Complete Genome Sequencing and Comparative Genomic Analysis of Helicobacter Apodemus Isolated From the Wild Korean Striped Field Mouse (Apodemus agrarius) for Potential Pathogenicity

Abstract

The Helicobacter bacterial genus comprises of spiral-shaped gram-negative bacteria with flagella that colonize the gastro-intestinal (GI) tract of humans and various mammals (Solnick and Schauer, 2001). In particular, Helicobacter pylori was classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) in 1994, and has been shown to occur with a high prevalence in humans, although this varies between geographical regions, ethnic groups, and various populations (Kusters et al., 2006; Goh et al., 2011). To date, more than 37 Helicobacter species have been identified in addition to H. pylori (Péré-Védrenne et al., 2017). Furthermore, non-H. pylori Helicobacters (NHPH) have been shown to infect both humans and animals, and NHPH infections are associated with intestinal carcinoma, and mucinous adenocarcinoma (Swennes et al., 2016). Despite the demonstrated association between NHPH and disease, most studies to date have investigated H. pylori in humans; thus, it is necessary to characterize NHPH and elucidate its role in the GI tract of wild rodents which are potential Helicobacter carriers (Taylor et al., 2007; Mladenova-Hristova et al., 2017). Helicobacter apodemus, a spiral curved rod bacterium with a single flagella, was first identified in the GI tract of the Korean striped field mouse (Apodemus agrarius) in Korea, and shown to exhibit urease, oxidase, and catalase activity (Jeon et al., 2015). Since then, rodents colonized with H. apodemus have been found all over the world, including within the Xinjiang-Uygur Autonomous Region of China, Cambridge in the United States, and animal facilities in Sweden (Goto et al., 2004; Johansson et al., 2006; Miller et al., 2014). A previous study suggested that H. apodemus has the potential to cause rectal prolapse and colorectal cancer in rodents (Miller et al., 2014; Zhang et al., 2017), while another suggested that it may act as a rodent pathobiont, normally activating regulatory T-cells to maintain immune tolerance, but activating effector T-cells to contribute to inflammation and disease pathogenesis (Chai et al., 2017). Rodent H. apodemus colonization has been shown to be significantly decreased after treatment with azithromycin (compared to other antibiotics such as amoxicillin, or cefaclor), and similarly, after administration of Lactobacillus casei Zhang, and vitamin K2 (Khan et al., 2016; Zhang et al., 2017). Nevertheless, continued research is essential to elucidate the molecular mechanisms by which H. apodemus alternately causes GI tract inflammation and GI tolerance in rodents, depending upon host health. The current study was therefore conducted to identify the genomic characteristics and specificity of H. apodemus, and to reveal its potential role in the rodent GI tract. Specifically, the genome of H. apodemus str. SCJK1 isolated from Apodemus agrarius was completely sequenced, and subjected to a comparative genomic analysis with 17 genome sequences of other Helicobacter species. It is hoped that the data in this study will serve as the basis for further studies of H. apodemus-related bacterium, and furthermore, enable future in-depth biomedical research regarding the immunological and pathological role of H. apodemus in the rodent GI tract. In May 2015, fresh fecal samples from wild A. agrarius were collected, and transported to the laboratory at 4°C. The fecal samples were homogenized in PBS, spread onto modified Charcoal-Cefoperazone-Deoxycholate agar (mCCDA) with a selective supplement (Oxoid), and micro-aerobically incubated at 42°C for 6 days. After incubation, suspected colonies were transferred to blood agar, and micro-aerobically incubated at 42°C for 2 days. Genomic DNA was extracted from each colony confirmed to be H. apodemus (via Polymerase Chain Reaction (PCR) (Miller et al., 2014), and 16S rRNA sequencing analyses) using MG^TM Genomic DNA Purification kit (Macrogen, Korea). The quality of the extracted genomic DNA was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The whole-genome sequencing of H. apodemus str. SCJK1 was carried out using a PacBio RS α sequencer (Pacific Biosciences, Menlo Park, CA, USA). A 20 kb DNA library was prepared using a SMRTbell^TM template Prep Kit, and sequenced using a P6-C4 chemistry reagent kit (Pacific Biosciences, Menlo Park, CA, USA). The obtained sub-reads were assembled de novo using Hierarchical Genome Assembly Process v. 3.0 and SMRT Analysis v. 2.3 (default options) software (Pacific Biosciences, Menlo Park, CA, USA) (Chin et al., 2013). The reads were polished using Quiver v. 1.0 software (Pacific Biosciences, Menlo Park, CA, USA) to ensure a higher level of accuracy and lower error rate (Chin et al., 2013). Genes were annotated according to the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP, https://www.ncbi.nlm.nih.gov/genome/annotation_prok/), and “Clusters of Orthologous Group (COG)” categories were assigned using the NCBI COGs database (2014 version, https://www.ncbi.nlm.nih.gov/COG/). A summary of the generated sequencing data is included in Supplementary Table 1. Sequences for Helicobacter-related virulence genes presented in the Virulence Factor Database (VFDB, www.mgc.ac.cn/VFs/) were used to predict the H. apodemus str. SCJK1 virulence factor. A total of 17 genome sequences of other Helicobacter spp. were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/genome/), and used to conduct a comparative genomic analysis, including JRPC_s (GCA_ 000765745.1), H. himalayensis (GCA_ 001602095.1), H. mustelae (GCA_ 000091985.1), H. cinaedi (GCA_ 000349975.1), H. bilis...