Fungal dysbiosis of the gut microbiota is associated with colorectal cancer in Chinese patients

Abstract

Changes in bacteria and virions are associated with colorectal cancer (CRC). However, the fungal microbiota in the intestines of CRC patients remains largely unexamined. We identified differences in the intestinal fungal microbiota between healthy persons and patients with colorectal polyps or CRC. Using second-generation sequencing technology, we sequenced and aligned the ITS1 regions of fungi collected from fecal samples. We found a significant increase in the Candida albicans levels in the guts of CRC patients. Dectin-1 is a C-type lectin receptor that recognizes β-1,3-glucan in the cell walls of most fungi and is expressed by many cell types, including dendritic cells, macrophages, and monocytes. However, the mechanisms controlling the expressions and functions of dectin-1 in intestinal epithelial cells (IECs) remain unclear. Furthermore, the putative effects of C. albicans on IECs are unknown. C. albicans induces the proliferation of IECs by activating the Wnt signaling pathway, and the Wnt pathway contributes to the development of CRC. Mice infected with C. albicans show an activation of the Wnt pathway. Therefore, IECs may recognize the activation of the Wnt pathway by C. albicans through dectin-1 to promote the development of CRC.

Keywords: Candida albicans; Fungi; Wnt; intestinal epithelial cells; proliferation; sequencing.

Figure 1

Intestinal fungal ecological disorders related to CRC. A. Relative abundance of dominant enteric fungi phyla in N (n=14), P (n=28) and T (n=13). The dominant phyla are Ascomycota and Basidiomycota in both groups. B. The rarefaction curves show that a sufficient sequencing depth was reached and that an increased number of intestinal fungal species were detected in the T group compared with the N and P groups. C. The alpha-diversity (Chao1 plot) of the gut fungus communities in the N, P, and T groups. D. The alpha-diversity (Simpson plot) of the gut fungus communities in N, P, and T groups. E. The alpha-diversity (Shannon plot) of the gut fungus community in N, P, and T groups.

Figure 2

Taxonomic changes in the intestinal fungi in CRC. A. Venn diagrams were used to show the overlap of the OTUs of the study groups. The samples were divided into three groups: N, T, P. B. The fungal structures at the samples’ phylum levels. Only the relative abundances of the 10 most abundant genera are plotted. C. The fungal structures at the samples’ genus levels. Only the relative abundances of the 10 most abundant genera are plotted. The others are concatenated in the group named ‘others’. D. β-diversity is represented by a weighted UniFrac distance between the intestinal fungal communities. Wilcoxon rank-sum test, *P<0.05, **P<0.01, ***P<0.001. E. PCA analysis on N, P, and T samples based on core OTUs. F. NMDS ordination analysis of the fungal OTU community distance based on the Bray-Curtis dissimilarity metrics of the relative abundance profiles.

Figure 3

The abundance of Candida in the CRC is increased. A. A two-dimensional heatmap depicting the rank normalized abundances (scaled between -1 and 1) of the 35 fungal genera in the N, P, and T group patients of gut fungi. B. A linear discriminant analysis (LDA) revealed significant bacterial differences between the N and P groups. The LDA scores (log10)>2 and P<0.05 are listed. C. Our linear discriminant analysis (LDA) revealed significant bacterial differences between the P and T groups. The LDA scores (log10)>2 and P<0.05 are listed. D. Our linear discriminant analysis (LDA) revealed significant bacterial differences between the N and T groups. The LDA scores (log10)>2 and P<0.05 are listed. E. Our linear discriminant analysis (LDA) revealed significant bacterial differences between the N, P, and T groups. The LDA scores (log10)>2 and P<0.05 are listed. F. A histogram of the increased abundances of Candida in the T group patients compared with the N and P groups.

Figure 4

The abundance of Candida albicans in the CRC is increased. A. A comparative analysis of the phage taxonomy in the three group samples, N, P, and T. The phages identified from the samples are represented by different colors. B. Colon sections from the N, P, and T patients were stained with an anti-Candida albicans antibody (green) and counterstained with DAPI. The magnification is 200 times and the scale bar represents 50 microns.

Figure 5

The IECs express Dectin-1, and C. albicans induces a proliferation of the NCM460 cells through the Wnt/β-catenin signaling pathway. A. The Dectin-1 expression counterstained with DAPI by NCM460 was measured using immunofluorescence. The magnification is 200 times and the scale bar represents 50 microns. B. Western blot analysis of the cell lysates from primary human NCM460 cells revealed signals referring to Dectin-1 at 23 KDa. C. NCM460 cells were incubated with PBS, or heat-inactivated C. albicans (10:1 ratio). The cell proliferation rates were evaluated using cell number counting after treatment at 6, 24, and 48 hours (*P<0.05; **P<0.001, unpaired Student t-test). D. NCM460 cells were incubated for 24 h in the absence (N) or presence of heat-inactivated C. albicans (10:1 ratio). The effect of cell proliferation was assessed using MTT assays. E. Western blots showed that C. albicans upregulates the expression of Ki-67 and activates the Wnt pathway activation in NCM460. F. The effect of C. albicans and the Wnt signaling inhibition ( ICG-001) on cell proliferation was assessed using MTT assays. G. The expression of Ki-67 and the Wnt signaling-related proteins in the groups: N (not exposed ICG-001 or C. albicans), C. albicans (exposed to C. albicans) and ICG-001 (exposed to ICG-001 and C. albicans). H. NCM460 cells were incubated for 24 h in the absence (N) or presence of heat-inactivated C. albicans (10:1 ratio). The effect of the C. albicans and Dectin-1 inhibition on the cell proliferation was assessed using MTT assays. I. The expressions of Ki-67 and the Wnt signaling-related proteins in the groups: N (not exposed ICG-001 or C. albicans), C. albicans (exposed to C. albicans) and Anti-dectin-1 mAb (exposed to Anti-dectin-1 mAb and C. albicans).

Figure 6

C. albicans induces the proliferation of colorectal tissues through the Wnt/β-catenin signaling pathway. A. Colorectal tissues from the WT mice (N) and the WT mice were administered stomah with C. albicans (CA) stained with an anti-Candida albicans antibody (red) and counterstained with DAPI. The magnification is 200 times and the scale bar represents 50 microns. B. The colorectal tissues from WT mice (N) and WT mice were administrated stomach (CA) with C. albicans for four doses, the mRNA expressions of C. albicans in the colorectal tissues were measured using qPCR. C. Western blots illustrated that C. albicans activated Ki-67 and the Wnt pathway activation in colorectal tissues.