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

Citation: Kim J, Kim W, An J-U, Suh JG, Seong JK, Jeon B-Y and Cho S (2018) Complete Genome Sequencing and Comparative Genomic Analysis of Helicobacter Apodemus Isolated From the Wild Korean Striped Field Mouse (Apodemus agrarius) for Potential Pathogenicity. Front. Pharmacol. 9:838. doi: 10.3389/fphar.2018.00838 Complete Genome Sequencing and Comparative Genomic Analysis of Helicobacter Apodemus Isolated From the Wild Korean Striped Field Mouse (Apodemus agrarius) for Potential Pathogenicity


INTRODUCTION
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 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.

H. apodemus Isolation and DNA Extraction
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).

Whole-Genome Sequencing, Genome Assembly, and Gene Annotation
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 2). The orthologous Average Nucleotide Identity (OrthoANI) algorithm was used to measure the genetic relatedness between H. apodemus str. SCJK1 and the other Helicobacter spp., and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram was constructed based on the OrthoANI value (Lee et al., 2016). Pan-genome Orthologous Groups (POGs) were determined using the BIOiPLUG Comparative Genomics Database (https://www. bioiplug.com/), and a heat map and UPGMA dendrogram were constructed based on these data (i.e., the presence/absence of a POG).

Quality Assurance
To ensure a pure culture, a single H. apodemus colony was transferred three times. Furthermore, contamination was excluded by comparing three 16S rRNA gene fragments found in the H. apodemus str. SCJK1 genome using EzBioCloud DB software (https://www.bioiplug.com/).

Ethics Approval
Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Yonsei University Wonju Campus (YWC-151203-1).

Virulence Genes
In Helicobacter spp., a total of 91 virulence genes were identified, and classified into seven categories, and 17 subcategories. Specifically, these comprised of acid resistance (urease), adherence (adherence-associated lipoprotein (alp) A, alpB, blood group antigen binding adhesins, H. pylori adhesin (hpa) A, hopZ, horB, peb1, and sialic acid binding adhesins), immune evasion (lipopolysaccharide Lewis antigens), immune-modulator (neutrophil-activating protein and outer inflammatory protein), motility (flagella), secretion system (cag pathogenicity island (cag-PAI)-type IV secretion system), and toxin (cytolethal distending toxin, vacuolating cytotoxin) genes (Supplementary Table 3). H. apodemus str. SCJK1 was found to harbor 47 of these 91 virulence genes, including all of the urease-related "acid resistance" genes, 30 of the 38 flagella-related "motility" genes, and three of the "toxin" genes. Of the 10 possible "adherence" genes, H. apodemus str. SCJK1 carried only peb1 (PEB1-related gene). In addition, the strain harbored only one (futA) of the three "immune evasion" genes, and one (napA) of the two "immune modulator" genes. The H. apodemus str. SCJK1 plasmid was shown to include the CagX, V, E, and C (virB 9, 8, 4, and 2, respectively) "secretion system" genes, known to be associated with the cag-PAI type IV secretion system. Three Helicobacter strains, H. acinonychis str. Sheeba, H. hepaticus ATCC 51449, and H. pylori HPAG1, were used to infer virulence factors in the H. apodemus str. SCJK1 genome (Supplementary Table 3). Of the identified virulence genes, peb1 ("adherence" category), and cdtA and B ("toxin" category, and "cytolethal distending toxin" subcategory) were shown to be  (Tomb et al., 1997). Furthermore, peb1 has been previously reported to be expressed on the surface of all Campylobacter jejuni and C. coli bacteria, and to thereby mediate intestinal colonization, indicating that it is a prominent target for the immune system (Pei and Blaser, 1993). Consistent with these observations, peb 1 of H. hepaticus was expected to be involved in colonization of the intestine, and according to the results of this study, it may also be involved in intestinal colonization of H. apodemus (Suerbaum et al., 2003). In addition, the cytolethal distending toxin gene, consisting of cdtA, cdtB, and cdtC, has been shown to be expressed by GI pathogens including Campylobacter spp., Escherichia coli, and Helicobacter spp., and associated with irreversible G2/M cell-cycle arrest, which results in the gradual expansion of the nucleoli, and corresponding loss of the cytoplasm (Young et al., 2000;Taylor et al., 2003). Accordingly, H. hepaticus, which is known to carry the cdt gene, has been shown to be associated with chronic GI tract inflammation, and the onset of irritable bowel disease (IBD) in rodents (Whary and Fox, 2004;Young et al., 2004;Ge et al., 2007). The present study showed that H. apodemus also harbors the cdtA and cdtB genes; thus, further study should be conducted to investigate whether H. apodemus exerts similar impact on the rodent GI tract. In addition, both H. pylori and H. apodemus carry genes known to be associated with the cag-PAI type IV secretion system, responsible for horizontal gene transfer between bacterial cells (Rohde et al., 2003). H. pylori has been previously reported to mediate the pathogenesis of gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma, by injecting cagA (which is a bacterial gene that promotes cell proliferation and differentiation) into gastric epithelial cells using the cag-PAI type IV secretion system (Odenbreit et al., 2000;Cascales and Christie, 2003). However, the H. apodemus genome did not include cag A; thus, the role of the cag-PAI type IV secretion system in the H. apodemus genome should be studied further.  Figure 1B). The calculated relatedness between these species was consistent with the findings of previous studies, in which primers used in an H. rodentium-specific PCR assay cross-reacted with both H. apodemus and H. rodentium (Shen et al., 1997;Miller et al., 2014). However, the results of the present study were not consistent with the data presented in phylogenetic tree (based on 16S rRNA sequence comparisons) in which H. apodemus was clustered with H. mesocricetorum, H. ganmani, and H. rodentium, but not with H. pullorum or H. canadensis (Jeon et al., 2015). In the heat map and UPGMA dendrogram (based on POG analysis), H. apodemus str. SCJK1 was clustered with JRPC_s, H. pullorum, H. canadensis, H. rodentium, and JRPB_s, consistent with orthoANI value-based results ( Figure 1C). The consistency in results from orthoANI values-based and POG-based analysis indicated that H. apodemus was genetically similar to the above Helicobacter species (H. pullorum, H. canadensis, H. rodentium, and JRPB_s). In addition, the six strains shared 1,197 POGs, of which 143 were identified only in the strains analyzed in the present study ( Figure 1D). H. apodemus strains (i.e., those isolated in the current study, and JRPC_s) shared 1,647 POGs, which were expected to have specific characteristics of H. apodemus.

FUTURE DIRECTIONS
Studies using whole-genome sequencing technology have made important advances in rodent GI microbial research; however, the present study is the first to analyze the complete genome sequence of H. apodemus from wild A. agrarius, which acts as a pathobiont in wild rodents. It is hoped that the results of this analysis, together with those of the conducted comparative genomic analysis, will serve as the basis for further biomedical studies investigating the immunological and inflammatory effects of H. apodemus on the rodent GI tract.

DATA ACCESS
The genome sequence of Helicobacter apodemus str. SCJK1 was deposited in the GeneBank under accession number CP021886-CP021887.

AUTHOR CONTRIBUTIONS
SC conceived and designed the study. J-UA and WK analyzed the genome sequencing data. JGS, JKS, and B-YJ performed sampling, and prepared the manuscript. JK was a major contributor, both in experiments and writing the manuscript. All authors have read and approved the final manuscript.