Polymicrobial interactions in Crohn's Disease

克罗恩病中的多种微生物相互作用

• 批准号: 10441347
• 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/10441347
• 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; 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; 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; diagnostic strategy; experimental study; fungus; gut inflammation; gut microbiota; healing; host microbiota; in vivo; in vivo evaluation; in vivo imaging; indoleacetic acid; inhibitor; insight; interleukin-22; intestinal injury; metabolomics; microbial; microbiome; microorganism; microorganism interaction; mouse model; murine colitis; 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（“真菌组”，MYC）的作用以及细菌组（BM）和真菌之间的相互作用 克罗恩病（CD）社区相对被忽视。最近，我们比较了肠道BM和MYC CD 患者与其健康亲属的比较，结果显示真菌热带念珠菌 (CT) 的丰度 与粘质沙雷氏菌（SM）和大肠杆菌（EC）呈正相关。 我们分析了 患有全部 3 种疾病的 CD 患者数量；表现出所有 3 种生物体的 CD 患者人数为 30%， 相比之下，健康对照组的这一比例为 9%。至关重要的是，CD 患者中所有三种微生物的丰度均高出 6.2 倍 与控制。因此，这种微生物组合在克罗恩病患者胃肠道中的流行率是显着的。 我们进一步表明，在体外，这些多微生物三重物种合作形成强大的生物膜（TSB）， 与单物种（SSB）或双物种（CT+EC或 CT+SM、DSB）。支持数据表明 CT、SM 和 EC 之间的相互作用是特定的；替代 毛孢子菌属(TC) 或 Saccharomyces fibuligera (SF) 不会增加生物膜的形成。不过，初步 使用白色念珠菌 (CA) 作为对照比较的数据（据其他人报告，在 CD 患者中升高）确实 导致 TSB 生物膜形成增加，表明这是念珠菌特有的作用。重要的是，我们验证了 3 种病原体使用葡聚糖钠在体内（厌氧环境）形成坚固的生物膜 小鼠溃疡性结肠炎硫酸盐（DSS）模型。 TSB 上清液的代谢组学分析鉴定出 11 与 SSB 或 DSB 相比，代谢显着增加。其中，5-氧代脯氨酸（5-OP）最为重要。 代谢物升高（TSB 是 SSB 或 DSB 的 131 倍）。此外，还形成了吲哚-3-乙酸 (IAA) 乳杆菌代谢色氨酸的结果，色氨酸可以作为芳基烃的内源配体 受体 (AhR)，介导先天淋巴细胞 (ILC) 产生 IL-22，进而触发 CA 的抑制 定殖也显着增加。这 2 种代谢物影响念珠菌毒力因子 （丝状、粘附和自动聚集）。尽管这些数据很重要并且表明了强有力的 尽管 CD 中存在多种微生物相互作用，但他们并没有建立这些微生物相互作用的潜在机制。 该提案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） 生物膜的形成，协同相互作用，加剧肠道炎症，加剧症状 为了解决机制，我们将： (1) 确定机制 TSB 中 CT、EC 和 SM 形成的代谢物会改变肠道结果； (2) 识别基因变化 使用微生物突变菌株和 RNAseq 进行潜在念珠菌修饰以增加致病性 方法； (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。 通过多物种相互作用。