Dynamics of Campylobacter jejuni and host responses in the porcine intestinal-loop infection model

项目摘要 感染空肠弯曲杆菌每年导致数百万例腹泻，是一种常见的 格林-巴利综合征的前驱，这是导致迟缓性瘫痪的主要原因。感染C. 空肠病人每天排10次以上的大便，而其他人则患上 炎症性腹泻，小量粘液便含血。目前还不确定是否会 传染性空肠弯曲菌菌株，个人的免疫反应，或两者都导致症状的变化， 空肠弯曲菌感染疾病的严重程度和持续时间。 我们的中心假设是，与空肠弯曲菌菌株相关的临床表现的变化在一定程度上是 由于不同菌株间空肠弯曲菌基因表达的差异。空肠弯曲杆菌侵袭性菌株导致出血 腹泻，而侵袭性较小的菌株导致无血腹泻(这些细菌较少入侵和 在肠道细胞中繁殖)。我们进一步假设，感染侵袭性空肠弯曲菌将导致 宿主促炎细胞因子和Th17细胞因子(影响肠道通透性)比感染 侵袭性较小的菌株。最后，我们假设空肠弯曲菌感染(不考虑表型)会导致 宿主-肠道代谢的变化，至少在开始时，有利于其在肠道中的生长。 该方案的创新之处在于利用猪结扎-肠环模型建立离散复制模型 四种不同的(经测序的)空肠弯曲菌在肠腔中的生态位。空肠弯曲杆菌的两个菌株将是 在我们的模型中测试会引起猪的出血性腹泻，而两个菌株会导致没有血液的腹泻。我们的 初步研究表明：1)四种空肠弯曲菌之间存在显著的遗传多样性 菌株；和2)空肠弯曲菌在生长条件下专一或优先合成多种蛋白质 宿主样条件(在存在上皮细胞或具有生理上相关浓度的胆汁时 通过基因芯片(已发表)和RNA-Seq分析判断)与营养液的比较 (未发表)。需要进行体内研究来确定空肠弯曲菌毒力基因之间的关系 表达和临床疾病表现，并提供关于宿主复杂性的信息 对不同菌株的反应。 这项建议的具体目的是： 1)利用从培养的空肠弯曲菌中提取的RNA来鉴定独特和/或差异表达的基因 实验室与从猪结扎肠环收集的空肠弯曲菌进行转录组学比较。 2)表征宿主对感染引起血性和水样腹泻的空肠弯曲杆菌菌株的反应。 3)对未感染和感染空肠弯曲菌的肠腔内容物进行比较代谢组学研究。

Project Summary Infection with Campylobacter jejuni is responsible for millions of cases of diarrhea per year and is a common precursor to Guillain-Barré syndrome, the leading cause of flaccid paralysis. Some individuals infected with C. jejuni experience watery diarrhea consisting of more than 10 stools per day whereas others suffer from inflammatory diarrhea with small-volume mucoid stool containing blood. It is currently uncertain whether the infectious C. jejuni strain, an individual's immune response, or both contribute to the variation in symptoms, severity, and duration of C. jejuni-mediated disease. Our central hypothesis is that the variation in clinical presentation associated with C. jejuni strains is, in part, due to differences in C. jejuni gene expression between strains. Invasive strains of C. jejuni cause bloody diarrhea whereas less-invasive strains cause diarrhea without blood (fewer of these bacteria invade and multiply in intestinal cells). We further hypothesize that infection with invasive strains of C. jejuni will result in greater host pro-inflammatory cytokines and Th17 cytokines (influence gut permeability) than infection with less-invasive strains. Finally, we hypothesize that C. jejuni infection (regardless of phenotypes) will result in a shift in the host-gut metabolism that, at least initially, favors its growth in the intestine. The novelty of this proposal is the use of the pig ligated-intestinal loop model to establish discrete replicative niches for four distinct (sequenced) C. jejuni strains in the intestinal lumen. Two of the C. jejuni strains to be tested in our model cause bloody diarrhea in pigs whereas two strains result in diarrhea without blood. Our preliminary studies have revealed that: 1) Significant genetic diversity exists amongst these four C. jejuni strains; and 2) C. jejuni synthesize a number of proteins either exclusively or preferentially when grown under host-like conditions (in the presence of epithelial cells or with a physiologically relevant concentration of the bile acid deoxycholate) as compared to nutrient broth, as judged by microarray (published) and RNA-Seq analysis (unpublished). In vivo studies are needed to determine the relationship between C. jejuni virulence gene expression and clinical disease presentation and to provide information regarding the complexity of the host response to different strains. The Specific Aims of this proposal are to: 1) Identify unique and/or differentially expressed genes using RNA extracted from C. jejuni cultured in the laboratory versus from C. jejuni collected from pig ligated-intestinal loops by transcriptomics. 2) Characterize the host response to infection with C. jejuni strains that cause bloody versus watery diarrhea. 3) Perform comparative metabolomics of luminal contents from uninfected and C. jejuni-infected loops.