Extracellular Replication of Listeria in Vivo

描述(申请人提供)：单核细胞增多性李斯特菌是一种主要的食源性病原体，在全球范围内引起严重的发病率和死亡率。它目前还在作为一种抗癌治疗剂进行临床试验。单核细胞增多性李斯特菌致病机制的许多分子细节已被揭示，它是最全面的免疫模式之一。尽管如此，仍有许多事情有待发现，特别是关于宿主病原体在活体动物或人类对象中的相互作用。我们最近在有症状和无症状感染的小鼠中发现了胆囊是一个潜在的细菌储存库，并确定了它在这个组织中的复制是细胞外的，而细胞内的复制是这种细菌在体内生长和发病的主要模式。胆汁中的复制可能是这种病原体在人类体内无症状携带的来源，并可能成为导致食物污染的蓄水池。在这里，我们试图进一步描述胆囊李斯特菌的遗传学和独特的生理学特征，以此作为预防食品污染和改进抗菌治疗的手段。我们提出了三个具体目标。首先，我们将在图像引导下对小鼠胆囊单核细胞增多性李斯特菌的细胞内复制进行定量分析。我们以前的结果表明，在胆汁中的复制不需要典型的毒力因子，这表明由细胞内复制介导的毒力不同于在胆汁中的生长。PRFA基因的突变是李斯特菌毒力的中心控制元件，不会影响在胆汁中生长的能力。胆汁是胆汁的主要耐药部位，胆汁对于胆汁的复制是必不可少的。这些结果提示了感染该部位的一组独特的基因，因此我们提出了一种全面和定量的功能基因组学分析。其次，我们将确定胆囊腔和纯胆汁培养中单核细胞增多性李斯特菌的基因表达模式，并与普通培养中观察到的模式进行比较。我们假设，基因表达模式将与纯胆汁培养中诱导的基因表达模式非常相似，但又不同。然后将构建高诱导基因的突变体。最后，我们将使用质谱仪测量从感染和未感染的动物身上提取的胆汁和从纯胆汁培养中提取的胆汁的成分。我们将使用腔内容物的质谱仪来评估感染的胆汁中胆汁成分的状态，并确定细菌生长对纯净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将被用于第一个和第三个目标的分析，以表征在胆囊中复制的遗传学和细菌生理学。 与公共卫生相关：单核细胞增生性李斯特菌是最被了解的感染源之一，它提供了关于细菌如何感染人类的关键信息。我们发现这种细菌生长在胆囊内，这在任何动物模型中都是非常不寻常和独特的，类似于人类伤寒。我们建议使用分子成像技术来了解这种细菌如何定植在胆囊部，这构成了目前唯一的人类伤寒携带者状态的动物模型。

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.