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

形成孢子的细菌病原体艰难梭菌导致约450，000例肠道感染和约30，000例 在2017年死亡，使其成为美国医疗相关感染的主要原因。C.艰难生长在 因为一个完整的常驻微生物菌群对抗它的生长。作为C.艰难生长在 结肠，它使葡萄糖基化毒素负责引起疾病病理和孢子所必需的， 传播疾病。最近的研究表明，毒素的产生促进了C。在肠道中艰难生长， 炎症，产生宿主衍生的代谢物，C.具体来说，就是利用。例如，毒素- 介导的炎症刺激宿主降解胶原并产生山梨醇。C. difficile然后分解代谢 产生的代谢产物，使其在肠道中生长到更高的水平。因为这些代谢物很可能 由于它们集中在产生它们的肠上皮细胞附近，因此接近该细胞层可能有益于C。 艰难感染与这一假设相一致，C.对粘蛋白衍生的糖的艰难化学税，及其 这些糖的利用增强了其在感染期间的生长。粘液层也阻碍毒素结合到 靶细胞，因此C.在上皮层附近的艰难梭菌可能会促进对这一层的损害， 管腔细菌 虽然这些研究表明，上皮近端C。difficile亚群可能在 infection，C.艰难梭菌最初被认为是一种肠腔病原体。例如，以前的作者 荧光原位杂交研究定位C.小鼠感染期间的艰难梭菌得出结论， 很少与肠上皮细胞相关。然而，使用新的C.艰难组成型荧光报告菌株 我们发现C.艰难梭菌经常生长在靠近肠道上皮，即使大多数的 人口是鲁米诺。此外，毒素的产生似乎促进了C。靠近上皮的艰难生长。 结合我们的发现，上皮相关的基因表达水平，但不是管腔，C。 艰难梭菌与疾病结果相关，我们假设毒素基因在上皮近端表达， 亚群可以驱动小鼠疾病预后。为了验证这一假设，我们使用转录报告基因 菌株定位C.艰难梭菌，并在单细胞水平上定量其毒素基因表达， 小鼠和类结肠衍生的单层系统。通过对C.艰难的突变体， 不同数量的毒素或有代谢缺陷，我们将确定上皮细胞之间的关系- 近端生长、宿主代谢物利用和损害。因为我们的初步数据进一步表明 基因的表达与孢子形成基因的表达呈负相关，我们也将检验C. 艰难梭菌在突变体中的产毒素细胞与产孢子细胞之间建立了感染期间的"劳动分工" 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动我们的 了解C。艰难梭菌建立感染，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.