Comparing the genomes of Helicobacter pylori clinical strain UM032 and Mice-adapted derivatives
- Yalda Khosravi1,
- Vellaya Rehvathy1,
- Wei Yee Wee2,
- Susana Wang3,
- Primo Baybayan3,
- Siddarth Singh4,
- Meredith Ashby3,
- Junxian Ong5,
- Arlaine Anne Amoyo5,
- Shih Wee Seow5,
- Siew Woh Choo2,
- Tim Perkins6,
- Eng Guan Chua7,
- Alfred Tay7,
- Barry James Marshall7,
- Mun Fai Loke1,
- Khean Lee Goh8,
- Sven Pettersson5, 9, 10 and
- Jamuna Vadivelu1Email author
© Khosravi et al.; licensee BioMed Central Ltd. 2013
Received: 2 July 2013
Accepted: 16 August 2013
Published: 19 August 2013
Helicobacter pylori is a Gram-negative bacterium that persistently infects the human stomach inducing chronic inflammation. The exact mechanisms of pathogenesis are still not completely understood. Although not a natural host for H. pylori, mouse infection models play an important role in establishing the immunology and pathogenicity of H. pylori. In this study, for the first time, the genome sequences of clinical H. pylori strain UM032 and mice-adapted derivatives, 298 and 299, were sequenced using the PacBio Single Molecule, Real-Time (SMRT) technology.
Here, we described the single contig which was achieved for UM032 (1,599,441 bp), 298 (1,604,216 bp) and 299 (1,601,149 bp). Preliminary analysis suggested that methylation of H. pylori genome through its restriction modification system may be determinative of its host specificity and adaptation.
Availability of these genomic sequences will aid in enhancing our current level of understanding the host specificity of H. pylori.
KeywordsHelicobacter pylori PacBio Single Molecule Real-Time (SMRT) technology Clinical H. pylori Mice-adapted
Helicobacter pylori persistently colonizes the human stomach to cause chronic gastritis, peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma . The mechanisms involved in the pathogenesis of H. pylori infections are still not fully established . Thus, experimental animal models that mimic human diseases are essential to provide information on etiopathogeny, immunity and therapy, as well as to improve our understanding on ways H. pylori can induce a diverse range of gastric pathologies [3, 4]. Among various animal models available, mouse remains the most readily used animal model for studying H. pylori-induced diseases and have played important roles in the elucidation of factors required for colonization, distribution and persistence of infection .
Mice adaptation study
Adopting a similar strategy as in previous studies [2, 5], a pool consisting of twelve clinical strains of H. pylori isolated from patients presenting for gastroscopy at the University of Malaya Medical Centre (UMMC) was inoculated intragastrically into five 4–6 weeks old male C57BL/6 mice. Multiple colonies of H. pylori were successfully recovered from the gastric tissue sample of a mouse (1/5) following necropsy two weeks post-infection. Random amplification of polymorphic DNA (RAPD) fingerprinting was used to trace back the mice-adapted isolates to its parental clinical strain, UM032. H. pylori UM032 was isolated from a patient presenting with peptic ulcer disease. Mice-adapted isolates from the first mouse passage were designated as 298 and were used for the second round of mouse passage to access the stability and infectivity of this mice-adapted strain. All three mice inoculated with the mice-adapted 298 strain were successfully infected and H. pylori isolated from the second passage were designed as 299. The animal study was performed with the approval of the SingHealth Institutional Research Committees (SHS IBC) and the Ethical Committee for Animal Research (Form No. SHS-IBC-201, January 2010).
In this study, H. pylori DNA was isolated using the RTP Bacteria DNA Mini Kit (Invitek GmbH, Berlin, Germany). The extracted DNA samples were sequenced using Pacific Biosciences RS sequencing technology (Pacific Biosciences, Menlo Park, CA), yielding >20× average genome coverage. Each sample was prepared as a 10-kb insert library using C2 chemistry and sequenced on 8 Single-Molecule Real-Time (SMRT) cells.
Assembly and annotation
De novo assembly of the read sequences was created using the continuous long reads (CLR) following the Hierarchical Genome Assembly Process (HGAP) workflow (http://pacbiodevnet.com/) as available in SMRT Analysis v2.0. The genomes were annotated with the NCBI (National Center for Biotechnology Information) Prokaryotic Genomes Automatic Annotation Pipeline and NMPDR (National Microbial Data Resource) Rapid Annotation using Subsystem Technology (RAST) . The SEED-Viewer was used to visualize the genome annotation and comparison generated by RAST .
Submission of genome sequence
The genome sequence of the Helicobacter pylori strains UM032, 298 and 299 are available in DDBJ/EMBL/GenBank under Accession numbers CP005490, CP006610 and CP005491 respectively.
The genomic DNA was isolated from pure bacterial isolate (positive for urease, catalase and oxidase tests) and was further confirmed with 16SrRNA sequencing and genotyping of bacterial virulence factors. Bioinformatic assessment of potential contamination of the genomic library by allochthonous microorganisms was done using PGAAP and RAST annotation systems.
Type of base modifications and associated motifs detected
# of motifs detected
The availability of complete sequences of mice-adapted strains and their parental clinical isolate will provide important information that contributes towards our understanding of the host specific and adaptation of H. pylori. In addition, it will help in extrapolate results obtained using mice model to the natural human host of H. pylori. H. pylori 298 strain will be used for H. pylori colonizing studies in mice.
Putative gene clusters responsible for survival and virulence of H. pylori
To our knowledge, this is the first genome sequence of H. pylori isolated from human and mouse using PacBio SMRT Technology. Comparative genomic and more-detailed methylomic analysis of these data is in process and will be included in future publications. Mice-adapted H. pylori described here will be used in future H. pylori infection studies in mice.
Availability of supporting data
The data sets supporting the results of this article are included within the additional files.
Funding was provided by the University of Malaya-Ministry of Higher Education (UM-MOHE) High Impact Research (HIR) grant (reference UM.C/625/1/HIR/MOHE/CHAN-02; “Molecular Genetics”). SW, PB, SS and MA are full-time employees at Pacific Biosciences, the commercial company for the SMRT sequencing technologies. We thank the NCBI (National Center for Biotechnology Information) rapid annotation pipeline team for providing the genome annotation services.
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