Gut microbiota ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2019-319635 on 12 February 2020. Downloaded from Southern Chinese populations harbour non- nucleatum Fusobacteria possessing homologues of the colorectal cancer- associated FadA virulence factor Yun Kit Yeoh ,1,2 Zigui Chen ,1,2 Martin C S Wong ,1,3 Mamie Hui,1,2 Jun Yu ,1,4 Siew C Ng ,1,4 Joseph J Y Sung,4 Francis K L Chan ,1,4 Paul K S Chan1,2 ► Additional material is ABSTRact published online only. To view Objective Fusobacteria are not common nor relatively Significance of this study please visit the journal online abundant in non-colorectal cancer (CRC) populations, (http:// dx. doi. org/ 10. 1136/ What is already known about this subject? gutjnl- 2019- 319635). however, we identified multiple Fusobacterium taxa nearly absent in western and rural populations to be ► Fusobacterium nucleatum are specifically 1Centre for Gut Microbiota comparatively more prevalent and relatively abundant in enriched in gut microbiomes of individuals with Research, The Chinese University colorectal cancer (CRC). of Hong Kong, Shatin, Hong southern Chinese populations. We investigated whether Kong these represented known or novel lineages in the ► The FadA adhesin and Fap2 lectin are 2Department of Microbiology, Fusobacterium genus, and assessed their genomes for implicated in the association between F. The Chinese University of Hong features implicated in development of cancer. nucleatum and CRC. Kong, Shatin, Hong Kong 3Jockey Club School of Public Methods Prevalence and relative abundances of What are the new findings? fusobacterial species were calculated from 3157 CRC Health and Primary Care, The ► Non- CRC southern Chinese populations carry Chinese University of Hong and non-­CRC gut metagenomes representing 16 multiple known and novel fusobacterial taxa Kong, Shatin, Hong Kong populations from various biogeographies. Microbial 4Department of Medicine and phylogenetically distinct from F. nucleatum in Therapeutics, The Chinese genomes were assembled and compared with existing their guts; these taxa are nearly absent in other University of Hong Kong, Shatin, reference genomes to assess novel fusobacterial surveyed populations. Hong Kong diversity. Phylogenetic distribution of virulence genes ► Several fusobacterial taxa other than F. implicated in CRC was investigated. nucleatum are enriched in CRC cohorts relative Correspondence to Results Irrespective of CRC disease status, southern to non- CRC controls. http://gut.bmj.com/ Professor Paul K S Chan, Chinese populations harboured increased prevalence Department of Microbiology, ► Homologues of the FadA adhesin were detected The Chinese University of Hong (maximum 39% vs 7%) and relative abundances in several species of Fusobacterium including Kong, Shatin, Hong Kong; (average 0.4% vs 0.04% of gut community) of multiple F. varium and F. ulcerans, suggesting potential paulkschan@ cuhk. edu. hk recognised and novel fusobacterial taxa phylogenetically associations with CRC and/or disease. distinct from Fusobacterium nucleatum. Genomes Received 12 August 2019 assembled from southern Chinese gut metagenomes How might it impact on clinical practice in the Revised 29 January 2020 Accepted 1 February 2020 increased existing fusobacterial diversity by 14.3%. foreseeable future? on September 26, 2021 by guest. Protected copyright. Homologues of the FadA adhesin linked to CRC were ► These findings indicate that CRC in southern consistently detected in several monophyletic lineages Chinese populations may be linked to F. varium sister to and inclusive of F. varium and F. ulcerans, but and other fusobacterial species in addition to F. not F. mortiferum. We also detected increased prevalence nucleatum. and relative abundances of F. varium in CRC compared ► Use of microorganisms as disease biomarkers with non-­CRC cohorts, which together with distribution or targets for therapeutic intervention needs of FadA homologues supports a possible association with to be tailored according to discrepancies in gut disease. gut microbiome composition among human Conclusion The proportion of fusobacteria in guts of populations. southern Chinese populations are higher compared with several western and rural populations in line with the © Author(s) (or their notion of environment/biogeography driving human gut cancer (CRC) in humans. Irrespective of biogeog- employer(s)) 2020. Re- use microbiome composition. Several non-nucleatum taxa raphy, multiple studies have consistently reported permitted under CC BY-­NC. No possess FadA homologues and were enriched in CRC enrichment of F. nucleatum in the guts1–7 and commercial re- use. See rights cohorts; whether this imposes a risk in developing CRC 8–10 and permissions. Published tumour tissue of CRC subjects compared with by BMJ. and other gut diseases deserves further investigation. non- CRC cohorts. Furthermore, the association between F. nucleatum and CRC has been demon- To cite: Yeoh YK, Chen Z, strated through cell model studies, implicating Wong MCS, et al. Gut Epub 11 12 13 14 ahead of print: [please two proteins FadA and Fap2 in facilitating include Day Month Year]. INTRODUCTION adherence, invasion and induction of oncogenic doi:10.1136/ Fusobacterium nucleatum is a bacterial pathogen and inflammatory responses in CRC cells by F. gutjnl-2019-319635 most well- known for its association with colorectal nucleatum. Yeoh YK, et al. Gut 2020;0:1–10. doi:10.1136/gutjnl-2019-319635 1 Gut microbiota In contrast, relatively less is known about the biology of (Illumina NovaSeq 6000). A mock community sequencing fusobacterial species other than F. nucleatum and their roles control (ZymoBIOMICS Microbial Community DNA Standard, Gut: first published as 10.1136/gutjnl-2019-319635 on 12 February 2020. Downloaded from in human health, if any. According to the List of Prokaryotic catalogue number D6305, Zymo Research, Irvine, California) names with Standing in Nomenclature (LPSN), there are 21 was included. recognised species in the Fusobacterium genus at the time of writing. Apart from F. nucleatum, a few other species such as 15 16 17 Prevalence and relative abundances of fusobacterial species F. necrophorum, F. gonidiaformans, F. periodonticum, F. 18 based on gut metagenomes mortiferum, F. ulcerans and F. varium have been reported in To examine prevalence and relative abundances of fusobac- human-associated samples. For example, F. necrophorum are terial species in guts of human populations from different often associated with thrombophlebitis of the internal jugular geographical backgrounds, we included non- CRC gut metag- vein (termed Lemierre’s syndrome), F. gonidiaformans found in 16 enome sequence data generated by previous studies in Hong urogenital and intestinal tracts, F. periodonticum in oral cavi- 2 29 30 31 3 27 4 32 19 Kong, China, USA, Austria, Denmark, France, ties associated with squamous cell carcinoma, F. ulcerans in 5 32 33 34 35 36 20 Germany, Spain, Israel, Sweden, El Salvador, Peru, skin ulcers and F. varium in human guts associated with ulcer- 37 38 37 40 21 22 Fiji, Mongolia and Tanzania (online supplementary ative colitis (UC). Apart from cases of disease, their preva- table S1). The Hong Kong, Austria, France, Germany and one lence and relative abundances in guts of healthy individuals are 3 23–28 USA cohort were comprised of CRC and non- CRC subjects. relatively low, often below detection thresholds consistent These gut metagenome data sets were chosen because they have with the notion that the presence of Fusobacterium in human 42–44 26 been binned for microbial genomes. Together with data guts is specifically associated with CRC. generated from the HKGutMicMap cohort (present study), We initially produced shotgun metagenomes using stools raw sequences were quality- filtered using Trimmomatic V.0.38 collected from 556 self- reported healthy individuals recruited for to remove adapter and low quality regions. Next, microbial establishing a gut microbiota databank in Hong Kong (HKGut- community compositional profiles were inferred from quality- MicMap project). These data were analysed together with filtered sequences (forward reads) using MetaPhlAn245 V.2.6 publicly- available stool metagenomes of other healthy subjects with the v20 database. For each fusobacterial species identified from Hong Kong,2 29 Austria,4 China,30 31 Denmark,32 France, 5 33 32 34 35 3 27 by MetaPhlAn2, their prevalence rates were calculated based on Germany, Israel, Spain, Sweden and the USA, as well the number of samples each species was detected in (ie, relative as several rural populations from El Salvador, Peru,36 Fiji,37 38 39 40 abundance >0%) divided by the total number of samples in the Mongolia and Tanzania to assess variation in gut micro- respective cohorts. bial community composition across different biogeographies. We serendipitously observed a consistent increase in prevalence and relative abundance of multiple fusobacterial species in the Binning fusobacterial population genomes from Hong Kong, Chinese and Spanish but not American, European metagenomes and other rural cohorts, concordant with the idea of variation in To explore genomic diversity of fusobacterial species in the human gut microbiomes primarily driven by environment/geog- Hong Kong population, we assembled metagenomes from the 41 Hong Kong cohorts and binned population genomes (termed raphy. Here, we reconstructed fusobacterial genomes from http://gut.bmj.com/ the Hong Kong gut metagenomes and showed that F. varium, F. metagenome-