Polymicrobial interactions in Crohn's Disease

克罗恩病中的多种微生物相互作用

• 批准号: 10223109
• 负责人: Mahmoud A Ghannoum
• 金额: $ 61.74万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-08 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223109
• 关键词: Address; Adhesions; Affect; Anaerobic Bacteria; Anti-Bacterial Agents; Antifungal Agents; Bacteria; Candida; Candida albicans; Candida tropicalis; Chronic; Clinical Research; Coculture Techniques; Colitis; Colon; Communities; Crohn' s disease; Data; Diagnostic; Environment; Escherichia coli; Exhibits; Exposure to; Family; Feces; Filament; Flare; Gastrointestinal tract structure; Gene Expression; Generations; Genes; Goals; Habits; Hyphae; Immune response; In Vitro; Indoles; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interruption; Intestinal Mucosa; Intestines; Knock-out; Lactobacillus; Letters; Lymphoid Cell; Mediating; Microbe; Microbial Biofilms; Modeling; Modification; Mucous Membrane; Mus; Oral; Organism; Outcome; Pathogenicity; Patients; Phase; Phenotype; Play; Prevalence; Process; Production; Pyrrolidonecarboxylic Acid; Recovery; Relapse; Reporting; Resolution; Role; Saccharomyces; Sampling; Serratia marcescens; Severities; Sodium Dextran Sulfate; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Symptoms; Testing; Therapeutic; Thick; Tissues; Trichosporon; Tryptophan; Ulcerative Colitis; Virulence Factors; Yeasts; aryl hydrocarbon receptor ligand; bacteriome; design; dextran sulfate sodium induced colitis; experimental study; fungus; gut microbiota; healing; host microbiota; in vivo; in vivo evaluation; in vivo imaging; indoleacetic acid; inflammatory disease of the intestine; inhibitor/antagonist; insight; interleukin-22; intestinal injury; metabolomics; microbial; microbiome; microorganism; microorganism interaction; mouse model; mutant; mycobiome; novel therapeutics; pathogen; polymicrobial biofilm; preclinical study; response; transcriptome sequencing

The role of gut fungal community (“mycobiome”, MYC) and interactions between the bacteriome (BM) and fungal communities in Crohn’s Disease (CD) have been relatively ignored. Recently, we compared the gut BM and MYC of CD patient’s to their healthy relatives and showed that abundance of the fungus Candida tropicalis (CT) was positively correlated with the bacteria Serratia marcescens (SM) and Escherichia coli (EC). We analyzed the number of CD patients that had all 3 species; the number CD patients exhibiting all 3 organisms was 30%, compared to 9% in healthy controls. Critically, abundance of all three organisms is 6.2 fold higher in CD patients vs. controls. Thus, the prevalence of this combination of microorganisms in the GI tracts of CD patients is significant. We further showed that ex-vivo, these polymicrobial triple species cooperated to form robust biofilms (TSBs) that were significantly increased compared to those formed by single-species (SSBs) or double-species (CT+EC or CT+SM, DSBs). Supporting data showed the interaction between CT, SM, and EC was specific; substitution of Trichosporon spp. (TC) or Saccharomyces fibuligera (SF) did not increase biofilm formation. However, preliminary data using Candida albicans (CA) as a control comparator reported by others to be elevated in CD patients, did cause increased TSB biofilm formation, indicating that this is a Candida specific effect. Importantly, we validated that the 3 pathogens formed robust biofilms in vivo (an anaerobic environment) using a dextran sodium sulfate (DSS) model of murine ulcerative colitis. Metabolomic analyses of supernatants from TSBs identified 11 significantly increased metabolites compared to SSBs or DSBs. Of these, 5-oxoproline (5-OP), was the most elevated metabolite (131-fold higher in TSBs compared to SSBs or DSBs). Also, Indole-3-acetic acid (IAA), formed as a result of Lactobacilli metabolizing tryptophan, which can act as an endogenous ligand for aryl hydrocarbon receptors (AhR), mediating innate lymphoid cell (ILC) production of IL-22, that in-turn triggers the inhibition of CA colonization, was also significantly increased. These 2 metabolites influence candidal virulence factors (filamentation, adhesion and auto-aggregation). Although these data are significant and indicate a strong polymicrobial interaction in CD, they do not establish the underlying mechanisms of these microbial interactions. This proposal focuses on determining the mechanism/s underlying the interaction of mixed species microbes using complementary in vitro and in vivo approaches. Our hypothesis is that SM, EC and Candida (CT and CA), in the setting of biofilms, interact cooperatively to exacerbate intestinal inflammation and exacerbate symptoms To address the mechanism(s) we will: (1) Determine the mechanism(s) metabolites formed by CT, EC, and SM in TSBs alter intestinal outcomes; (2) Identify gene changes underlying Candida modifications that increase pathogenicity using microbial mutant strains and RNAseq approaches; and (3) Determine whether exposure of TSBs to antibacterial, antifungal or metabolite inhibitors modulates the severity of intestinal inflammation in vivo. via multispecies interactions.

肠道真菌群落（“真菌群落”，MYC）的作用以及细菌群落（BM）和真菌群落之间的相互作用 克罗恩病（CD）的社区相对被忽视。最近，我们比较了肠道BM和MYC 的CD患者的健康亲属，并表明真菌热带念珠菌（CT）的丰度是 与细菌粘质沙雷氏菌（SM）和大肠杆菌（EC）呈正相关。 我们分析了 具有所有3种微生物的CD患者数量;显示所有3种微生物的CD患者数量为30%， 相比之下，健康对照组为9%。至关重要的是，CD患者中这三种微生物的丰度高出6.2倍 vs.对照因此，CD患者胃肠道中这种微生物组合的患病率是显著的。 我们进一步表明，离体，这些多微生物三重物种合作形成坚固的生物膜（TSB）， 与单种（SSB）或双种（CT+EC或 CT+SM、DSB）。支持性数据显示CT、SM和EC之间的相互作用是特异性的; 丝孢酵母属物种(TC)或扣囊酵母（SF）不增加生物膜形成。然而，初步 使用白色念珠菌（CA）作为对照比较的数据，其他人报告在CD患者中升高， 导致TSB生物膜形成增加，表明这是念珠菌特异性效应。重要的是，我们验证了 使用葡聚糖钠，3种病原体在体内（厌氧环境）形成了坚固的生物膜， 硫酸盐（DSS）小鼠溃疡性结肠炎模型。TSB上清液的代谢组学分析鉴定了11个 与SSB或DSB相比，代谢物显著增加。其中5-氧代脯氨酸（5-OP）含量最高 代谢产物升高（TSB中的代谢产物是SSB或DSB的131倍）。此外，吲哚-3-乙酸（IAA），形成 作为乳酸杆菌代谢色氨酸的结果，色氨酸可以作为芳烃的内源性配体 受体（AhR），介导先天淋巴细胞（ILC）产生IL-22，进而触发CA的抑制 殖民化也显着增加。这2种代谢物影响念珠菌毒力因子 （粘附、粘附和自动聚集）。虽然这些数据是重要的，并表明一个强大的 尽管它们在CD中存在多种微生物相互作用，但它们没有建立这些微生物相互作用的潜在机制。 该建议的重点是确定混合物种微生物相互作用的机制， 互补的体外和体内方法。 我们的假设是SM，EC和念珠菌（CT和CA），在 生物膜的形成，相互作用，加剧肠道炎症和症状 为解决机制问题，我们将：（1）确定机制 TSB中由CT、EC和SM形成的代谢物改变肠道结果;（2）识别基因变化 使用微生物突变菌株和RNAseq增加致病性的潜在念珠菌修饰 方法;和（3）确定TSB是否暴露于抗菌剂、抗真菌剂或代谢物 抑制剂调节体内肠道炎症的严重程度。 通过多物种的相互作用。