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.

Public Health Relevance

The current application is designed to gain insight into the mechanism(s) of bacterial/fungal polymicrobial interactions in the setting of intestinal inflammation, similar to that observed in inflammatory bowel diseases such as colitis and Crohn?s. Specifically, we propose to examine how metabolites produced as a result of this interaction may exacerbate pathogenicity. In addition, we will localize the metabolites in the intestinal mucosae and stool using in vivo imaging, and quantify them using MALDI MS/MS approaches. Alternatively, substituting mutant non- pathogenic organisms will determine genes implicated in increased inflammation associated with mixed species infection. Moreover, we will determine whether interrupting mixed species biofilm formation using antibacterial, antifungal or metabolite inhibitors reduce the observed pathogenicity and inflammation associated with polymicrobial infection.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Rothermel, Annette L
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Case Western Reserve University
Schools of Medicine
United States
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