Subscribe Now Subscribe Today
Abstract
Fulltext PDF
References
Research Article
 
Association of H. pylori Virulence Genes CagA, VacA and Ure AB with Ulcer and Nonulcer Diseases in Iranian Population



Shohreh Farshad, Abdolvahab Alborzi and Amin Abbasian
 
ABSTRACT

To evaluate the association of virulence genes CagA, VacA and UreAB of H. pylori with the development of different gastric disorders, polymerase chain reaction was performed on H. pylori organisms isolated from biopsy samples of stomach of patients with ulcerative disease and nonulcerative disease. The difference between the groups was statistically significant (p<0.05) only for VacA gene. We detected 8 phenotypes, characterized as CagA+-VacA+-UreAB+ (phe 1), CagA¯-VacA¯-UreAB¯ (phe 2), CagA+-VacA+- UreAB¯ (phe 3), CagA+-VacA¯-UreAB+ (phe 4), CagA¯-VacA+-UreAB+ (phe 5), CagA+-VacA---UreAB¯ (phe 6), CagA¯-VacA+-UreAB¯ (phe 7), CagAVacA¯-UreAB+ (phe 8). The prevalence of phenotype 1 was significantly higher in the patients with UD than that in the patients with NUD (p<0.05). These results suggest that in the population under our study, being infected by a H. pylori strain with the genotype CagA+-VacA+-UreAB+ may be associated with an increased risk of acquiring an ulcer disease.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Shohreh Farshad, Abdolvahab Alborzi and Amin Abbasian, 2007. Association of H. pylori Virulence Genes CagA, VacA and Ure AB with Ulcer and Nonulcer Diseases in Iranian Population. Pakistan Journal of Biological Sciences, 10: 1185-1189.

DOI: 10.3923/pjbs.2007.1185.1189

URL: https://scialert.net/abstract/?doi=pjbs.2007.1185.1189

INTRODUCTION

Helicobacter pylori organisms colonize approximately half of the world’s human population (The EUROGAST Study Group, 1993). It is considered the etiological agent of Chronic Gastritis (CG) and peptic ulcer and their complications (Marshall, 1986; Cover and Blaser, 1995; Sipponen, 1997; Maaroos et al., 1999). Histological gastritis is essentially universal among H. pylori infected individuals, but only a minority develops a clinically significant outcome, such as peptic ulcer disease or gastric cancer.

The process by which different disease patterns develop has not been fully elucidated. However two putative virulence determinants of H. pylori have been identified as markers which may influence the pathogenicity of different H. pylori isolates, the cytotoxin associated gene A (CagA) and the vacuolating cytotoxin gene A (VacA) (Xiang et al., 1995; Censini et al., 1996). The CagA gene encodes a 120-140 kDa protein of unknown function in about 60-70% of H. pylori strains. Overall, the data support the notion that infection with a CagA positive isolate increases the risk but does not predict the presence of a clinically significant outcome (Konraethsson et al., 2003; Ali et al., 2005; Shibata et al., 2006). VacA codes for another important virulence factor that induced vacuolization on eukaryotic cells in vitro. Differences in VacA gene (the mosaic combination of signal[s] regions and middle[m] region allelic types) have been identified and attempts have been made to associate specific VacA genotypes with different outcomes, especially with Ulcer Disease (UD) (Atherton et al., 1997; He et al., 2000; Ruzsovics et al., 2001; Correa, 2005).

The aim of this study was to evaluate the distribution of CagA and VacA genes in H. pylori strains isolated from Iranian patients. Besides that, we studied the prevalence of another pathogenic factor, UreAB gene, as it has been cited that intense urease activity is a key pathogenicity factor (Shen et al., 1998).

MATERIALS AND METHODS

Patients and samples: We performed this study in Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, during November 2004 to October 2005. In this study 116 patients undergoing endoscopy, at endoscopy ward of Nemazee Hospital of Shiraz University of Medical Sciences were enrolled (average age 41.3±14, range 16-80, 60 males and 56 females). Histologically, antral biopsy specimens were embedded in paraffin, stained and examined by a central study pathologist for diagnosis of H. pylori infection and confirmation of gastric disease. Another piece of antrum biopsy from each patient was obtained and transferred to the lab for isolation of H. pylori by previously described culture methods (Farshad et al., 2004a, b).

Table 1: PCR primers for amplification of CagA, VacA and UreAB sequences

Briefly, the homogenates of the biopsy specimens were cultured on Brucella agar base (Merk, Germany) containing 10% lysed horse blood and appropriate antibiotics. The cultures were kept in a microaerophilic atmosphere (6% O2, 7.1% CO2, 7.1% H2, 79.8% N2) (ANOXOMAT Mark II, Mart Microbiology BV, Netherlands) at 37°C for 5-10 days, the organisms were identified as H. pylori by gram staining, colony morphology and positive oxidase, catalase and urease reactions.

Preparation of genomic DNA: Pure isolates were collected from the surface of the plates, washed with sterile PBS buffer and pelleted in 1.5 mL tubes. The pellets were resuspended in 383 mL of TE Buffer [10 mM Tris-HCl [PH, 8.0], 1 mM EDTA, 10% SDS] and 2 μL of 20 mg mL-1 solution of proteinase K and incubated at 56°C for 2 h in a hot block. DNA was then extracted using phenol- chloroform method (Wilson, 1994).

Detection of CagA, VacA and UreAB by PCR: The primers sequences were previously reported and obtained from TIB MOLBIOL Syntheselabor Gmb H (Berlin, Germany) (Han et al., 1998). Description and sequences of the PCR primers used in this study are given in Table 1. Amplifications were carried out in a gradient thermal cycler (Eppendorf, Germany) as described earlier by Han et al. (1998). Individual PCR products were electrophoresed on agarose gels, stained with ethidium bromide and photographed.

Data analysis: Fisher’s exact test was used for analysis of data for different groups and diseases. An amount of <0.05 was accepted for p-value as statistically significant.

RESULTS

Patient groups and prevalence of H. pylori infection: According to endoscopic and pathologic findings the patients were categorized to 2 groups: Ulcerative (37)

and nonulcerative (77). Totally from antrum of ulcerative and nonulcerative patents 30 (81.08%) and 35 (45.45%) H. pylori strains were isolated, respectively.

Prevalence of CagA, VacA and UreAB among H. pylori positive patients: In polymerase chain reaction analysis totally from 65 H. pylori isolates 31 strains (47.69%) were CagA+, 37 strains (56.92%) were VacA+ and 42 (64.61%) strains were UreAB+.

Relation between CagA, VacA, UreAB status and ulcerative or nonulcerative disease: Thirteen of 35 (37.14%) H. pylori strains isolated from patients with Non Ulcerative Disease (NUD) and 24 of 30 (80%) H. pylori strains isolated from patients with Ulcerative Disease (UD) were VacA+. The presence of VacA in the patients with UD was significantly higher than that in the patients with NUD (p<0.05). 13 of 35 (37.14%) H. pylori strains isolated from patients with NUD and 18 of 30 (60%) H. pylori strains isolated from patients with UD were CagA+. CagA positivity was higher in the patients with UD than that in the patients with NUD, but this difference between the groups was not statistically significant (p>0.05). Twenty of 35 (57.14%) H. pylori strains isolated from patients with NUD and 22 of 30 (73.3%) H. pylori strains isolated from patients with UD were UreAB+. According to the Table 2 CagA, VacA and UreAB positivity was higher in the patients with UD (60, 80 and 73.3%, respectively) than that in the patients with NUD (37.14, 37.14 and 57.14%, respectively), but the difference between the groups was statistically significant (p<0.05) only for VacA gene. We detected 8 phenotypes, characterized as CagA+-VacA+-UreAB+ (phe 1), CagA¯-VacA¯-UreAB¯ (phe 2), CagA+-VacA+-UreAB¯ (phe 3), CagA+-VacA¯-UreAB+ (phe 4), CagA¯-VacA+-UreAB+ (phe 5) CagA+-VacA¯-UreAB¯ (phe 6), CagA¯-VacA+- UreAB¯ (phe 7) and CagA¯-VacA¯-UreAB+ (phe 8) (Table 2). Phenotype 1 was found in 26.15% of the patients (40% UD, 14.28% NUD). The prevalence of this phenotype was significantly higher in the patients with UD than that in the patients with NUD (p<0.05). However, phenotype 8 was only seen in the patients with NUD. There was not any significant difference between the groups according to the other phenotypes (p>0.05).

Table 2: Distribution of CagA, VacA and UreAB genes in H. pylori strains isolated from patients with non ulcerative and ulcerative diseases
NUD-Nun Ulcerative Disease, UD-Ulcerative Disease

DISCUSSION

It has been suggested that H. pylori may induce more or less severe gastroduodenal diseases according to the strain virulence. In this study, we employed VacA, CagA and UreAB genotyping to characterize 65 individual H. pylori isolates drived from two groups of patients. The presence of VacA, was significantly more prevelent in the patients with UD than those in patients with NUD (p<0.05). The positivity of CagA was higher in the patients with UD than that in the patients with NUD, but difference was not statistically significant (p>0.05). However, we found a significantly higher prevalence of VacA positive strains in UD than that in NUD patients (80 and 37.14%, respectively). It was reported from different centers that in patients with ulcer diseases, the positivity rates of CagA and VacA and both CagA, VacA were 71-100, 47.5-92, 37-75%, respectively (Kidd et al., 1999; Lamarque et al., 1999; Brito et al., 2003). In all of these studies, the positivity of CagA and VacA was higher in the patients with UD, however some was statistically significant (Hennig et al., 1999; Lin et al., 2000; Martin Guerrero et al., 2000; Leite et al., 2005) and some not when it was compared to patients without ulcer (Kodama et al., 1999; Mahachai et al., 1999; Audibert et al., 2000). In patients with NUD, the positivity rates of CagA and VacA were reported to be 37-89.7% (Hennig et al, 1999; Kodama et al., 1999; Lamarque et al., 1999; Tan et al., 2006) and 33.3-73% (Weel et al., 1996; Lamarque et al., 1999; Mahachai et al., 1999), respectively. Nearly in all of these studies, CagA and VacA positivity rate in the patients with NUD was found to be low compared to that in the patients with ulcer, however, this difference was statistically significant in some studies (Ito et al., 1997; Warburton et al., 1998; Martin Guerrero et al., 2000; Chen et al., 2005) but not in some others (Mitchell et al., 1996; Mahachai et al., 1999; Zheng et al., 2000; Bulent et al., 2003). In this research, the VacA positivity in the patients with NUD was significantly lower than that in the ulcer patients (p<0.05). Although CagA positivity was higher in the patients with ulcer than that in the patients with NUD, this was not statistically significant and did not seem to be an important risk factor for the development of ulcer in our patients. However, determination the VacA genotypes and the presence of CagA gene together may contribute to potential clinic determination of patients who have different levels of risk. It has been shown that VacA type s1/m1 strains produce more cytotoxins than type s1/m2 and that type s2/m2 strains do not produce active cytotoxins (Yamaoka et al., 1997). Many studies have confirmed these findings (Evans et al., 1998; Stephens et al., 1998; Rudi et al., 1999) but some other reports have found no association between the presence of CagA or s1 allelic variant of VacA and the clinical outcome of an H. pylori infection (Faundez et al., 2000). In the literature, it has been controversial that CagA and VacA positive isolates cause more serious gastroduodenal lesions (Mitchell et al., 1996; Martin Guerrero et al., 2000). The results for UreAB gene showed that UreAB gene doesn’t have any role in ulceration process without any combination with VacA genes. Positivity of UreAB in NUD patients was significantly higher than that in UD patients, when there was no other virulence gene. On the other hand, in our study significant difference between distribution of CagA+-VacA+-UreAB+ phenotype in UD patients and NUD patients (40% vs 14.28%) showed that the most virulent strains of H. pylori harbor these three virulence genes together. Consequently, it was seen that duodenal ulcer incidence increased in the patients with CagA, VacA and UreAB positive.

In conclusion, these results suggest that in the population under our study, being infected by a H. pylori strain with the genotype CagA+- VacA+- UreAB+ may be associated to an increased risk of acquiring an ulcer disease. The genetic predisposition of the population and local environmental factors, may also be important factors in the development of diseases caused by H. pylori, which may explain the differences observed with respect to other countries.

ACKNOWLEDGMENTS

This study was supported by Prof. Alborzi Clinical Microbiology Research Center, Shiraz, Iran, grant No. 81-5. The authors wish to thank Dr. Davood Mehrabani at Center for Development of Clinical Research of Nemazi Hospital for editorial and statistical analysis assistant.

REFERENCES
Ali, M., A.A. Khan, S.K. Tiwari, N. Ahmed, L.V. Rao and C.M. Habibullah, 2005. Association between cag-pathogenicity island in Helicobacter pylori isolates from peptic ulcer, gastric carcinoma and non-ulcer dyspepsia subjects with histological changes. World J. Gastroenterol., 11: 6815-6822.

Atherton, J.C., R.M. Peek, K.T. Tham, T.L. Cover and M.J. Blaser, 1997. Clinical and pathological importance of heterogeneity in Vac A, the vacuolating cytotoxin gene of Helicobacter pylori. Gastroenterology, 112: 92-99.
Direct Link  |  

Audibert, C., B. Janvier, B. Grignon, L. Salaun, C. Burucoa, J.C. Lecron and J.L. Fauchere, 2000. Correlation between IL-8 induction, cagA status and vacA genotypes in 153 French Helicobacter pylori isolates. Res. Microbiol., 151: 191-200.
Direct Link  |  

Brito, C.A., L.M. Silva, N. Juca, N.C. Leal and W. de Souza et al., 2003. Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil. Mem. Inst. Oswaldo. Cruz, 98: 817-821.
Direct Link  |  

Bulent, K., A. Murat, A. Esin, K. Fatih and H. Murat et al., 2003. Association of CagA and VacA presence with ulcer and non-ulcer dyspepsia in a Turkish population. World J. Gastroenterol., 9: 1580-1583.
Direct Link  |  

Censini, S., C. Lange, Z. Xiang, J.E. Crabtree and P. Ghiara et al., 1996. Cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc. Natl. Acad. Sci. USA., 93: 14648-14653.
PubMed  |  Direct Link  |  

Correa, P., 2005. New strategies for the prevention of gastric cancer: Helicobacter pylori and genetic susceptibility. J. Surg. Oncol., 90: 134-138.
CrossRef  |  PubMed  |  Direct Link  |  

Cover, T.L. and M.J. Blaser, 1995. Helicobacter pylori: A bacterial cause of gastritis, peptic ulcer disease and gastric cancer. ASM News, 61: 21-26.

Evans, D.G., D.M. Queiroz, E.N. Mendes and D.J. Evans, 1998. Helicobacter pylori cagA status and s and m alleles of vacA in isolates from individuals with a variety of H. pylori-associated gastric diseases. J. Clin. Microbiol., 36: 3435-3437.
Direct Link  |  

Farshad, S.H., A. Alborzi, S.A. Malek Hosseini, B. Oboodi, M. Rasouli, A. Japoni and J. Nasiri, 2004. Identification of Helicobacter pylori DNA in Iranian patients with gallstones. Epidemiol. Infect., 132: 1185-1189.
Direct Link  |  

Farshad, S.H., M. Rasouli and A. Alborzi, 2004. Simultaneous detection of Helicobacter genus and Helicobacter pylori species using a Multiplex PCR method. Iranian. Biomed. J., 8: 205-209.

Faundez, G., M. Troncoso and G. Figueroa, 2002. cagA and VacA in strains of Helicobacter pylori from ulcer and nonulcerative dyspepsia patients. BMC Gastroenterol., 10: 2-20.
Direct Link  |  

Han, S.R., H.J. Schreiber, S. Bhakdi, M. Loos and M.J. Maeurer, 1998. VacA genotypes and genetic diversity in clinical isolates of Helicobacter pylori. Clin. Diag. Lab. Immunol., 5: 139-145.
Direct Link  |  

He, Y., P. Hu, X. He, Z. Zeng, Y. Cui and C. Li, 2000. [Prevalence of cag A and vac A subtypes of Helicobacter pylori in Guangzhou]. Zhonghua. Nei. Ke. Za. Zhi, 39: 818-820.
Direct Link  |  

Hennig, E.E., L. Trzeciak, J. Regula, E. Butruk and J. Ostrowski, 1999. VacA genotyping directly from gastric biopsy specimens and estimation of mixed Helicobacter pylori infections in patients with duodenal ulcer and gastritis. Scand. J. Gastroenterol., 34: 743-749.
Direct Link  |  

Ito, A., T. Fujioka, K. Kodama, A. Nishizono and M. Nasu, 1997. Virulence-associated genes as markers of strain diversity in Helicobacter pylori infection. J. Gastroenterol. Hepatol., 12: 666-669.
Direct Link  |  

Kidd, M., A.J. Lastovica, J.C. Atherton and J.A. Louw, 1999. Heterogeneity in the Helicobacter pylori vacA and cagA genes: Association with gastroduodenal disease in South Africa? Gut, 45: 499-550.
Direct Link  |  

Kodama, K., A. Ito, A. Nishizono, T. Fujioka and M. Nasu et al., 1999. Divergence of virulence factors of Helicobacter pylori among clinical isolates does not correlate with disease specificity. J. Gastroenterol., 34: 6-9.

Konraethsson, A., L.P. Andersen, E. Oddsson, H. Guethjonsson and B. Thornjoethleifsson, 2003. [Prevalence of Helicobacter pylori and Cag-A strains in patients with duodenal ulcer in Iceland]. Laeknabladid, 89: 595-597.
Direct Link  |  

Lamarque, D., T. Gilbert, F. Roudot-Thoraval, L. Deforges, M.T. Chaumette and J.C. Delchier, 1999. Seroprevalence of eight higher rate of seroreactivity against CagA and 35-kDa antigens in patients with peptic ulcer originating from Europe and Africa. Eur. J. Gastroenterol. Hepatol., 11: 721-726.
Direct Link  |  

Leite, K.R., E. Darini, F.C. Canavez, C.M. Carvalho, C.A. Mitteldorf and L.H. Camara-Lopes, 2005. Helicobacter pylori and cagA gene detected by polymerase chain reaction in gastric biopsies: Correlation with histological findings, proliferation and apoptosis. Sao Paulo. Med. J., 123: 113-118.
Direct Link  |  

Lin, C., S.C. Wu, S.C. Lee and K.S. Cheng, 2000. Genetic analysis and clinical evaluation of vacuolating cytotoxin gene A and cytotoxin-associated gene A in taiwanese Helicobacter pylori isolates from peptic ulcer patients. Scand. J. Infect. Dis., 32: 51-57.
PubMed  |  

Maaroos, H.I., T. Vorobjova, P. Sipponen, R. Tammur and R. Uibo et al., 1999. An 18-year follow-up study of chronic gastritis and Helicobacter pylori: Association of CagA positivity with development of atrophy and activity of gastritis. Scand. J. Gastroenterol., 34: 864-869.
CrossRef  |  PubMed  |  Direct Link  |  

Mahachai, V., P. Tangkijvanich, N. Wannachai, P. Sumpathanukul and P. Kullavanijaya, 1999. CagA and VacA: Virulence factors of Helicobacter pylori in Thai patients with gastroduodenal diseases. Helicobacter, 4: 143-147.
Direct Link  |  

Marshall, B.J., 1986. Campylobacter pyloridis and gastritis. J. Infect. Dis., 153: 650-658.

Martin Guerrero, J.M., P. Hergueta Delgado, J. Esteban Carretero, R. Romero Castro, F.J. Pellicer Bautista and J.M. Herrerias Gutierrez, 2000. Clinical relevance of Helicobacter pylori CagA-positive strains: Gastroduodenal peptic lesions marker. Rev. Esp. Enferm., 92: 160-173.
Direct Link  |  

Mitchell, H.M., S.L. Hazell, Y.Y. Li and P.J. Hu, 1996. Serological response to specific Helicobacter pylori antigens: Antibody against CagA antigen is not predictive of gastric cancer in a developing country. Am. J. Gastroenterol., 91: 1785-1788.
Direct Link  |  

Rudi, J., A. Rudy, M. Maiwald, D. Kuck, A. Sieg and W. Stremmel, 1999. Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases. Am. J. Gastroenterol., 94: 1525-1531.
Direct Link  |  

Ruzsovics, A., B. Molnar, Z. Unger, Z. Tulassay and L. Pronai, 2001. [Determination of cagA, vacA genotypes of Helicobacter pylori with real-time PCR-method]. Orv. Hetil., 142: 509-514.
Direct Link  |  

Shen, Z., D.B. Schauer, H.L. Mobley and J.G. Fox, 1998. Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes UreAB. J. Clin. Microbiol., 36: 2447-2453.
Direct Link  |  

Shibata, W., Y. Hirata, S. Maeda, K. Ogura and T. Ohmae et al., 2006. CagA protein secreted by the intact type IV secretion system leads to gastric epithelial inflammation in the Mongolian gerbil model. J. Pathol., 210: 306-314.
Direct Link  |  

Sipponen, P., 1997. Helicobacter pylori gastritis-epidemiology. J. Gastroenterol, 32: 273-277.
Direct Link  |  

Stephens, J.C., J.A. Stewart, A.M. Folwell and B.J. Rathbone, 1998. Helicobacter pylori cagA status, vacA genotypes and ulcer disease. Eur. J. Gastroenterol. Hepatol., 10: 381-384.
Direct Link  |  

Tan, H.J., A.M. Rizal, M.Y. Rosmadi and K.L. Goh, 2006. Role of Helicobacter pylori virulence factor and genotypes in non-ulcer dyspepsia. J. Gastroenterol. Hepatol., 21: 110-115.
CrossRef  |  PubMed  |  Direct Link  |  

The EUROGAST Study Group, 1993. Epidemiology of and risk factors for, Helicobacter pylori infection among 3,154 asymptomatic subjects in 17 populations. Gut, 34: 1672-1676.

Warburton, V.J., S. Everett, N.P. Mapstone, A.T. Axon, P. Hawkey and M.F. Dixon, 1998. Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis. J. Clin. Pathol., 51: 55-61.
Direct Link  |  

Weel, J.F., R.W. van der Hulst, Y. Gerrits, P. Roorda and M. Feller et al., 1996. The interrelationship between cytotoxin-associated gene A, vacuolating cytotoxin and Helicobacter pylori-related diseases. J. Infect. Dis., 173: 1171-1175.
Direct Link  |  

Wilson, K., 1987. Preparation of Genomic DNA from Bacteria. In: Current Protocols in Molecular Biology, Ausubel, F.A., R.E. Brent, D.D. Kingston, J.G. Moore and J.A. Seidman (Eds.). John Wiley and Sons, New York, pp: 2.4.1-2.4.5.

Xiang, Z., S. Censini, P.F. Bayeli, J.L. Telford, N. Figura, R. Rappuoli and A. Covacci, 1995. Analysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin. Infect. Immun., 63: 94-98.
Direct Link  |  

Xue-Jun, C.H., Y. Jie and S.H. Yue-Fang, 2005. Dominant cagA/vacA genotypes and coinfection frequency of Helicobacter pylori in peptic ulcer or chronic gastritis patient in Zhejiang province and correlations among different genotypes, coinfection and severity of the diseases. Chin. Med. J., 118: 460-467.
Direct Link  |  

Yamaoka, Y., M. Kita, T. Kodama, N. Sawai, K. Kashima and J. Imanishi, 1997. Induction of various cytokines and development of severe mucosal inflammation by cagA gene positive Helicobacter pylori strains. Gut, 41: 442-451.
Direct Link  |  

Zheng, P.Y., J. Hua, K.G. Yeoh and B. Ho, 2000. Association of peptic ulcer with increased expression of Lewis antigens but not cagA, iceA and VacA in Helicobacter pylori isolates in an Asian population. Gut, 47: 18-22.
Direct Link  |  

©  2018 Science Alert. All Rights Reserved
Fulltext PDF References Abstract