Host Specificity of Flagellins from Segmented Filamentous Bacteria Affects Their Patterns of Interaction with Mouse Ileal Mucosal Proteins

Abstract

Segmented filamentous bacteria (SFB) are known modulators of the mammalian immune system. Currently, the technology for investigating SFB culture in vitro is immature, and as a result, the mechanisms of SFB colonization and immune regulation are not yet fully elucidated. In this study, we investigated the gene diversity and host specificity of SFB flagellin genes. The fliC1 and fliC2 genes are relatively conserved, while the fliC3 and fliC4 genes are more variable, especially at the central and C-terminal regions. Host specificity analysis demonstrated that the fliC1 genes do not cluster together based on the host organism, whereas the fliC3 and fliC4 genes were host specific at the nucleotide and deduced amino acid levels. SFB flagellin protein expression in the ileum mucosa and cecal contents was detected by using fluorescence in situ hybridization (FISH) combined with immunohistochemical (IHC) analysis, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the purified SFB FliC3 protein originating from both mouse and rat was able to activate Toll-like receptor 5 (TLR5)-linked NF-B signaling, no host specificity was observed. Interestingly, the patterns of interaction with mouse ileum mucosal proteins were different for mouse FliC3 (mFliC3) and rat FliC3 (rFliC3). Gene Ontology (GO) and KEGG analyses indicated that more adherence-related proteins interacted with mFliC3, while more lysosome-and proteolysis-related proteins interacted with rFliC3. In vitro degradation experiments indicated that the stability of rFliC3 was lower than that of mFliC3 when they were incubated with mouse ileum mucosal proteins. In summary, the gene diversity and host specificity of SFB flagellin genes were investigated, and SFB flagellin expression was detected in gut samples.IMPORTANCE Since SFB genomes contain only one copy of each FliC gene, the diversity of FliC is representative of SFB strain diversity. Currently, little is known regarding the diversity and specificity of members of the group of SFB. The work presented herein demonstrates that select SFB strains, exhibiting unique FliC patterns, are present in a variety of mammalian hosts. SFB fliC genes were found to interact with a number of unique targets, providing further evidence for SFB host selection. Together, this work represents a major advancement in identifying SFB and delineating how members of the group of SFB interact with the host. Future examination of FliC genes will likely enhance our knowledge of intestinal colonization by the gut microbiota.KEYWORDS segmented filamentous bacteria, flagellin, host specificity, gene diversity, ileum mucosal proteins

Microbiology and immunology: An ideal partnership for a tango at the gut surface—A tribute toPhilippe Sansonetti

“…The presence of SFB in the colonic lumen was confirmed using in situ hybridization and mass spectrometry detection of SFB‐derived peptides (Chen et al, ). Interestingly, SIgA concentrations in the colonic fluid and ileal expression of immune transcripts and, notably IL‐17 mRNA, were significantly increased in children with colonic fluid positive for SFB‐16S DNA (Chen et al, ). These exciting results encourage cooperation between microbiologists and immunologists to further characterize the putative human SFB, to define whether and how it may contribute to a healthy and robust gut immune barrier in humans, and if so, to learn how to use it to boost gut defences against pathogens.…”

“…A first mechanism may involve the production of flagellin(s). Indeed, SFB harbour a full set of flagella genes in their genome (Kuwahara et al, ; Pamp et al, ; Prakash et al, ; Sczesnak et al, ), and recombinant SFB flagellins are TLR5 stimulatory (Chen, Yin, Wang, Wang, & Xiang, ). Moreover, a recent work led by Pamela Schnupf has demonstrated SFB flagellation at the single‐cell stage during both in vivo and in vitro SFB growth conditions (unpublished).…”

“…Although SFB have been identified in the microbiota of many vertebrates, their presence in the human microbiota has remained debated due to the difficulty to identify relevant 16S DNA in human faeces (Sczesnak et al, ). SFB‐related 16S DNA sequences have, however, been detected in the human faeces of Chinese children and, more recently, of U.S. children (Chen et al, ; Yin et al, ). The presence of SFB in the colonic lumen was confirmed using in situ hybridization and mass spectrometry detection of SFB‐derived peptides (Chen et al, ).…”

“…SFB‐related 16S DNA sequences have, however, been detected in the human faeces of Chinese children and, more recently, of U.S. children (Chen et al, ; Yin et al, ). The presence of SFB in the colonic lumen was confirmed using in situ hybridization and mass spectrometry detection of SFB‐derived peptides (Chen et al, ). Interestingly, SIgA concentrations in the colonic fluid and ileal expression of immune transcripts and, notably IL‐17 mRNA, were significantly increased in children with colonic fluid positive for SFB‐16S DNA (Chen et al, ).…”

“…Gradient colouring (ranging from purple for dim phalloidin staining to yellow for intense staining) shows the characteristic accumulation of polymerised actin at the site of attachment of SFB to the epithelial cell. Photographs provided by the courtesy of I. Nkamba and P. Schnupf genome (Kuwahara et al, 2011;Pamp et al, 2012;Prakash et al, 2011;Sczesnak et al, 2011), and recombinant SFB flagellins are TLR5 stimulatory (Chen, Yin, Wang, Wang, & Xiang, 2017 (Ivanov et al, 2009), or monocolonisation by SFB (Gaboriau-Routhiau, unpublished observations), indicating that TLR5 is dispensable for this response.…”