Background
Gut mycobiota is associated with visceral hypersensitivity (VH) in irritable bowel syndrome (IBS) patients, but there have been few studies of role of colon specific single fungus in IBS development. We aim to evaluate the effects of specific genus screened out from the mucosal mycobiota of IBS patients on VH and investigate the possible mechanism through animal and cell experiments.
Methods
Colonic mucosal mycobiota of 14 IBS-D patients and 12 healthy volunteers was identified by ITS-1 sequencing, and the visceral sensitivity (VS) was assessed by IBS- severity scoring system (IBS-SSS). The biomarker fungus was screened out and the correlation with VS was evaluated. Then the biomarker fungus was administered to VH mice model induced by TNBS, to investigate the influence on VH by colorectal distension- abdominal withdrawal reflex (CRD-AWR). 16s and ITS sequencing were used to detect alteration of gut flora. Western blot was used to evaluate colonic dectin-1 pathway in mice.
Results
Altered mucosal mycobiota and negative correlation between specific genus Cladosporium and VS in IBS patients
The overall mucosal mycobiota α diversity and β diversity of IBS patients was different from that of healthy volunteers as indicated by simpson index (Fig. 1A), PCoA analysis (Fig. 1B) and PLS-DA analysis (Fig. 1C). LEfSe analysis indicated that genus Cladosporium and Fomitopsis was the biomarker for IBS patients (Fig. 1D-E). Furthermore, Wilcoxon test showed the most significantly increased mucosal fungi in IBS patients was Cladosporium screened by fold change > 2 and FDR < 0.1 (Fig. 1F). Next, the spearman correlation showed that abundance of Cladosporium was negatively associated with IBS-SSS score (r = -0.73, P < 0.01, Fig. 1G).
Cladosporium cladosporioides (C. cladosporioides) ameliorates VH induced by TNBS through downregulated the dectin-1/NF-κB pathway in mice
To explore the role of genus Cladosporium in VH, we firstly established a mouse model of VH induced by TNBS. The schematic diagram was depicted in Fig. 2A. After 4 weeks recovery from TNBS enema, the VS was increased compared to control group at the pressure level of 40mmHg, 60mmHg (Fig. 2C-D). We selected a typical species C. cladosporioides to characteristic the role of genus Cladosporium. After 4 weeks gavage of C. cladosporioides, the VS in TNBS+Fungi group was lower than TNBS group at 60mmHg (Fig. 2B). Gavage of C. cladosporioides also restored the gut microbiota (Fig. 2F.H) and mycobiota (Fig. 2G.I) which was altered in VH mice. Meanwhile, the abundance of C. cladosporioides in TNBS group was lower than Control group (Fig. 2J). Furthermore, C. cladosporioides downregulated the colon dectin-1/NF-κB pathway in TNBS mice (Fig. 2K-L).
Conclusion
Gut fungi Cladosporium was related to lower VH in IBS-D patients and could impove the VH induced by TNBS in mice through inhibiting the dectin-1/NF-κB.

$Fig. 1. Altered mucosal mycobiota and negative correlation between specific genus Cladosporium and visceral sensitivity in IBS patients. (A) α diversity of simpson index. (B) β diversity depicted by PCOA based on unweighted unifraction. (C) β diversity depicted by PLS-DA. (D) LDA score of LEfSe analysis. (E) Cladogram of LEfSe analysis. (F) Wilcoxon test between IBS patients and healthy controls. (G) Spearman correlation berween abundance of Cladosporium and IBS-SSS score.$

Fig. 1. Altered mucosal mycobiota and negative correlation between specific genus Cladosporium and visceral sensitivity in IBS patients. (A) α diversity of simpson index. (B) β diversity depicted by PCOA based on unweighted unifraction. (C) β diversity depicted by PLS-DA. (D) LDA score of LEfSe analysis. (E) Cladogram of LEfSe analysis. (F) Wilcoxon test between IBS patients and healthy controls. (G) Spearman correlation berween abundance of Cladosporium and IBS-SSS score.

$Fig. 2. Cladosporium cladosporioides ameliorates VH induced by TNBS through downregulated the dectin-1/NF-κB pathway in mice. (A) Schematic representation of VH model establishment and subsequent fungal gavage experiments. (B-E) Visceral hypersensitivity in mice evaluated by CRD- AWR. Data were presented as (P25, P75), one-way ANOVA followed by Tukey's test and Student's t-test. (F) α diversity of fecal microbiota. (G) α diversity of fecal mycobiota. (H) β diversity of fecal microbiota depicted by CPCOA based on bray curtis. (I) β diversity of fecal mycobiota depicted by CPCOA based on unweighted unifraction. (J) Abundance of genus Cladosporium in four groups. (K) Western blot of colon dectin-1/NF-κB pathway. (L) Statistical analysis of dectin-1 protein expression. Data were presented as means±SD, one-way ANOVA and followed by Tukey's test. N=7. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.$

Fig. 2. Cladosporium cladosporioides ameliorates VH induced by TNBS through downregulated the dectin-1/NF-κB pathway in mice. (A) Schematic representation of VH model establishment and subsequent fungal gavage experiments. (B-E) Visceral hypersensitivity in mice evaluated by CRD- AWR. Data were presented as (P₂₅, P₇₅), one-way ANOVA followed by Tukey's test and Student's t-test. (F) α diversity of fecal microbiota. (G) α diversity of fecal mycobiota. (H) β diversity of fecal microbiota depicted by CPCOA based on bray curtis. (I) β diversity of fecal mycobiota depicted by CPCOA based on unweighted unifraction. (J) Abundance of genus Cladosporium in four groups. (K) Western blot of colon dectin-1/NF-κB pathway. (L) Statistical analysis of dectin-1 protein expression. Data were presented as means±SD, one-way ANOVA and followed by Tukey's test. N=7. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Chenglin Guo

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