Overall virome community composition in conditional VDR knockout mice
In all fecal samples, 4,048 viral species were identified. Distinct viral compositions and communities, which differed in both diversity and composition, were present between the control mice and the conditional VDR knockout mice (Figure 1). We presented the 10 most abundant viral species for individual samples in each group (Figure 1). The most abundant species in all the groups were Vibrio phage JSF5, Vibrio phage JSF6, bovine viral diarrhea virus 1, Escherichia coli O157 typing phage 7, human alphaherpesvirus 1, Lactobacillus prophage Lj771, influenza A virus, Lactobacillus phage KC5a, and viruses incertae sedis unidentified phage (Figure 1A). When individually analyzing the species, Vibrio phages JSF5 and JSF6 were significantly more abundant in all three VDR KO mouse models compared to control VDRLoxP mice. Escherichia coli O157 typing phage 7 was found to be significantly more abundant in VDRΔPC mice, compared to the control mice. Meanwhile, Lactobacillus phage phiadh was considerably less abundant in VDRΔLyz mice compared to control VDRLoxP mice (Figure 1B) (P<0.01).
Altered diversity of the virome in conditional VDR knockout mice
Shannon diversity is commonly used to characterize species diversity in a community [38]. We found that the three conditional VDR knockout groups had lower Shannon diversities than the control mice. The Shannon diversity at the viral species level was significantly lower in VDRΔIEC, VDRΔPC, and VDRΔLyz mice than in VDRLoxP control mice (P=0.05, P=0.05, and P=0.04, respectively) (Figure 2A).
The Bray-Curtis dissimilarity index was used in this study to measure the dissimilarities of samples. We first performed PCoA and found viral dissimilarities between conditional VDR knockout mice and control mice (Figure 2B). VDRΔIEC mice partially overlapped with control VDRLoxP mice, whereas VDRΔPC and VDRΔLyz mice were completely separated from VDRLoxP mice. The group differences can be explained by a total of 37.2% (29.3% + 7.9%) of the variations among the animals.
Next, we performed nonparametric PERMANOVA to evaluate whether VDR status impacts the overall intestinal viral profile. The sequential test “Group and Gender” showed that the dissimilarities among groups were significantly different (P=0.011). Because the overall dissimilarity among groups was significantly different, we performed a pairwise PERMANOVA and found that the Bray-Curtis dissimilarities of viruses of the VDRΔIEC, VDRΔPC, and VDRΔLyz mice were significantly different from the dissimilarity in the VDRLoxP mice. Furthermore, the differences in dissimilarity among groups were confirmed by ANOSIM (analysis of similarity), where the rank dissimilarities between and within groups were significantly different (P=0.001). VDRΔIEC, VDRΔPC, and VDRΔLyz mice had higher dissimilarities (i.e., lower similarity) than VDRLoxP mice (Figure 2C).
Altered abundance of the virome in conditional VDR knockout mice
We observed that a total of 12 viral species were differential in the conditional VDR knockout mice compared to the control VDRLoxP mice, of which 6 had q-values <0.001, 2 had q-values <0.01 and 4 had q-values <0.05 (Figure 3A). The log-ratio of fold change with significant differences (q<0.05) is shown with colored histograms. There were two enriched viral species in the comparison of VDRΔIEC/VDRLoxP, Vibrio phage JSF5 (q<0.001) and bovine viral diarrhea virus 1 (q<0.05), and one enriched viral species in the comparison of VDRΔPC/VDRLoxP, Vibrio phage JSF5 (q<0.01). Vibrio phage JSF5 was more abundant in both VDRΔIEC and VDRΔPC mice, whereas enriched BVDV1 was only found in VDRΔIEC compared with the other groups (Figure 3A). For the comparison of VDRΔLyz and VDRLoxP, nine virus species were found to be significantly altered (5 with a q value <0.001, 1 with q<0.01, and 3 with a q value <0.05). Of these species, three were enriched (Vibrio phage JSF5, bovine viral diarrhea virus 1 and Vibrio phage JSF6), while six were depleted (Lactobacillus prophage phiadh, Cherry green ring mottle virus, Lactobacillus phage KC5a, avian avulavirus 1, Mycobacterium virus Phayonce, and one unidentified species in the Podoviridae family) (Figure 3A).
Sex-based differences in the gut virome altered by VDR status
To investigate the impact of sex on the alteration of the virome community in the intestines of the studied mice, we illustrated significantly different virus species abundances in male and female mice with logarithmic fold-changes and q-values (Figure 3B). Seven species were detected to be significantly altered in the female VDRΔIEC/VDRLoxP comparison but not when comparing males of the same two groups, the altered species included 2 enriched species, Vibrio phages JSF5 and BVDV1, and 5 depleted species, Lactobacillus prophage phiadh, Lactobacillus prophage KC5a, Lactobacillus prophage Lj771, macacine alphaherpesvirus 1 and Catovirus CTV1. However, only Vibrio phage JSF5 was enriched in female VDRΔPC mice compared to control VDRLoxP mice (Figure 3B), which is the same as in the group-factor analysis. Five species in the female VDRΔLyz/VDRLoxP comparison were found to be significantly differential (q<0.05), including 2 enriched species:, Vibrio phage JSF5 and bovine viral diarrhea virus 1, and 3 depleted species, Lactobacillus prophage phiadh, Cherry green ring mottle virus and Lactobacillus prophage KC5a, while in males, only Vibrio phage JSF5 was enriched and only Lactobacillus prophage phiadh was depleted (Figure 3B). Overall, more altered viral species in all conditional VDR knockout mice were found in the female mice than in male mice, which indicated the impact of VDR status on sex differences.
VDR deletion led to significantly enriched Vibrio phages
Vibrio phages target Vibrio cholerae bacteria, which can secrete cholera toxin and cause watery diarrhea in patients [44, 45]. In this study, we found a markedly enriched abundance of Vibrio phage JSF5 and Vibrio phage JSF6 in VDRΔIEC, VDRΔPC, and VDRΔLyz mice compared with control VDRLoxP mice (Figure 1). Moreover, differential analysis found that Vibrio phage JSF5 was significantly enriched in all three conditional VDR-knockout mice (q<0.01) compared to the control mice, and Vibrio phage JSF6 was also more enriched in VDRΔLyz mice in comparison to VDRLoxP mice (q<0.01) (Figure 3). When the sex factor was included in the analysis, the fold-changes of Vibrio phage JSF5 were found to be significant in both female and male VDRΔLyz mice but only in female VDRΔIEC and VDRΔPC mice (Figure 3).
Altered bacterial abundance in conditional VDR knockout mice
We reasoned that the abundance of bacteria in the intestines of the mice should be altered due to the dysbiosis of bacteriophages. In this study, we found that Vibrio cholerae, the host of Vibrio phage JSF5 and Vibrio phage JSF6, which were largely enriched in conditional VDR knockout mice (Figure 1B), was detected at low concentrations in knockout mice compared with control mice (Figure 4A). Significantly lower abundance of this bacterium was found in VDRΔLyz mice, which matches the finding that both Vibrio phage JSF5 and Vibrio phage JSF6 were depleted in VDRΔLyz mice. A similar situation was also found in Lactobacillus gasseri, the bacterial host of Lactobacillus prophage phiadh and Lactobacillus prophage KC5a (Figure 1B; Figure 4A). Furthermore, as the target host of Escherichia coli O157 typing phage 7, E. coli was found to have the opposite phage species abundance in the mice studied here (Figure 1B; Figure 4A). The altered bacterial abundance in the microbial community supports our findings of virome changes.
To further evaluate the altered bacteria, we showed the altered bacterial species with q-values <0.1 in differential analysis in our three mouse models (Figure 4B). We found that Haemophilus ducreyi and Mesorhizobium huakuii were significantly depleted in VDRΔIEC mice compared with control VDRLoxP mice (Figure 4B). Meanwhile, five bacterial species were significantly depleted, and three bacterial species were significantly enriched in VDRΔPC mice, compared with the control. These depleted species were Haemophilus ducreyi Kushneria konosiri, Microbacterium sp. LKL04, Isosphaera pallida, and Actinomyces radingae. Three enriched bacterial species were Bacteroides uniformis, Faecalibaculum rodentium, and Cutibacterium acnes (q<0.01. Figure 4B). Furthermore, we found that nine bacterial species were significantly depleted, and three bacterial species were enriched in VDRΔLyz mice. These depleted bacterial species were Bifidobacterium pseudolongum, Bifidobacterium choerinum, Bifidobacterium animalis, Bordetella pseudohinzii, Haemophilus ducreyi, Clostridium perfringens, Streptomyces peucetius, and Bacteroides acidifaciens. The three enriched bacterial species were Ralstonia solanacearum, Chroococcidiopsis thermalis, and Cutibacterium acnes (Figure 4B). The less abundant Haemophilus ducreyi is a gram-negative bacterium and causative agent of genital ulcer disease chancroid [46] and was detected in the three conditional VDR knockout mice. Similar to viruses, more bacterial species were changed in VDRΔPC and VDRΔLyz mice compared to the control mice (Figure 4B).
Correlation of viral and bacterial alterations in conditional VDR KO mice
Bacteria and viruses are essential for protective, metabolic, and physiological functions. We found that the viral (consisting mostly of bacteriophages) and bacterial species abundances were altered in conditional VDR KO mice. To investigate the interactions between bacteria and viruses, we performed a correlation analysis of viruses and bacteria. All the bacterial and viral species with significant fold changes in the differential analysis (q<0.05) were included in the correlation analysis (Figure 4C and Supplement Table 1).
In VDRΔIEC mice, bovine viral diarrhea virus 1 was significantly negatively correlated with the bacteria Mesorhizobium huakuii (correlation coefficient (r)=-0.63; P=0.05) (Figure 4C). M. huakuii induces the formation of nitrogen-fixation nodules on its host plant Astragalus sinicus and has been assigned to a new biovariety based on its host range and taxonomic characteristics [47]. M. huakuii isolates were also found to have endotoxic activity against lipopolysaccharides [48]. In VDRΔPC mice, the Vibrio phage JSF5 was dramatically positively correlated with the bacteria Cutibacterium acnes (r=0.98; P<0.0001) (Figure 4C). Cutibacterium acnes, formerly known as Propionibacterium acnes, is an anaerobic, aerotolerant, bacillus-shaped bacterium. It is ubiquitously found as a commensal on the surface of skin in areas rich in oleic and palmitic acids [49]. Moreover, variable results for the association between C. acnes and ulcerative colitis were found [50]. In VDRΔLyz mice, 11 pairs of virus-bacteria were found to be significantly correlated, including Vibrio phage JSF5 and Bordetella pseudohinzii (r=0.65; P=0.04), Vibrio phage JSF5 and Cutibacterium acnes (r=0.68; P=0.03), bovine viral diarrhea virus 1 and Bifidobacterium animalis (r=-0.64; P=0.05), bovine viral diarrhea virus 1 and Haemophilus ducreyi (r=0.65; P=0.04), bovine viral diarrhea virus 1 and Bacteroides acidifaciens (r=-0.66; P=0.04), bovine viral diarrhea virus 1 and Bifidobacterium sp. (r= -0.66; P=0.04), Lactobacillus phage phiadh and Clostridium perfringens (r= -0.71; P=0.02), Vibrio phage JSF6 and Bifidobacterium animalis (r=-0.67; P=0.03), Vibrio phage JSF6 and Ralstonia solanacearum (r=0.65; P=0.04), Vibrio phage JSF6 and Haemophilus ducreyi (r=0.75; P=0.01), and Podoviridae (family level) and Chroococcidiopsis thermalis (r=-0.75; P=0.01) (Figure 4C). In addition, we found a positive correlation between Vibrio phage JSF5 and Cutibacterium acnes in VDRΔPC mice and VDRΔLyz mice. Taken together, these data indicate the critical role of viral and bacterial interactions in intestinal microbial homeostasis with the support of VDR.
VDR status altered the expression of PRRs in colonic epithelial cells
To examine the impact of virome dysbiosis, virus-related receptors in the colon were evaluated (Figure 5). TLR3 and TLR7 are transmembrane PRRs located in endosomes that recognize nucleic acids and mediate cell extrinsic virus recognition [51]. We observed that expression of TLR3 and TLR7 was upregulated in the conditional VDR knockout mice compared with the control mice, with a significant difference in the VDRΔLyz group vs. the control group (P<0.05) (Figure 5).
NLRs characterized by the presence of a conserved NOD motif, comprise a large receptor family, including NOD1, NOD2, and NLRPs [52, 53]. NLRs are activated not only in response to viruses but are also important modulators of other virus sensing pathways [51]. Here, we observed upregulated expression of NOD1 and NLRP6 and significantly increased NOD2 RNA in VDRΔLyz mice compared with control VDRLoxP mice (P<0.05) (Figure 5). C-type lectin receptors (CLRs) also mediate cell-extrinsic sensing of specific viruses by binding viral glycans [54]. Compared with the control VDR group, the expression of CLEC4L, one of the CLRs, was significantly upregulated in all three conditional VDR knockout mice, especially in VDRΔIEC and VDRΔPC mice (Figure 5). Overall, the significant alterations of PRRs in conditional VDR knockout mice suggest the influence of VDR on intestinal homeostasis and expression of PRRs.
VDR status altered metabolites related to bacteriophage infection in feces
Microbiota-derived metabolites are chemical messengers that elicit a profound impact on host physiology. Thus, we investigated bacterial metabolites that are related to bacteriophage infection (Figure 6). We found that glycolysis glucose was significantly decreased in VDRΔIEC mice compared with the control (P<0.05), while ribulose/xylulose and xylose were significantly increased in VDRΔLyz mice compared with the control (P<0.05) (Figure 6A). Moreover, most of the long-chain fatty acids were significantly increased in both VDRΔIEC and VDRΔLyz female mice compared to control mice, while some fatty acids were only increased in VDRΔIEC mice. 10-Hydroxystearate, which is related to phage infection, was decreased in VDRΔLyz mice and increased in female VDRΔIEC mice compared to control mice (P<0.05) (Figure 6B). Metabolic alterations of nucleotides were also detected in both conditional knockout mice, such as uridine in VDRΔIEC mice and 3-ureidoisobtyrate in VDRΔLyz mice (P<0.05) (Figure 6C). Similarly, we observed amino acid alterations in the feces of conditional VDR knockout mice when compared to those of the control mice. For instance, we observed decreased phage infection-related serine in both VDRΔIEC and VDRΔLyz female mice and decreased cellular virus infection-related glutamine in VDRΔIEC and VDRΔLyz female mice compared to the control (Figure 6D).