Bacterial surface layer-specific immune protection against gut microbial infection

 描述(由申请人提供)：针对胃肠道(GI)感染的有效保护性和受控免疫需要由平衡的肠道微生物区系和相关代谢物诱导的关键调节信号，从而导致功能稳态，以防止病理性肠道疾病的表现。我们证明，与给早产儿使用的配方奶相比，人类母乳严重地重塑了肠道微生物组成，增加了有益细菌属(如丙酸杆菌)。这些新鉴定的益生菌菌株之一的初步基因组序列分析，暂定为P.UF1，与已知的丙酸杆菌物种密切相关，但又截然不同(85%的序列同源性)，包括P.freudenreichii。已获得的数据表明，P.UF1显著调控了无菌(GF)小鼠体内诱导性炎症细胞因子(如IL-1β)的调节，这些小鼠通过从配方奶喂养的早产儿粪便中获得的不平衡微生物群进行转运。P.UF1不仅调节Th17细胞对病原体感染的应答，而且增强了肠粘膜屏障功能。这种免疫调节是由细菌的表层(S层)紧密控制的，该细菌与结肠树突状细胞(DC)表达的特异性细胞间黏附分子-3-抓取非整合素相关1(SIGNR1)结合，导致S层特异的Th17T细胞分化。这种动态平衡在Signr1-/-小鼠中显著恶化，进一步强调了这种分子相互作用在保护性肠道免疫中的关键作用。TH17 在H2-AB1-/-(MHC II-/)小鼠中，形成明显被消融，表明S依赖于层的Th17分化。给予P.UF1的单基因相关的GF-小鼠表现出特异性的Th17分化和参与调节Th17功能的色氨酸分解代谢的关键代谢物(如犬尿氨酸)。此外，在P.UF1中缺失了二氢硫酰胺酰基转移酶(DlaT)基因，命名为ΔdlaT P.UF1，以阐明这一至关重要的S层成分在S蛋鸡特异性Th17T分化、肠道微生物组成和相关诱导代谢产物中的作用。初步数据有力地表明，在P.UF1中去除dlaT基因从根本上削弱了该细菌控制诱导的肠前炎症(例如，IL-1β)的调节功能。这项研究建议的目标是清楚地描绘出 P.UF1的S层在诱导受调控的保护性S层特异性Th17应答中的作用因此，我们推测，新发现的UF1菌株不仅通过与SIGNR1相互作用而诱导S层特异性Th17分化以提供对病原体的保护，而且细菌S层dlaT基因显著参与了这些抗原特异性Th17反应。其具体目的是：1)阐明S蛋鸡特异性Th1 7的分化及其受SIGNR1信号的调控；2)阐明SIGNR1信号转导的关键 细菌二氢硫酰胺酰基转移酶(DlaT)在S蛋鸡特异性Th17分化中的作用这样的机制洞察将揭开参与调节诱导保护性S蛋鸡特异性免疫反应的完整机制，以抵御病原体感染。

 DESCRIPTION (provided by applicant): Efficacious protective and controlled immunity against gastrointestinal (GI) infection requires critical regulatory signals induced by a balanced gut microbiota and associated metabolites resulting in functional homeostasis that prevents the manifestation of pathologic intestinal disorders. We demonstrated that human breast milk, when compared to formula given to preterm infants, critically reshapes the gut microbial composition and increases beneficial bacteria genera (e.g., Propionibacterium). First draft genome-sequence analysis of one of these newly identified probiotic strains, tentatively designated, P. UF1, is closely related to, and yet distinct, (85% sequence homology) from known Propionibacterium species, including P. freudenreichii. Obtained data demonstrate that P. UF1 significantly governs the regulation of induced inflammatory cytokines (e.g., IL-1β) in germ-free (GF) mice transfaunated with imbalanced microbiota derived from the feces of formula-fed preterm infants. P. UF1 not only regulated Th17 responses against pathogen infection, but also fortified the intestinal mucosal barrier function. Such immune regulation is tightly controlled by the surface layer (S-layer) of the bacterium binding to specific intercellular adhesion molecule-3-grabbing nonintegrin-related 1 (SIGNR1) expressed by colonic dendritic cells (DCs), resulting in S-layer-specific Th17 differentiation. This homeostasis significantly deteriorated in Signr1-/- mice, further emphasizing the crucial role of this molecular interaction in protective gut immunity. Th17 formation was significantly ablated in H2-Ab1-/- (MHC II-/) mice, suggesting S- layer-dependent Th17 differentiation. Mono-associated GF-mice given P. UF1 exhibited specifically Th17 differentiation and key metabolites (e.g., kynurenine) involved in tryptophan catabolism that regulates Th17 function. Additionally, the dihydrolipoamide acyltransferase (dlaT) gene was deleted in P. UF1 designated, ΔdlaT P. UF1, to elucidate the role of this critically important S-layer component in S-layer-specific Th17 differentiation, intestinal microbial composition, and related induced metabolites. Preliminary data strongly indicate that ablation of the dlaT gene in P. UF1 fundamentally diminishes the regulatory function of this bacterium to control induced gut proinflammation (e.g., IL-1β). The objective of this research proposal is to clearly delineate the role of the S-layer of P. UF1 in the induction of regulated protective S-layer-specific Th17 responses against intestinal infection. Thus, we hypothesize that the newly identified P. UF1 species not only induces S-layer-specific Th17 differentiation upon interaction with SIGNR1 to confer protection against pathogens, but that the bacterial S-layer dlaT gene is significantly involved in these antigen-specific Th17 responses. The Specific Aims are: 1) Elucidate S-layer-specific Th17 differentiation and its regulation by SIGNR1 signaling, and 2) Delineate the critical involvement of bacterial dihydrolipoamide acyltransferase (DlaT) in S-layer-specific Th17 differentiation. Such mechanistic insights will unravel integral mechanisms involved in the regulation of induced protective S-layer-specific immune responses against pathogen infection.