Helicobacter pylori cytotoxin-associated gene A protein among adult dyspeptic patients in South-western Nigeria

1 Department of Microbiology, University of Lagos, Lagos State, Nigeria. 2 Molecular Biology and Biotechnology Division, Nigerian Institute of Medical Research, Lagos, Lagos State, Nigeria. 3 College of Medicine, University of Lagos, Lagos State, Nigeria. 4 College of Medicine, Lagos State University, Ikeja, Lagos State, Nigeria. 5 Department of Medicine, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. 6 University College Hospital, Ibadan, Oyo State, Nigeria.


INTRODUCTION
Helicobacter pylori is a bacterial pathogen that colonizes exclusively the mucous layer of the human stomach and classified as carcinogenic to humans by the International Agency for Research on Cancers (IARC, 1994).However, H. pylori organisms vary in their carcinogenic potential and the common type of gastric cancers is  (Higashi et al., 2002;Suzuki and Mori, 2016).Since the discovery of H. pylori by Marshall and Warren (1984), it has been confirmed that H. pylori infection results in chronic active gastritis and can lead to gastric ulcer, duodenal ulcer and gastric cancer (Malfertheiner et al., 2007;Yamaoka, 2010).Infection with H. pylori is more common and acquired at an earlier age (30 to 50%) in children and attain over 90% during adulthood in developing countries (Salih, 2009).In South-west, Nigeria, studies reported high prevalence rates (60.5 to 73%) of H. pylori among dyspeptic patients (Ndububa et al., 2001;Adesanya et al., 2002;Ola et al., 2006).Cytotoxin associated gene A (cagA) is one of the important virulence factors of H. pylori.The CagPAI contains 31 genes, including CagA gene, which is one of the markers for the cag pathogenecity island (CagPAI).CagA codes for 120 to 145 kDa protein that is associated with cytotoxin production and more severe clinical outcomes such as peptic ulcer and gastric cancer (Arents et al., 2001;Hatakeyama, 2006).Previous studies have reported the prevalence of H. pylori and the virulence genes in relation to disease outcome among dyspeptic patients in Nigeria (Smith et al., 2002;Olokoba et al., 2013).However, data relating to the information on the association of the cagA gene with the disease outcome is still controversial and there is dearth of information on protein expression of putative cagA genes in relationship to clinical outcomes.This information is important as CagA protein is one of the major virulence factors of H. pylori.Hence, this study was aimed at confirming whether any correlation exists between the CagA protein of these strains and the endoscopic findings among dyspeptic patients from South-western, Nigeria and also to investigate the evolutionary relationships among H. pylori cagA gene with the GenBank strains.

MATERIALS AND METHODS
A total number of one hundred and twenty-four isolates of H. pylori from dyspetic patients were obtained from the laboratory of Dr. S. I. Smith of the Nigeria Institute of Medical Research, Yaba.Medical history and the endoscopic findings of the patients were obtained from four different hospitals based on the records provided by the gastroenterologist between April 2012 -December 2015, by which approval to conduct the study was obtained from Ethics Committee of the Nigerian Institute of Medical Research Yaba, Lagos.

Culturing of H. pylori
The isolates were sub-cultured on Columbia blood agar base (Oxoid), containing laked horse serum (7%) and Dent's antibiotic supplement (Oxoid) in a microaerophilic atmosphere conditions at 37°C as described previously (Toledo and Lopez-Solis, 2010).Colonies were identified by Gram staining and biochemical tests such as oxidase, catalase and urease reactions.

DNA extraction and PCR amplification
Genomic DNA of the isolates were extracted with the QIAamp® DNA kit (Qiagen, Germany) according to the manufacturer's instructions.The extracted DNA was stored at -20°C until use.The PCR amplification of H. pylori 16s rRNA was carried out in 25 µl reaction mixture and PCR conditions were: 35 cycles at 94°C for 1 min, annealing 60°C for 1 min, 72°C for 1 min, and a final elongation at 72°C for 5 min.The DNAs of J99 and P12 were used as positive controls.H. pylori 16S rRNA gene-positive samples were subjected to PCR to detect cagA gene using the primers and PCR conditions as described previously by Smith et al. (2002) and the PCR primers are listed in Table 1.

Protein expression of cagA gene
Antigens were prepared from lysates of strains of H. pylori positive for cagA genes.Single gel system for protein separation was prepared according to a protocol of Ahn et al. (2001) and the procedures for Western blotting were performed following the methods of Figueroa et al. (2002) with some modifications.Briefly, the proteins from the lysates were electrophoretically separated in polyacrylamide gel electrophoresis (SDS-PAGE) according to molecular weight (MW) of the proteins.Proteins were then blotted to nitrocellulose membrane (PVDF).For the development of immunoblots, polyvinylidene difluoride (PVDF) filters were blocked in TBS containing 3% (w/v) bovine serum albumin.CagA protein expression was analyzed using a 1:2,000 dilution of CagA specific polyclonal antibodies.Anti-rabbit antibody was used as a secondary *Corresponding author: E-mail: stellaismith@yahoo.com.Tel: +2348037058989.

Sequencing and bioinformatic analysis
The amplified PCR products of cagA gene from twelve selected strains based on each centre used were purified and subjected to sequencing reactions by inqaba™ biotech, South Africa (BigDye® Terminator v3.1 Cycle Sequencing).Blasting and Genebee (http://www.genebee.msu.su/services/phtree_reduced.html) were used for phylogenetic prediction.

Statistical analysis
The data obtained were subjected to descriptive statistical analysis using EPI info 7. Chi squared test was used to determine associations between the protein status of the isolates and the endoscopic findings and the differences were considered to be statistically significant when the p value obtained was less than 0.05.

RESULTS
One hundred and twenty four H. pylori isolates were amplified for the detection of H. pylori 16S rRNA and cagA gene by polymerase chain reaction (PCR) and all were 100% positive for both H. pylori and cagA gene.
Immunoreactive bands for CagA protein expression is shown in Figure 1.

DISCUSSION
From these findings, CagA gene was found in all the isolates (100%) studied and the result is in agreement with previous studies in Africa and East Asia countries where cagA genes were present in more than 90% of cases (Tanih et al., 2010;Yamaoka et al., 1999).However, this result is higher than those observed from western populations where cagA were present in about 60 to 70% of H. pylori causing infections (Podzorski et al., 2003;Ribeiro et al., 2003).This indicates that cagA prevalence varies in every part of the world (Ozbey and Aygun, 2012).Several studies have demonstrated that the genotype varies among H. pylori strains isolated from different geographic regions (Kersulyte et al., 2000;Faundez et al., 2002), indicating that there are important geographic differences.In this study, CagA proteins was expressed in 88.7% (110/124) isolates and expression was observed with CagA specific antibodies by SDS-PAGE and Western blot (WB) analysis.This study showed that most H pylori strains isolated were positive for Cytotoxin associated gene (CagA) proteins.Also, Hatakeyama (2004) reported that many strains of H.  pylori produce cagA protein, which is a 120 to 145 kDa hydrophilic protein that induces several alterations in the signalling pathways.From these findings, there was no significant association between expressed cagA and the clinical outcomes and this is in agreement with earlier reports on cagA genotypes (Smith et al., 2002;Ozbey et al., 2013).Whereas in Western countries, the occurrence of vacAs1 and cagA genotypes have been reported to be significantly associated with peptic ulcers (Ribeiro et al., 2003).However, the reported studies were based on the association of cagA genes with the endoscopic findings, but this present study focuses on the association of CagA protein with the endoscopic findings.
From these findings, none of the patients studied were diagnosed with gastric cancer, more so, with the presence of cagA positive isolates.Abdulkareem et al. (2015) reported that, there is usually low incidence of gastric cancer in Africa (Africa enigma) despite a high prevalence of H. pylori with most strains positive for the virulent genes such as cagA gene and vacA gene.A lot of factors may contribute to this, and one of which could be mixed infection of cagA negative and cagA positive strains which could lower disease burden, and diets rich in anti-oxidants which reduces risk of gastric cancer (Abdulkareem et al., 2015).A study from Soweto, South Africa indicated that host response to H. pylori among Africans may be protective against virulent form of H. pylori (Segal et al., 2001).Also, the ability CagA secreted by different strains of H. pylori to perturb hostcell functions and to trigger gastric cancer is determined by the binding potential of Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) which is high in H. pylori strains from East Asian countries than those from Western countries (Higashi et al., 2002).The phylogenetic tree of cagA genes revealed that the strains were clearly delineated into 2 phylogenetic clades with strains LA2 and LA4 distantly separated from others.However, blasting of the edited sequences of strains LA2 and LA4 in NCBI platform, revealed that they were 89 and 87% respectively, most similar to the western type of cytotoxin associated protein A (cagA) gene, partial cds of H. pylori strain 01-1086 from Colombia (Accession no: JQ318034.1).The other strains clustered in the second clade but with significant genetic variability.This clade further subdivided into 3 minor clades with strain LU15 most distant and stands as a bridge linking the clade with strains LA 2 and LA4.Strains LA1, IB5 and IB6 clustered to form a minor clade with IB 5 and IB6 more related.Also, the results from blasting indicated that IB 5 and IB6 were 95 and 96%, respectively, most similar to H. pylori cag Pathogenicity Island, strain LSU2003-1 originated from African with Accession number of FR666843.1 (Olbermann et al., 2010).Strains from Ife (IF 9, IF 10 and IF 11) clustered forming a minor clade and were all related (96, 95 and 95%).While cluster from LU 14, LU 16 and LU 17 were related to Western cagA gene of H. pylori strain PHL121 (accession no: GU173869) obtained primarily from gastritis patients in philippines (Cortes et al., 2010).

Conclusion
This study demonstrated a high prevalence of H. pylori infection of the cagA genotype and the phylogenetic analysis revealed that cagA gene was related to the western cagA.However, there was no significant association between CagA protein and the clinical outcomes.
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Figure 2 .
Figure 2. Phylogenetic tree of cagA genes of twelve strains from southwest Nigeria and four strains from the GenBank.

Table 1 .
PCR primers used in this study.

Table 2 .
Association between expressed cagA and the endoscopic findings.

Table 3 .
Association between expressed cagA and positive endoscopic lesions.