Analyzing the Biogeography of Virulence and Transmission Gene Expression during Clostridioides difficile infection

芽胞形成细菌艰难梭状芽胞杆菌造成约450,000例腹泻感染和约30,000例腹泻 2017年死亡人数增加，使其成为美国医疗保健相关感染的主要原因。艰难梭菌在 肠道内的微生物群完好无缺，会阻碍肠道的生长。随着艰难梭菌在 结肠，它使葡萄糖化毒素负责导致疾病病理和必要的孢子 传播疾病。最近的研究表明，毒素的产生通过诱导艰难梭菌在肠道中的生长来促进其生长。 炎症，产生艰难梭菌专门利用的宿主衍生的代谢物。例如，毒素- 介导性炎症刺激宿主降解胶原蛋白并产生山梨醇。艰难梭菌随后分解代谢 由此产生的代谢物，使其在肠道中生长到更高的水平。因为这些代谢物很可能 集中在产生它们的肠道上皮细胞附近，靠近这一细胞层可能有利于C. 艰难梭菌感染。与这一假设一致，艰难梭菌对粘蛋白衍生的糖具有趋化作用，其 在感染期间，对这些糖的利用可以促进其生长。粘液层也会阻碍毒素与 靶细胞，所以艰难梭菌在上皮层附近的生长对这一层的损害可能比 管状细菌。 虽然这些研究表明，艰难梭菌上皮近端的亚群可能在 艰难梭菌主要被认为是一种肠道病原体。例如，以前的一本书的作者 针对艰难梭菌在小鼠感染过程中定位的荧光原位杂交研究得出结论：它 很少与肠道上皮结合。然而，使用新的艰难梭菌结构性荧光报告菌株 我们发现艰难梭菌经常生长在靠近肠道上皮的地方，即使大多数 人口是平凡的。此外，毒素的产生似乎促进了艰难梭菌在上皮附近的生长。 结合我们的发现，上皮相关的基因表达水平，而不是腔上皮，C. 艰难梭菌与疾病结局相关，我们假设毒素基因在上皮近端的表达 亚群可能影响小鼠的疾病预后。为了验证这一假设，我们使用了转录记者 艰难梭菌感染过程中单细胞水平定位及其毒素基因表达的研究 小鼠和结肠素衍生的单层系统。通过分析艰难梭菌突变株中的这些记者，这些突变株使 不同数量的毒素或有代谢缺陷，我们将确定上皮之间的关系- 近端生长、寄主代谢物利用和损害。因为我们的初步数据进一步表明毒素 基因表达与产孢子基因表达呈反向调节，我们还将检验C. 艰难杆菌在突变株的产毒细胞和产孢细胞之间的感染过程中建立了“分工”。 改变了这两个过程的分布。总的来说，这些分析将从根本上推进我们的 了解艰难梭菌如何建立感染，并可能确定疾病严重程度的新决定因素。

The spore-forming bacterial pathogen Clostridioides difficile caused ~450,000 diarrheal infections and ~30,000 deaths in 2017, making it the leading cause of healthcare-associated infections in the US. C. difficile thrives in the dysbiotic gut because an intact resident microflora antagonizes its growth. As C. difficile grows in the colon, it makes the glucosylating toxins responsible for causing disease pathology and the spores necessary to transmit disease. Recent work has shown that toxin production promotes C. difficile growth in the gut by inducing inflammation, which generates host-derived metabolites that C. difficile specifically exploits. For example, toxin- mediated inflammation stimulates the host to degrade collagen and produce sorbitol. C. difficile then catabolizes the resulting metabolites, allowing it to grow to higher levels in the gut. Since these metabolites are likely concentrated close to the gut epithelium where they are produced, proximity to this cell layer may benefit C. difficile infection. Consistent with this hypothesis, C. difficile chemotaxes towards mucin-derived sugars, and its utilization of these sugars enhances its growth during infection. The mucus layer also impedes toxin binding to target cells, so the growth of C. difficile near the epithelial layer may promote more damage to this layer than luminal bacteria. While these studies suggest that an epithelium-proximal C. difficile sub-population may play key roles during infection, C. difficile is primarily thought of as a gut luminal pathogen. For example, the authors of a previous fluorescent in situ hybridization study directed at localizing C. difficile during murine infection concluded that it rarely associates with the gut epithelium. However, using novel C. difficile constitutive fluorescent reporter strains we find that C. difficile frequently grows in close proximity to the gut epithelium, even though most of the population is luminal. Furthermore, toxin production appears to promote C. difficile growth close to the epithelium. Combined with our finding that gene expression levels in epithelium-associated, but not luminal, C. difficile correlates with disease outcome, we hypothesize that toxin gene expression in the epithelium-proximal sub-population may drive disease prognosis in mice. To test this hypothesis, we are using transcriptional reporter strains to localize C. difficile and quantify its toxin gene expression at the single-cell level during infection of both mice and a colonoid-derived monolayer system. By analyzing these reporters in C. difficile mutants that make varying amounts of toxin or have metabolic defects, we will determine the relationship between epithelium- proximal growth, host metabolite utilization, and damage. Since our preliminary data further suggest that toxin gene expression is inversely regulated with sporulation gene expression, we will also test the hypothesis that C. difficile establishes a “division of labor” during infection between toxin-producing vs. sporulating cells in mutants with altered distributions of these two processes. Collectively, these analyses will fundamentally advance our understanding of how C. difficile establishes infection and may identify novel determinants of disease severity.