ARTÍCULO ORIGINAL

¹Department of Research and Development, Division of Production and Development of Equipments and Diagnostics, National Centre for Scientific Research, Ciudad de La Habana, Cuba

²Department of Gastroenterology, Medical and Surgery Research Centre (CIMEQ), Ciudad de La Habana, Cuba

³Department of Molecular Biology, Biotechnology Division, National Centre for Scientific Research, Ciudad de La Habana, Cuba

⁴Departments of Medicine and Microbiology, New York University, School of Medicine, USA

Author contributions:

L. E. Torres and B. L. Rodríguez contributed equally to this work.

L. E. Torres, L. González, J. Campos and B. L. Rodríguez designed the research.

L. González, L. E. Torres, O. Reyes, A. Moreno, M. Hernández, L. Bermúdez collected and processed the samples and performed the research.

K. Melián, J. Alonso performed the endoscopy and managed the patient data.

L. E. Torres, L. González, G. Pérez-Pérez and B. L. Rodríguez analyzed the data and wrote the paper.

Recibido: 26/05/11; aceptado:14/09/11

Introduction. It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies.

Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies.

Materials and methods. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced

Results. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia).

Conclusion. The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

Key words: Helicobacter pylori, gastric neoplasms, virulence factors, Cuba.

Patrón de los motivos EPIYA de cepas cubanas de Helicobacter pylori positivas para CagA

Introducción. Se sabe que el polimorfismo en la región C-terminal de la citotoxina asociada al gen A (CagA) influye en el desarrollo de la enfermedad gástrica durante la infección por Helicobacter pylori.

Objetivo. Determinar el número y el tipo de patrones de fosforilación de CagA (patrón EPIYA) en aislamientos cubanos de H. pylori, y estudiar su asociación con las enfermedades gástricas.

Materiales y métodos. Se empleó el ADN de 95 cepas de H. pylori positivas paraCagA, para amplificar la región 3´ variable del gen cagA por PCR, mediante el empleo de diferentes estrategias. Además, se diseñaron nuevos cebadores para clasificar por PCR los aislamientos según el tipo de CagA, occidental o del este asiático. Los productos de PCR obtenidos de 14 aislamientos representativos se purificaron y secuenciaron para confirmar los resultados de la PCR.

Resultados. La distribución de los patrones EPIYA encontrada, fue: 2 AB (2,1 %), 1 AC (1,1 %), 1 BC (1,1 %), 70 ABC (73,6 %), 19 ABCC (20 %), y 2 ABCCC (2,1 %). El análisis de la secuenciación confirmó las clasificaciones hechas por PCR en las 14 cepas estudiadas y demostró tres cepas con secuencias únicas de nucleótídos, no reportadas anteriormente. La distribución del patrón EPIYA-ABC fue equivalente en todas las enfermedades encontradas: 78,9 % en úlcera gástrica, 72,5 % en úlcera duodenal y 72,2 % en dispepsia no ulcerada.

Conclusión. La mayoría de los aislamientos cubanos presentaron las combinaciones de motivos EPIYA menos virulentas (ABC). Los resultados del empleo de los nuevos cebadores y el análisis de la secuenciación, confirmaron que todas las cepas estudiadas portaban el gen cagA de tipo occidental. Ninguno de los patrones específicos de EPIYA se asoció con úlcera péptica. Este es el primer reporte que muestra la distribución de los motivos EPIYA en los aislamientos de H. pylori de la región del Caribe.

Palabras clave: Helicobacter pylori, neoplasias gástricas, factores de virulencia, Cuba.

H. pylori strains producing the cytotoxin associated gene A protein (CagA) have been related with the development of severe gastroduodenal diseases, atrophic gastritis and gastric adenocarcinoma (1,2). Gastric carcinoma (GC) is one of the leading causes of cancer-related deaths in many parts of the world (3). The CagA protein is delivered into the epithelial cells for a Type IV Secretion System(4) and it is phosphorylated by host kinases on the tyrosine phosphorylation motifs (TPMs) called EPIYA (Glu-Pro-Ile-Tyr-Ala), located in the carboxyl-terminal region (5,6). Upon phosphorylation, CagA interacts with the phosphatase SHP-2 (7), activating the Erk MAP kinase cascade eliciting a cell-morphological transformation, which is characterized by a dramatic cytoskeletal rearrangements of epithelial cells (1,8). Additionally, phosphorylated CagA can stimulate multiple motility-inducing signaling cascades such as the activation of cAbl (9) and Crkll cascades(10). Therefore, deregulation of SHP-2 by CagA plays an important role in gastric carcinoma development (1). On the other hand, CagA also disturbs cell functions in a tyrosine phosphorylation-independent manner, activating a nuclear factor that induces transcription of several inflammatory genes (11). Furthermore, CagA is also capable of activating NF-kb, which in turn induces interleukin-8 expression(12) and more recently, it has been reported that this bacterial protein deregulate the b-catenin signaling (13).

CagA TPMs are classified as type A, B, C and D based on specific amino acid sequences surrounding the EPIYA motif (7). The H. pylori strains isolated from Western countries mainly consist of EPIYA-A, B, and a single or multiple repeat of C segment, while East Asian strains (Eastern strains) mainly have a combination of EPIYA-A, B and D motifs (7). SHP-2 binds both EPIYA-C and D motifs, but EPIYA-D exhibits greater SHP-2 binding activity and therefore a stronger ability to induce severe changes in epithelial cells (7,14). Consequently, the predominant presence of strains carrying an EPIYA-ABD type has been associated with the higher GC rate in East Asia (1,15,16). Furthermore, Western strains harboring multiple EPIYA-C motifs have been more frequently isolated from patients with gastric adenocarcinoma (17,18). The above observations support the hypothesis that specific TPM patterns could determine the carcinogenic potential of H. pylori isolates, and partly explaining the geographic differences in GC incidence (1). In fact, very recently CagA have been recognized as the first bacterial oncoprotein which acts in mammalian cells (19) and its carcinogenic activity is modulated by specific TPM patterns (20). Therefore, accurate detection of the EPIYA pattern may become a useful prognostic tool for gastric malignancy due to H. pylori infection. Despite the relevance of TPM patterns in CagA protein as virulence markers, there are not reports about the EPIYA types among Cuban H. pylori isolates, or from strains isolated in the Caribbean region, despite that gastric cancer death rate varies significantly between countries in this region (21). In this disease, the highest mortality rates are exhibited by the Haitian males and Jamaican females with 25.1/100 000 and 11.6/100 000, respectively, while 9.7 and 5.6/100 000 are the lowest rates in the area, reported for Cuban males and females, respectively (21, http://www.sld.cu/servicios/estadisticas/). Therefore, the aim of this work was to study the EPIYA motif pattern of CagA gene in H. pylori isolates from a Caribbean population.

Materials and methods

Patients and H. pylori isolates

A total of 95 cagA positive H. pylori strains were isolated from antrum biopsies in a previous study involving 130 consecutive patients (77 male and 53 female) with a mean age of 49.1 (range 18 to 88) who underwent routine endoscopy due to dyspeptic complaints at CIMEQ Hospital, Havana, Cuba (22). Patients were classified in three groups (40 with duodenal ulcer, 19 with gastric ulcer, and 36 with non-ulcer dyspepsia) according to endoscopic and histological observations. H. pylori strains J99 and CCUG-17874 were used as reference strains, and kindly provided by Professor Francis Megraud from Pellegrin Hospital, Bordeaux, France and Professor Ann-Mary Svennerholm, Gothenburg University, Sweden, respectively. All the strains were cultured as previously described (22).

DNA extraction and PCR amplification

Genomic DNAs of the strains were extracted by CTAB method with phenol/chloroform and isopropanol precipitation as previously described (22). Purified DNAs were stored at -20ºC until use. PCR amplifications were carried out in a 25 µl reaction mixture containing 2.5 µl 10X PCR buffer, 0.2 mM of each deoxynucleotide triphosphate, 0.6 mM sense and antisense primers, 4 mM magnesium chloride, 1.25 U Taq DNA polymerase (CIGB, Cuba) and 100 ng genomic DNA. The PCR had an initial step at 94ºC for 1 min, followed by 40 cycles at 94ºC for 1 min, 60ºC for 1 min and 72ºC for 1 min, and a final extension at 72ºC for 5 min, using a Master Cycler apparatus (Eppendorf, Germany). Several primers were used for subtyping cagA variable region (table 1). PCR products were separated on 1.6 % agarose gel electrophoresis containing ethidium bromide. Images were digitalized using the Gene Genius system (Syngene, England).

Sequence analysis of the 3´ region of cagA gene

DNAs amplified with primers cag2 and cag4 (table 1) from 14 representative strains of different EPIYA patterns were isolated by using the ADN-GFXTM purification kit (Amersham-Pharmacia, Germany) and sent for sequence analysis to Macrogen Inc., Korea. Sequencing reactions were performed for both DNA strands, and the ClustalW2 multiple-sequence alignment software was used to analyse sequencing results.

Specific subtyping of cagA gene into EPIYA-C or D

Two new reverse primers were synthesized, cagA-EpiC and cagA-EpiD (table 1), to differentiate EPIYA motifs C and D by PCR. These reverse primers were used with forward primer cag2 (Table 1) and were designed using cagA gene sequences from Western and East Asian isolates published in Genbank Database and employing the Genrunner Software. Professor Guillermo Pérez-Pérez, from New York University, USA, kindly provided us with four DNAs from Eastern CagA isolates which were used as controls in our experiments.

Accession numbers

The nucleotide sequences of the cagA variable region from H. pylori strains in this study have been published in the GenBank database under accession numbers EU443785 (strain Hpcnic-35) and EU444054 to EU444066 (strains Hpcnic-22, Hpcnic-47, Hpcnic-74, Hpcnic-8, Hpcnic-10, Hpcnic-14, Hpcnic-15, Hpcnic-16, Hpcnic-17, Hpcnic-27, Hpcnic-19, Hpcnic-28 and Hpcnic-3).

Statistical analysis

Differences between groups were tested by c2 test. P values < 0.05 were considered to be significant. The Statistic software, version 8 for Windows, was used for statistical analysis.

Results

Determination of EPIYA pattern by PCR

Detection of the EPIYA pattern was made first by PCR amplification of 3¢ variable region of cagA gene, by using primers originally designed to detect cagA gene (primers cag2 and cag4, table 1). The strategy renders an amplicon of varying sizes according to the number of EPIYA motifs (24). These PCR reactions generated DNA fragments of 400 to 750 bp that were visualized on agarose gel electrophoresis (figure 1). DNAs from the reference strains J99 and CCUG-17874 exhibited their expected sizes of the cagA gene fragment (figure 1). The appearance of a double band in the electrophoresis of PCR products may reveal the presence of a co-infection or microevolution of a single strain in a given patient. There were only three strains in which the presence of strains with more than one EPIYA pattern was confirmed (figure 1), and all of them carried a mixed BC-ABC EPIYA pattern. Although, the above PCR methods allow estimating the EPIYA pattern of H. pylori strains, the exact prediction of the EPIYA motifs of a given strain cannot be established. Therefore, the elegant PCR method from Argent et al, (25) was carried outto determine the exact EPIYA type and number in our isolates (data not shown). This methodology amplifies each EPIYA sequence in three different PCRs, using the forward primer cag2 with primers cagA-P1C, cagA-P2CG and cagA-P3 to amplify EPIYA motifs A, B and C, respectively (table 1).

The EPIYA motifs in 95 Cuban H. pylori strains shown that most of isolates (73.7%) carries three TPMs with a single copy of the EPIYA-C motif (table 2). Additionally, three of our strains had an EPIYA-C that differs from the normal size of this motif, and one of them also lacks most of its EPIYA-B segment (table 2). The nucleotide sequence of these three strains (Hpcnic-27, Hpcnic-35 and Hpcnic-47) were studied in detail by sequencing analysis (figure 2). Furthermore, nineteen (20 %) Cuban strains had the EPIYA-ABCC type and only two strains (2.1%) had the ABCCC pattern (table 2).

Subtyping of cagA gene by PCR

Despite of the capacity of the above mentioned PCRs to determine the EPIYA pattern of H. pylori strains, none of them can distinguish between EPIYA-C and D motifs because designed primers to amplify the C motif are unspecific, and also amplify EPIYA-D. Therefore, we designed two new reverse primers to be used with the forward primer cag2 (table 1), for the specific detection of the EPIYA-C and D motifs. The new specific primer for TPM-C anneals to nucleotide positions 2928 to 2945, in cagA gene (HP0547) from the reference strain 26695. The specific primer for EPIYA-D anneals to nucleotide positions 2890 to 2910 of cagA gene (AB090073) from the Eastern strain F-13. The generated PCR-products by the new primers for EPIYA-C and D were visualized as bands of 397 and 399 bp on agarose gel electrophoresis, respectively (figure 3, Panels A and B, respectively). In the case of Western strains containing multiple C-segments (figure 3, Panel C), there might be a 102-bp increment over the first C motif (397 bp band), resulting in a new 499-bp amplicon for CC type strains (figure 3, Panel C, line 2) and a third 602-bp amplicon for CCC type strains (figure 3, Panel C, line 3). From the 95 Cuban strains analyzed with primer cagAEpi-C (table 1), 93 carried the EPIYA-C motif and none of the 95 isolates rendered an amplicon when the cagAEpi-D primer was used. In contrast, the four DNAs from Eastern cagA isolates were positive with cagAEpi-D primer and negative with cagAEpi-C primer (figure 3). The two Cuban strains that lack the TPM-C were also C negative by the new specific primer to the EPIYA-C motif (data not shown) and one of them (Hpcnic-74) was confirmed by sequence analysis (figure 2).

Sequencing analysis of the cagA gene variable region

Nucleotide sequencing reactions were performed in the 3¢ variable region of cagA gene from 14 representative strains using the cag2 and cag4 primers (table 1) to amplify this region. The PCR predictions perfectly matched the EPIYA pattern in the fourteen strains. In figure 2, we showed the variability found in the deduced peptide sequences of 9 studied strains. Three strains showing an EPIYA-C of different sizes in PCR analyses (table 2) were sequenced twice, in order to be sure of the results. The strain Hpcnic-47 had a deletion of 15 aa just after the EPIYA-C motif (figure 2) and the strain Hpcnic-27 had an insertion of 22 aa between the second and third EPIYA repeats. Furthermore, the strain Hpcnic-35 showed almost full deletion of the EPIYA-B sequence and a 21 aa deletion of EPIYA-C motif, coding for an unusually short cagA variableregion. These specific mutations in the EPIYA-C fragment had not been reported before in Database, and reflect the high intragenic recombination in the cagA variable region.

The 95 H. pylori strains were isolated from 19 cases of gastric ulcer, 40 from duodenal ulcer and 36 from non-ulcer dyspepsia, and the EPIYA-ABC motifs were detected in 78.9 %, 72.5 % and 72.2 %, respectively (Table 2). Thus, there was not a specific EPIYA motifs associated with peptic ulcer. Furthermore, no correlation was observed between the type and number of the EPIYA motifs and any other gastroduodenal diseases (table 2).

Discussion

Characterization of the EPIYA motif types seems to be a better virulence marker than detection of cagA gene prevalence alone to predict the pathogenic potential of H. pylori (1). The prevalence of CagA protein and its corresponding gene in Cuban H. pylori strains has already been detected (22,26). However, the EPIYA motif patterns of Cuban H. pylori isolates has never been investigated before.

PCR methodology is simpler and cheaper than sequencing analysis to determine EPIYA combinations in H. pylori strains. There are several set of primers that hybridized into cagA gene around the 3´ variable region and also generates a single band (27). Thus PCR methodologies, the reported previously (27) and the developed with primers cag2 and cag4 (25) have the same usefulness. Also, both PCR strategies could detect the presence of strains with more than one EPIYA pattern in the same patient.

Most of analyzed Cuban H. pylori isolates (73.7%) carries a cagA gene with the common EPIYA- ABC pattern (table 2), that resembles the CagA type reported in various Western populations from Europe, Africa and America (17,18,28,29). The percentage of ABC type strains in our case is slightly higher than those above mentioned, suggesting a possible lesser degree of virulence among Cuban CagA positive strains. Further studies, including H. pylori strains from representative areas of our country are required to confirm this tendency.

The prevalence of multiple EPIYA-C motif in Cuban strains, was very low. This isolates have been reported to induce in vitro higher levels of tyrosine phosphorylation, SHP-2 binding activity and morphological damage to cells. Thus, CagA protein with a greater number of EPIYA-C repeats is considered to be pathophysiologically more virulent and carcinogenic (17,18,29). There are only a few studies in Latin-America that have reported EPIYA patterns of cagA-positive H. pylori isolates. Most of the strains from Latin-America also had the EPIYA-ABC pattern reported in this study. On the other hand, differences in the prevalence of strains with multiple EPIYA-C have been reported in Costa Rica 10/33 (30.3 %) (30), Peru 3/26 (11.5 %) (28) and Mexico 6/30 (20 %) (31). While, three different studies from Colombia reported more than 31 % multiple C strains (32-34). It is important to mentioned that Costa Rica and Colombia have higher prevalence of gastric cancer than the other countries.

The newly designed primers (cagAEpi-C and cagAEpi-C) allow determining by PCR the EPIYA status of cagA positive H. pylori isolates. The results obtained let all Cuban strains to be classified according to the Western CagA status and provide evidence that is possible to classify H. pylori strains in Western or Eastern CagA types by a simple PCR method, without perform sequencing analysis. However, both primers should be validated using further Eastern and Western H. pylori isolates. As far as we know, there are only two similar methods published to discriminate the EPIYA-C and D repeats using PCR (35,36), but the designed primers and methodology here are different from those already published. Our PCR strategy could be use in combination with the previous ones, since it has been reported the usefulness of different set of primers in the amplification of polymorphic genes in H. pylori, especially in studies from different populations (36,37).

The efficacy of PCR methods from this study was confirmed by sequencing analysis; we found a great correlation in the fourteen analyzed isolates between sequencing and PCR results. Three isolates showed variation in the EPIYA-C motif, reinforcing that this region at cagA gene is highly polymorphic. Previous studies have reported differences in the response induced in gastric epithelial cells, depending on the number and type of EPIYA motif, being EPIYA-C the most relevant TPM (7). Moreover, it has been reported that heterogenicity in this variable region, such as lack of EPIYA-B motif (31) or changes in the multimerization sequence (10) could explain the diversity in the response induced in gastric epithelial cells by infection with H. pylori strains. Taking these observations into account, it would be very interesting to study how the polymorphism found in our isolates influences the biological activity of CagA.

In our study there was no significant difference between the number of EPIYA motifs in H. pylori from patients with different disease. These findings are in agreement with several reports that only found association to gastric adenocarcinoma and not to ulcer diseases (17,18). Unfortunately, we could not study a relationship of EPIYA-C repeats to precancerous lesions or GC, since we have no patients having those pathologies. This fact, must be influenced by the low gastric cancer death rate (6.9 / 100 000 in 2009, http://www.sld.cu/servicios/estadisticas/) in Cuban population. We hypothesize that the absence and relatively low percentage of H. pylori isolates carrying the more carcinogenic D or multiple C EPIYA motifs could be the main factor that influences the low rate of GC in our country. The absence of East-Asian CagA type in our study was an expected result, since this type is almost restricted to East Asian patients (38-41). EPIYA-D motif represents the CagA genotype with the highest association to premalignant lesions and GC development in infected persons (15,16). The small percentage of multiple EPIYA-C strains found here also indicates a lower risk for carcinogenicity among our isolates. In fact, studies from Costa Rica and Colombia showed higher percentages of multiple C isolates (30,32-34) as mentioned above, particularly in populations having a gastric cancer death rate over 20 / 100 000 (42); whereas Mexico, with a similar multiple C percentages as Cuba (31), have GC rates below 10 / 100 000 (42). However, two other molecular-epidemiological studies have found that the number of EPIYA-C motif was essentially equal in areas showing divergent incidence of GC. In the first study, strains from Nashville, EUA (low GC zone) and Colombia (high GC zone), no differences in proportion of multiple C motifs was found, indicating that other factors may influence differences in gastric cancer rates in these regions (33). Similar results were reported by Sicinschi et al., who found that the distribution of strains with one, two or three EPIYA-C motifs was not significantly different in two areas from Colombia with different gastric cancer levels (34). Due to the small number of strains characterized in the previous reports and that both reports came from the same research group, new molecular-epidemiological studies are needed to determine whether geographic differences in premalignant lesions and GC levels are influenced by the number of EPIYA-C motifs carried by infecting H. pylori strains.

In conclusion, the EPIYA motif pattern of Cuban H. pylori isolates is reported for the first time. All Cuban isolates characterized harbor the Western cagA gene type, with a predominant pattern of EPIYA-ABC and a relatively low incidence of multiple C motifs among the strains. These results assume that Cuban H. pylori circulating strains could have low carcinogenic potential, a finding that requires further studies. The new specific primers designed in this study are capable of classifying H. pylori cagA positive strains into Western or Eastern type. Additionally, mutations not reported before were found in the EPIYA-C motif of the cagA gene.

Conflicts of interest

The authors have declared no conflicts of interest.

Financing

This work was supported by a grant from the National Center for Scientific Research, Havana, Cuba.

Corresponding author: Boris Luis Rodríguez, Development and Research Department, Division of Equipments and Diagnostics, National Centre for Scientific Research, Avenida 25 and 158, Cubanacán, Playa, AP 6412, Ciudad de La Habana, Cuba. Teléfono: (537) 208-0959; fax: (537) 208-7538 boris.rodriguez@cnic.edu.cuboris.rodriguez@cnic.edu.cu

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