DETECTION OF cagA GENE AND TYPING vacA GENE OF Helicobacter pylori IN BIOPSIES OF PATIENTS WITH GASTRIC SYMPTOMS IN CUMANA, SUCRE STATE, VENEZUELA

DETECCIÓN DEL GEN cagA Y TIPIFICACIÓN DEL GEN vacA DE Helicobacter pylori EN BIOPSIAS DE PACIENTES CON SINTOMATOLOGÍA GÁSTRICA EN CUMANÁ, ESTADO SUCRE, VENEZUELA

Luz Bettina Vill alobos^1,2, María Eugenia Cavazza³, Diana Ortiz-Princz³

¹Universidad de Oriente, Núcleo de Sucre, Postgrado en Biología Aplicada, Cumaná, Venezuela,

²Universidad Simón Bolívar, Doctorado Interdisciplinario en Ciencias, Caracas, Venezuela,

³Universidad Central de Venezuela, Instituto de Biomedicina, MPPS-UCV, Laboratorio de Microbiología Molecular, Caracas, Venezuela E-mail: lbvillalobosb@yahoo.com

ABSTRACT

Helicobacter pylori has been associated with gastric diseases such as gastritis, ulcers, lymphoma gastroduodenal and is considered a risk factor for the development of gastric cancer. The aim of this study was to characterize vacA genotypes and to determine the presence of cagA gene in biopsies from patients with gastric symptoms treated at the University Hospital Antonio Patricio de Alcalá of Cumaná, Venezuela. Sixty nine patients with endoscopic indication were evaluated using the polymerase chain reaction (PCR). Only 42 (60.86%) of those patients were positive for amplification of vacA and/or cagA and of these, 54.76% amplified for both genes. The predominant genotype was m1s1 (61.90%) followed by mixed infections (33.33%), being the most frequent associations among strains with genotypes m1s2, m2s2 and m1s1. From the biopsies performed 72.5% were negative for the cagA gene. However, this was found to be associated mainly to genotype m1s1 (17.39%). Statistically highly significant correlations were found among the allelic forms m1 and s1 (ρ = 0.732), s2 and m2 (ρ = 0.643), significant m1 and s2 (ρ = 0.279). Gene cagA showed highly significant correlations with m1 (ρ = 0.509), m2 (ρ = 0.447) and significant with s1 (ρ = 0.319) and s2 (ρ = 0.263). These results show the existence of co-infections with different genotypes of H. pylori in the studied population. They, would explain in part the low incidence of severe gastric pathologies despite being present in the region virulent genotypes of this bacterium. The high percentage of coinfections observed could be the result of the influence of geographic and ethnicity factors in the region where the study was conducted..

Key words: H. pylori, genotyping, gastritis.

RESUMEN

Helicobacter pylori, ha sido asociado a patologías gástricas y es considerado un factor de riesgo para el desarrollo de cáncer gástrico. El objetivo de este estudio fue caracterizar los genotipos de vacA y la presencia del gen cagA en biopsias provenientes de pacientes con sintomatología gástrica atendidos en el Hospital Universitario Antonio Patricio de Alcalá de Cumaná, Venezuela. Se evaluaron 69 pacientes con indicación endoscópica, empleando la reacción en cadena de la polimerasa. Sólo 42 de ellos (60,86%) mostraron ser positivos para la amplificación de vacA y/o cagA y de éstos el 54,76% amplificó para ambos genes. El genotipo predominante fue m1s1 (61,90%) seguido de infecciones mixtas (33,33%), siendo más frecuentes las asociaciones entre cepas m1s2 con los genotipos m2s2 y m1s1. El 72,5% de las biopsias fueron negativas para el gen cagA. Sin embargo, este se encontró asociado principalmente al genotipo m1s1 (17,39%). Estadísticamente, se encontraron correlaciones muy significativas entre las formas alélicas m1 y s1 (ρ = 0,732), s2 y m2 (ρ = 0,643), significativas m1 y s2 (ρ = 0,279). El Gen cagA mostró correlaciones muy significativas con m1 (ρ = 0,509), m2 (ρ = 0,447) y significativas con s1 (ρ = 0,319) y s2 (ρ = 0,263). Estos resultados muestran la existencia de coinfecciones por distintos genotipos de H. pylori en la población estudiada, que explicaría en parte la baja incidencia de patologías gástricas severas a pesar de estar presente en la región genotipos virulentos de esta bacteria. El elevado porcentaje de coinfecciones observado podría ser el resultado de la influencia de factores geográficos y de etnicidad de la región donde se realizó el estudio.

Palabras Clave:  H. pylori, genotipficación, gastritis.

Recibido: abril 2015. Aprobado: junio 2015. Versión final: junio 2015.

INTRODUCTION

Helicobacter pylori is a Gram negative bacteria, microaerophilic which persistently colonizes the human gastric mucosa. Over 50% of the world population is infected with the bacteria and in developed countries can reach 80%. Although most are asymptomatic, the presence of H. pylori is associated with diseases such as peptic ulcers, gastric adenocarcinoma and gastric lymphoma (Cavazza et al. 2001). The rapid changes observed in the epidemiology of gastric pathologies associated with H. pylori is likely due to the interaction between environmental factors and host factors or changes in the prevalence of more or less virulent strains (Jafari et al. 2008). Two phenotypic characteristics allow the identification of strains capable of virulence, the vacuolating cytotoxin encoded by vacA gene, which is present in most strains of H. pylori, and the high molecular weight protein associated with cagA gene which is also cytotoxic. The cagA gene is located on a pathogenicity island (PAI) of 40 kb that was horizontally transferred from a source not known (Censini et al. 1996). The cag PAI encodes the type IV secretion system, through which CagA is carried into the cell. The structure of the 3' cagA gene contains a motif EPIYA that phosphorylate the tyrosine of CagA once it enters the cell, variations in this region differ with strains of H. pylori and can, inclusive, differentiate their Asian or Western origin (Mane et al. 2010, Suzuki et al. 2012). Vacuolating cytotoxin VacA, is produced by most strains of H. pylori, besides being a cytotoxin which has no similarity with other bacterial proteins and eukaryotic proteins (Cover and Blanke 2005), once it is produced may remain on the surface of the bacteria or being secreted as a toxin of approx. 88 kDa (Ilver et al. 2004, El-Bez et al. 2005), dissociates upon exposure to nonneutral environments, which when exposed to conditions of alkalinity or acidity amplifies its activity (Cover et al. 1997, Yahiro et al. 1999).

Gene structure of vacA allows variations in the vacuolating activity of the strains, possesses the region signal (s1, s2), the middle region (m) and a third intermediate region newly assigned as (i). In the region s of vacA is found the p33 portion of the toxin that influence vacuolating activity and efficiency in forming anion channels, due to the hydrophobic nature of the amino acid residues that are close to the proteolytic cleavage site (McClain et al. 2001), the s1 variant contains more hydrophobic amino acids near the region of cleavage that s2 variant, which allow better integration into the membrane of the host cell (McClain et al. 2001).

The middle region (m1 and m2) (Atherton et al. 1997) is found in the p55 portion of the toxin and influence the tropism of H. pylori in the host cells: the region m1 is toxic to a wide range of host cells (Pagliaccia et al. 1998, Amieva and El Omar 2008). Combinations of these regions result in strains that may be more or less virulent m1s1 genotype produces the highest level of toxin, the s1m2 strains produce low to moderate level of toxin, the m2s2 are considered nontoxic while s2m1 while not been recognizes like toxic strains.

The aim of this study was to characterize genotypes vacA and the presence of cagA gene detected in gastric biopsies of patients with gastric pathologies positive for infection with H. pylori, who attended to the Gastroenterology Service of the University Hospital Antonio Patricio de Alcalá of Cumaná, Sucre state.

MATERIALS AND METHODS

Patients

120 patients attending the Gastroenterology Service, Hospital Universitario Antonio Patricio de Alcalá of Cumana, who expressed by prior written consent to the taking of blood samples and biopsies following the guidelines of the Bioethics Committee of the Autonomous Service, Institute of Biomedicine (Ministerio del Poder Popular de la Salud-Universidad Central de Venezuela) (MPPCTII-FONACIT 2011). Each patient provided its epidemiological data by filling out a form designed for that purpose by modified Graffar test (Méndez-Castellano y Méndez 1994). The exclusion criteria of this study were, treatment with antibiotics and drugs containing bismuth or omeprazole two weeks before the test.

Gastroscopy examination

The stomach endoscopy was performed based on guidelines protocols of the hospital work, for it biopsies of the lesser curvature of the antrum were taken. Antrum samples for molecular testing were placed in Eppendorf tubes, identified with the number of the patient and immediately frozen at -80°C.

DNA extraction from biopsy

The procedure followed the guidelines of Perrone et al. (2009), briefly: To the tubes containing the biopsy were added 50-100 μL of Proteinase K (100 μg/ μL) + 50 μL of Lysis Buffer (10 mM Tris-HCl, pH 8.1 + 0.1% Sarcosine.), them stirred in Vortex and placed in water bath at 55°C for two hours. The inactivation of the proteinase K was achieved by heating at 95ºC for 5 minutes. Subsequently 1 V of chloroform: phenol: isoamyl was added. Vorterising and centrifuging at 14,000 rpm for 10 minutes.

PCR amplification of 349 bp region of cagA

All PCR mixtures consisted of 1 μL of DNA, 1X PCR Buffer (Gibco BRL, Gaithersburg, MD), 1.5 mM MgCl2, 0.2 mM each deoxynucleotide (Gibco, BRL), 0.5 mM each specific primer and 1.25 U of Taq polymerase (Gibco, BRL) in a final volume of 25 μL. A 349 bp region of cagA gene was amplified by PCR using the F1/ B1 (Tummuru et al. 1993) primers. Aliquots of isolated DNA from each patient were taken and processed by performing the following amplification program in a thermocycler (Gene Amp 9700 Perkin Elmer Applied Biosystems, USA): 35 cycles of 94 for 1 minute, 55 for 1 minute and 72 for 2 minutes and final extension of 72°C for 6 minutes.

Amplification of the 335 bp region of cagA

The 335 bp region of cagA was amplified by PCR using the B7628/B7629 initiators (González-Valencia et al. 2000). Aliquots of DNA isolated from each patient were taken and were processed by performing the following amplification program in a thermocycler (Gene Amp 9700 Perkin Elmer Applied Biosystems, USA): 35 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes and final extension of 72 for 6 minutes. For previous experiences in the laboratory it was decided to use two sets of primers for the detection of cagA and taken as positive anyone that achieve amplification.

PCR amplification of vacA alleles s1/s2 and m1/m2

For identification of allelic variants of the signal sequence S1/S2-F primers VA1/VA1-R (Atherton et al. 1995) were used to amplify the conserved regions of vacA of 259 bp and 286 bp respectively. A second set of primers R-VAG/VAG-F (Atherton et al. 1997) was used to amplify the middle regions 567 bp BEEF (m1) or 642 bp (m2). Aliquots of DNA extracted from biopsies from each patient and processed in a thermocycler (Gene Amp 9700 Perkin Elmer Applied Biosystems, USA), by 35 cycles of 94°C for 1 minute, 52 for 1 minute and 72 for 1.5 minutes was taken, with a final extension of 72°C for 6 minutes for m1/m2. As positive controls were used specific strains, 8822 (vacA s2m2) and 8823 (vacA s1m1) (ATCC 49503) and 84183 (ATCC 53726) (vacA s1m1) and as negative control sample without DNA and DNA of Escherichia coli (ATCC 2225) (Table 1).

Table 1. Oligonucleotides used for typing cag A and vac A.

Statistical analysis

The correlation between alleles of vacA and cagA gene present were evaluated by Spearman correlation which is a measure of linear association using serial numbers of each group of alelles and compare these ranges (Spearman rho) using SPSS version 17.0.

RESULTS

In 120 symptomatic patients who underwent endoscopic study, only 69 met the requirements for entry in this study, of whom 21 (30.4%) were male and 48 (69.6%) female aged between 10 and 85 years (mean age 38.5 years). Selected patients were positive for at least two diagnostic tests for the detection of H. pylori, the data are not shown in this work. Patients came from the social strata C and D of the population according to modified Graffar (Méndez-Castellanos 1994). By provision of the Ethic Committee of the Institute of Biomedicine, endoscopic examinations were not performed in healthy people, which precluded the inclusion of control patients in the study.

Genotyping of Helicobacter pylori

Determination by PCR of the genes encoding for the vacuolating toxin VacA or cytotoxin CagA showed that 42 of the 69 patients (60.86%) were positive for one of two such genes. 23/69 (33.33%) of patients showed the vacA gene. The 19/69 (27.33%) were positive for both genes. Strains with the genotype cagA only were not observed. 27/69 (39.13%) of the biopsies did not amplify for any of the initiators used, testing of these strains by PCR technique were done in duplicate to rule out any error in its realization.

Analysis of vacA alleles genotype

In 60.86% (42/69) of patients amplification for different allelic forms of the vacA gene was observed. Percentages of allele isolation were: in 80.95% (34/42) of patients allelic form s1 and 28.57% in (12/42) allelic type s2 was detected. For the middle region of vacA analysis revealed that 95.23% (40/42) were vacA m1, while 33.33% (14/42) were vacA m2 (Table 2). The frequency of combination of the different allelic types of middle and signal vacA regions in the gastric biopsies evaluated, showed that the most frequent combination was m1s1 26/42 (61.90%). It is to be noted that 14 out of 42 patients (33.33%) presented infections with more than one strain of H. pylori; being the more frequent combination m1s2 + m2s2 with 16.66% (Table 3).

 

Amplification of cagA gene in samples from biopsypositive patients was 19/42 (45.23%). The predominance of negative cagA strains was observed only associated with vacA positive strains.

Table 4 shows the frequency of distribution of the various combinations of vacA and cagA gene. Prevalence of cagA negative 23/42 (54.76%) strains were observed. Also shown that 14/42 (33.33%) of patients positive by PCR had mixed infections, predominantly the combination m1s2 + m2s2cagA- with 5/42 (11.90%). The predominant genotype strains m1s1 12/42 (28.57%) were associated with the cagA.  

Less virulent strains m2s2 and m2s1, not associated with cagA gene were detected in low numbers 1/42 (2.38%). Fourteen of the 42 positive strains for either gene, presented mixed genotypes had average double vacA regions allelic combinations and of these only (7/14) mixed genotypes were associated with cagA gene, so that highlights the existence of co-infections with different genotypes in the same patient.

Statistically Spearman Rho showed significant correlations (p < 0.05) between alleles of the middle region of vacA, m1 with m2 (ρ = 0.255) and between m1 and s2 (ρ = 0.279). Very significant correlations were observed (p < 0.01) between the presence of cagA gene with the middle regions of vacA m1 (ρ = 0.509), m2 (ρ = 0.447) and significant (p < 0.05) with s1 (ρ = 0.319) and s2 (0.263), explaining that the presence of cagA gene may be associated with any of the combinations of these alleles. The cagA gene showed a direct and significant relationship (p < 0.05, ρ = 0.253) with genotype m1s1+m2s2, as the cagA gene tends to be associated with the genotype m1s1, it is possible that this combination be favored by this feature.

DISCUSSION

The vacA gene structure allows variations in the vacuolating activity of strains, possesses the region signal (s1, s2), the middle region (m) and a third intermediate region newly assigned as (i). The region s of vacA is associated to the domain p33 toxin and influence vacuolating activity and efficiency in forming anion channels, due to the hydrophobic nature of the amino acid residues that are close to the proteolytic cleavage site (McClain et al. 2001), the s1 variant contains more hydrophobic amino acids near the region of cleavage that s2 variant, which allow better integration into the membrane of the host cell (McClain et al. 2001). The middle region (m1 and m2) (Atherton et al. 1997) is in the domain p55 of the toxin and influence tropism of H. pylori in the host cells: the region m1 is toxic to a wide range of host cells (Amieva and El-Omar 2008). Combinations of these regions result in strains that may be more or less virulent. Among the genotypes s1, the strains s1m1 are more toxic that strains s1m2 (Letley et al. 2003). The s2 form has a short N-terminal peptide in the mature protein, which blocks the biological activity of VacA (Letley et al. 2003). Strains vacA m2s2 encoding proteins of low toxicity and are not often associated with peptic ulcer or gastric cancer. It has been described that vacuolating activity s1m1, depend on the type il, being associated with peptic ulcer or adenocarcinoma (Basso et al. 2008). The s2m1 combination is rare (Salama et al. 2007) but has been reported in Chile (Martínez et al. 2001), Colombia (Citelly et al. 2002) and Cuba (Torres et al. 2009). In this work was not detected the presence of unique vacA allelic genotypes, strains without a single allele were found, but combined, unlike those reported by Ghose et al. (2005), in strains of H. pylori solated from different Venezuelan populations, especially Amerindians Piaroas and Guahibos. Recently Chiurillo et al. (2013) found genotypic diversity of H. pylori in patients from western Venezuela with more than one allele of vacA.

The results of this study established that the allelic combination of vacA most frequent was s1m1 (69.90%; 26/42), lower than that reported by Ortiz-Princz et al. (2010), but higher than that observed by Perrone et al. (2009), both studies carried out in other regions of Venezuela. The genotype m1s1 has established itself as the most frequent in other Latin American countries (Martínez et al. 2001, Vega et al. 2010). In other Latin American countries such as Mexico, Argentina, Colombia, were found patients colonized with strains possessing multiple vacA genotypes. This may be a result of the acquisition of the bacterium from childhood, domestic infection and transmission route oral and fecal of different strains that can coevolve on the individual as demonstrated in murine models (Mane et al. 2010), establishing synergies between them favoring the permanence of the strains in the niche and a relationship with the host that allows colonization indefinitely. Ghose et al. (2005) and Salama et al. (2007) State that these variations can be innergeographics, i.e. as a result of isolation and/or progressive contact between different geographical locations.

In this study, 37.68% of biopsies not amplified for any gene despite the positivity of biopsies for the presence of H. pylori. This not genotyping has been observed in works by other authors (Gatti et al. 2005, Jafari et al. 2008, Torres et al. 2009) and has been suggested that nontypeable strains, is due to the existence of subfamilies allelic forms s and m that are not recognized by the primers available today. Atherton et al. (1999), failed to establish middle regions of 22 strains from patients from South America and Asia, by changes in the alignment in the middle region divider, so they suggested the design of a new genotyping for these populations.

In our study, the frequency of strains m2s2 was low compared with those reported by Ortiz-Princz et al. (2010) in another study in Venezuela. Furthermore, not only the presence of strains m1s2 was detected, but a percentage of mixed infections in 33.33% of biopsies and genotyping within these coinfections were observed m1s2 strains mainly associated to m1s1 and m2s2 genotypes. Detecting strains with genotype vacA m1s2, was consistent with other studies which detected this genotype (Kidd et al. 1999, Morales-Espinosa et al. 1999, Paniagua et al. 2009, Sugimoto and Yamaoka 2009, Torres et al. 2009). The pathogenicity of this genotype is not well defined, because it has a selective disadvantage to develop disease (Francesco et al. 2009), but it is known that the m1 allele is associated with high virulence strains (Atherton et al. 1995).

Co-infection with different strains, especially combinations of s1m1, with s1m2 strains in one patient, have been reported by several authors in different countries (Morales-Espinosa et al. 1999, González- Valencia et al. 2000). The results of this study establish that 33.33% of the genotyping showed that patients had multiple colonization, which is consistent with previous observations (Taylor et al. 1995, Chen et al. 2003), who state that the rates of co-infection are highest in countries with a high prevalence of H. pylori, as has been established for Venezuela (Cavazza et al. 2001, De Sousa et al. 2006). Another factor to consider for variations of strains and co-infection is ethnicity in Venezuela, in addition to being variable according to its historical process as a nation, geographical position places it at the gateway of people from other continents especially in the early twentieth century, they settled in the country or continued to other areas of the continent which would allow the exchange of strains of Helicobacter among different human groups (Atherton and Blaser 2009, Mahomed et al. 2009, Sugimoto and Yamaoka 2009, Yamaoka 2009).

Most biopsy specimens were negative for the presence of cagA gen (72.5%), similar results were observed in countries of Asia, Europe and Africa (Letley et al. 1999, Maeda et al. 1999, Yamaoka et al. 1999, 2008) rather than in Western countries. It has been noted that the populations of Western countries are exposed to antibiotic consumption from an early age which would have a cumulative effect and selection in strains of H. pylori colonizing the stomach (Perez-Perez et al. 1990, Marais et al. 1998) favorable for cagA negative strains that are less susceptible to treatment by antibiotics than cagA positive strains (Perez-Perez et al. 2001). The decline of the strains cag + according to Perez-Perez et al. (2002) in a study conducted in Finland, is due to socio-economic development which results in low transmissibility of strains especially in childhood and adulthood decline.

Another hypothesis is that the loss of cagA gene is an adaptive way to the bacteria remain in the host, because if the damage persists severely as to reach a metaplasia, the bacteria disappear for not having receptors for adhesion (Yahiro et al. 1997). cagA association was specially found with the middle regions of vacA m1 and m2 and signal sequences s1 and s2, being the most frequent combination of strains m1s1cagA +. These results coincide with those observed by Citelly et al. (2002) in Colombia, Martins et al. (2005) in northern Brazil, Torres et al. (2009) in Cuba. While this genotype m1s1cagA + is highly associated with severe gastrointestinal disease, its association with other genotypes allows is linked to inflammatory gastric diseases increasing virulence of these genotypes (Van Doorn et al. 1999, Kidd et al. 2001, Zambon et al. 2003), the association of cagA mainly observed with the combined genotype m1s1 + m2s2, explain in patients with chronic gastritis the virulence of the combined genotype.

The association among the most virulent vacA genotypes and cagA gene may be a coincidence or a preference of cagA for vacA genotypes. However, the idea that the cag pathogenicity island containing the cagA gene is a genomic island unstable and should prefer virulent vacA alleles during insertion into the genome (Nagiyev et al. 2009) is further assumed. In addition to the synergy of these virulence factors to produce pathogenic effect (Argent et al. 2008, Oldani et al. 2009), consider that there is no single polymorphism in vacA, but also in the cag PAI, as evidence of polymorphism among others, the IL-1B gene are associated factors for the initiation and amplification of the inflammatory response in chronic infection with H. pylori (Yamaoka et al. 1996).

Statistically it was showed significant positive correlations between alleles of the middle region vacA m1 and m2, between signal region s2, this result showed polymorphism having vacA in the population studied, where the observed genotypes showed allelic combinations middle regions and signal, being the most frequent genotype m1s1, followed by m2s2, these allelic combinations have been observed preferably in the Americas (Torres et al. 2009).Although it has been widely established that the region m2 of vacA has little toxic activity in vitro that affect the tropism of the bacteria. Pagliaccia et al. (1998) demonstrated in RK- 12 cells that this form of H. pylori induced vacuolation, thus the genotypes m1s2 and m2s2 and may have activity induce infectious disease processes. Correlations between vacA allelic regions with cagA and the combination of these genes can decrease the effects of each of the toxins by themselves, so as to lengthen the survival time in infected host cells (Argent et al. 2008). CagA and VacA are the most studied virulence factors of H. pylori, both toxins have a high degree of polymorphism and the evidence shows that this polymorphism, alone or together, is responsible for strains of H. pylori may affect patients with varying severity. This would explain why despite the presence of genotypes known by its virulence have not observed in this study patients with severe gastric diseases such as cancer or metaplasia.

ACKNOWLEDGES

To the medical staff and nurses of the Gastroenterology Service, University Hospital Antonio Patricio de Alcalá of Cumaná, by their valuable assistance in the endoscopic examination and taking biopsies. This project was funded in part by the Universidad de Oriente Nucleo de Sucre, Project No. CI 2-010101-1248/05.

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