Extracellular Replication of Listeria in Vivo

描述（由申请人提供）：单核细胞增生李斯特菌是一种主要的食源性病原体，在世界范围内引起严重的发病率和死亡率。它目前也在临床试验中作为抗癌治疗剂。单核增生乳杆菌发病机制的许多分子细节已经被揭示，它是最全面的免疫模型之一。尽管如此，仍有许多有待发现，特别是在活体动物或人类受试者中宿主病原体的相互作用方面。我们最近在有症状和无症状的感染小鼠中发现胆囊是潜在的细菌储存库，并确定其在该组织中的复制是细胞外的，而细胞内复制是这种细菌在体内生长和发病的主要模式。胆囊内的复制可能是人类无症状携带该病原体的来源，也是导致食品污染的潜在宿主。在这里，我们寻求进一步表征李斯特菌的遗传和独特的生理在胆囊作为一种手段，以防止食品污染和改善抗菌治疗。我们提出三个具体目标。首先，我们将对小鼠胆囊中单核增生乳杆菌的细胞内复制进行定量图像引导分析。我们之前的结果表明，胆囊中的复制不需要原型毒力因子，这表明细胞内复制介导的毒力与胆囊中的生长不同。prfA基因的突变，李斯特菌毒力的中心控制因素，不影响在胆囊中生长的能力。胆汁在胆囊内的复制是不可缺少的。这些结果表明该位点存在一组独特的感染基因，因此我们建议进行全面和定量的功能基因组学分析。其次，我们将确定单核增生乳杆菌在胆囊腔和纯胆汁培养中的基因表达模式，并与普通培养中观察到的模式进行比较。我们假设基因表达的模式将非常相似，但不同于那些在纯胆汁培养诱导。然后构建高度诱导的基因突变体。最后，我们将使用质谱法测量从感染动物和未感染动物以及纯胆汁培养物中取出的胆囊胆汁的成分。我们将使用质谱法来评估感染胆囊中胆汁成分的状态，并确定细菌生长对纯胆汁培养中胆汁成分的影响。我们假设细菌会改变管腔和纯胆汁培养中的胆汁成分，以避免胆汁毒性并获得营养。在第二个目标中构建的突变体，然后将用于第一和第三个目标的分析，以表征胆囊中复制的遗传学和细菌生理学。

DESCRIPTION (provided by applicant): Listeria monocytogenes is a major food-borne pathogen that is the cause of significant morbidity and mortality worldwide. It is also currently in clinical trials as an anti-cancer therapeutic agent. Many of the molecular details of the pathogenesis of L. monocytogenes have been revealed, and it is one of the most comprehensive models of immunity. Nonetheless, much remains to be discovered, particularly in regard to host pathogen interaction in the live animal or human subject. We have recently identified the gallbladder as a potential reservoir of bacteria in symptomatic and asymptomatic infected mice, and have determined that its replication in this tissue is extracellular, whereas intracellular replication is the primary pattern of growth and pathogenesis of this bacterium in vivo. Replication in the gall bladder could be the source of asymptomatic carriage of this pathogen in humans and a potential reservoir leading to food contamination. Here we seek to further characterize the genetics and unique physiology of Listeria in the gallbladder as a means to preventing food contamination and improving antimicrobial therapies. We propose three specific aims. First, we will perform a quantitative image-guided analysis of intracellular replication of L. monocytogenes in the murine gallbladder. Our previous results demonstrate that replication in the gall bladder does not require prototypical virulence factors, suggesting that virulence mediated by intracellular replication is distinct from growth in the gall bladder. Mutations of the prfA gene, the central control element of listerial virulence, do not affect the ability to grow in the gall bladder. The major bile resistance locus bilE is dispensable for replication in the gallbladder. These results suggest a unique set of genes for infection of this site, thus we propose a comprehensive and quantitative functional genomics analysis. Second, we will determine the gene expression patterns of L. monocytogenes in the gallbladder lumen and in pure bile culture in comparison to the pattern observed in ordinary culture. We hypothesize that patterns of gene expression will closely resemble, but be distinct from, those induced in pure bile culture. Mutants of highly induced gene will then be constructed. Lastly, we will measure the composition of gallbladder bile removed from infected and uninfected animals and from pure bile cultures using mass spectrometry. We will use mass spectrometry of luminal contents to assess the status of bile components in the infected gallbladder, and determine the effects of bacterial growth on bile composition in pure bile culture. We hypothesize that the bacterium will alter the bile components of the lumen and in pure bile culture, to avoid bile toxicity and obtain nutrients. The mutants constructed in the Second Aim, will then be employed in the assays of the First and Third Aims, to characterize the genetics and bacterial physiology of replication in the gallbladder. Public Health Relevance: The bacterium Listeria monocytogenes is one of the best understood infectious agents and has provided critical information about how bacteria infect people. We have discovered that this bacterium grows inside the gallbladder, which is highly unusual and unique in any animal model, and resembles human typhoid fever. We propose to use molecular imaging techniques to learn how this bacterium colonizes the gallbladder, constituting the only current animal model of the human carrier state of typhoid fever.