M. alligatoris uses sialidase (Nanl) and hyaluronidase (NagH) to generate fuel for glycolysis from host cell glycans. Its attenuated sibling species Mycoplasma crocodyli possesses NagH but not Nanl, so damage to the host's extracellular matrix alone cannot explain the particular virulence of M. alligatoris. While nan and nag systems could contribute to advantages in nutrient acquisition, our hypothesis is that virulence is a consequence of synergy of those glycosidases, co- localized by infection, that potentiates host CD44-transduced apoptosis, necrosis, and inflammation consistent with the fulminant disease observed. The corollary hypothesis is that a fitness-enhancing operon like nan can precipitate virulence after horizontal transfer to a genome like M. crocodyli's which is pre- adapted with nag, a significant paradigm for the emergence of pathogens. This proposal focuses on the nan and nag glycosidases and their interactions in naturally-simplified small-genome pathogens, and hosts which represent a significant stage in the history of co-evolution of microbial pathogens and vertebrate immunity. Our aims are to: 1) Demonstrate the advantages of the nan and nag systems to M. alligatoris for fitness reflected in growth rate and nutrient flux, by transposon-mediated knockout and complementation of genes for the key intermediary enzymes unique to their respective pathways; 2) Define the cytopathic effects of the glycosidases reflected in Fas (CD95), NO, and IFN expression, by inactivation and complementation of nanl and nagH using in vitro primary fibroblast infection models; and 3) Annotate the M. crocodyli genome and model the effects of horizontal transfer of nan genes on fitness and virulence of M. crocodyli as described for M. alligatoris. The results are expected to provide direct evidence of linkage between fitness and virulence factors of M. alligatoris, and support horizontal transfer of systems favorable by selection for nutritional fitness as an evolutionary mechanism precipitating the virulence of some bacteria. Relevance: Beyond explaining the remarkable pathogenicity of M. alligatoris, this work will investigate how CD44 signaling is modulated by bacteria during infection, which could suggest new treatment modalities for many invasive pathogens. Assessing the importance of genome pre-adaptation with modestly advantageous genes like hyaluronidase to precipitation of severe virulence following horizontal transfer of a system like nan could lead to predictive models for the emergence and evolution of pathogens.