Linked Virulence and Fitness of Mycoplasma alligatoris

描述（由申请人提供）：支原体病通常是微妙的，但短吻鳄支原体可引起易感宿主急性致死性原发性感染。这项工作的目的是解释这种毒性的机制和进化起源。基因组调查表明，短吻鳄利用唾液酸酶（Nanl）和透明质酸酶（NagH）从宿主细胞聚糖中产生糖酵解燃料。其减毒的兄弟物种鳄鱼支原体具有NagH，但不具有Nanl，因此仅对宿主细胞外基质的破坏不能解释短吻鳄支原体的特殊毒性。虽然nan和nag系统在营养获取方面具有优势，但我们的假设是，毒力是这些糖苷酶协同作用的结果，这些糖苷酶被感染共同定位，增强了宿主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。由此得出的假设是，像nan这样的适应性增强操纵子可以在水平转移到像鳄鱼m.s鳄鱼基因组这样的基因组后沉淀毒力，而鳄鱼基因组已经预先适应了nag，这是病原体出现的一个重要范例。本研究主要关注自然简化的小基因组病原体和宿主中nan和nag糖苷酶及其相互作用，它们代表了微生物病原体和脊椎动物免疫共同进化历史的重要阶段。我们的目标是：1)通过转座子介导的基因敲除和互补各自途径特有的关键中间酶基因，证明nan和nag系统对短吻鳄的适应性优势，反映在生长速度和营养通量上；2)利用体外原代成纤维细胞感染模型，通过nanl和nagH的失活和互补，确定糖苷酶在Fas （CD95）、NO和IFN表达中所反映的细胞病变作用；3)对鳄鱼基因组进行注释，并对鳄鱼基因水平转移对鳄鱼适应度和毒力的影响进行建模。研究结果有望为短吻鳄适合度与毒力因子之间的联系提供直接证据，并支持有利于营养适合度选择的系统水平转移作为促进某些细菌毒力的进化机制。相关性：除了解释短吻鳄短吻鳄的显著致病性外，这项工作还将研究细菌在感染过程中如何调节CD44信号，这可能为许多侵袭性病原体提供新的治疗方式。利用透明质酸酶等适度优势基因，评估基因组预适应在nan等系统水平转移后对严重毒力沉淀的重要性，可以为病原体的出现和进化建立预测模型。

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.