vacA genotypes and EPIYA motifs of Helicobacter pylori in patients with atrophic and non-atrophic gastritis

Background Helicobacter pylori is the main microorganism causing gastrointestinal diseases, such as chronic gastritis, peptic ulcer, MALT lymphoma, among others. The presence of the s1/m1 genotype of the vacA gene and EPIYA phosphorylation motifs of the cagA gene have been linked to the production of prolonged gastric inflammation. This study determines the presence of these virulence genotypes and their relationship with atrophic gastritis. Methods We included 231 patients with a history of dyspepsia undergoing upper gastrointestinal endoscopy. Samples of gastric tissue were taken to establish, through molecular techniques, the presence of H. pylori by amplifying the ureA and flaA2 housekeeping genes; in addition, the alleles of signal (s) and of the middle region (m) present in the vacA gene were amplified; and by sequencing the repeating patterns of the tyrosine phosphorylation motifs within the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs of the cagA gene were also amplified. A chi-square test was performed in order to establish the relationship between the virulence genes and the degrees of gastric injury. The histopathological study identified 113 patients (48.9%) with non-atrophic gastritis (NAG) and 118 patients with atrophic gastritis (AG); of 53 presented multifocal gastric atrophy (MGA) and 65 with intestinal metaplasia (IM). This study suggests that the ABCC genotype, increases the virulence potential of H. pylori, suggesting that the coexistence of these genes increases the severity of inflammation progression, which leads to pre-cancerous lesions such as intestinal metaplasia, which is a point of no return in carcinogenic progression. The findings from the Ecuadorian population present the first studies of the presence of virulent genotypes of H. pylori, evidencing a statistically significant relationship, p=0.02 , between the presence of the cagA and vacA genes, suggesting that their virulence mechanisms do not act independently.

virulence potential of the microorganism, suggesting that the coexistence of these genes could result in an increase in the severity of the progression of inflammation that leads to precancerous lesions.

Introduction
Helicobacter pylori is a Gram negative microaerophilic bacillus existing in between 35 and 70% of the population, depending on the geographical area (1); its ability to colonize in the acid environment of the gastrointestinal epithelium has allowed it to associate with different gastric and duodenal pathologies (2) that can range from gastric or duodenal peptic ulcer, to more severe cases where the infection contributes to the development of dysplasia, adenocarcinoma and lymphoma of lymphoid tissue associated with MALT mucosa (3).
Atrophic gastritis (AG) is a pre-neoplastic pathology; it usually starts with multifocal gastric atrophy (MGA), followed by type I or complete intestinal metaplasia, type II or incomplete intestinal metaplasia, dysplasia, and finally, the development of carcinoma (4), with a relative risk between 2.7 and 7 if they are associated with infection by H. pylori (5) .
VacA vacuolizing cytotoxin, encoded by the gene of the same name -vacA, is a virulence factor present in almost all circulating strains of H. pylori (6), related to induced apoptosis, immunomodulation and vacuolization in gastric epithelial cells (7,8). Its toxicity is associated with variation in its variable regions of signal (s-region), with its two variations, s1 and s2, in the middle one (m-region) with its variations, m1 and m2; and in the intermediate one (i-region) (9). The different combinations between variations have shown that the s1/m1 genetic variant is associated with diseases such as peptic ulcer; in addition, the prolonged inflammation produced by this cytotoxin leads to the development of gastric atrophy (10,11).
The strains of H. pylori that have a cagA gene, translate a protein of the same name-CagA; this is translocated inside the gastric epithelial cells thanks to a Type IV secretion system, inducing a cascade of tyrosine phosphorylation dependent on Src and Abl kinases. The carboxy-terminal region known as tyrosine phosphorylation motifs contains amino acid sequences Glu-Pro-Ile-Tyr-Ala (EPIYA).
The binding of different proteins to these EPIYA phosphorylation points allow the activation and inactivation of different signaling pathways that promote dynamic reorganization of the cytoskeleton and the activation of signaling pathways for cell proliferation (14,15).
To this date, four types of EPIYA motifs have been described and have been named according to the sequence of amino acids close to the motif: EPIYA-A, EPIYA-B, EPIYA-C and EPIYA-D (16). These four motifs can be combined and, depending on these combinations, their virulent potential is known; the strains of H. pylori isolated in Western countries consist mainly of EPIYA-A, B, and one or more repetitions of segment C; whereas the East Asian strains mainly have a combination of EPIYA-A, B and D motifs (17,18). In the West, strains with multiple repetitions of the EPIYA-C motif have been associated with the formation of gastric adenocarcinoma.
In Ecuador, despite the fact that gastric cancer is the second in lethality in men and the third in women, information about factors that predispose the development of this pathology is scarce. The evidence presented that associates H. pylori as a carcinogenic agent (19)  Histopathological diagnosis A biopsy sample was submitted to the Hospital's Pathology laboratory. Histological characteristics were determined by using the Sydney criteria (20).

DNA Extraction
The second biopsy was transported in saline at a temperature of 2-8°C (36-46°F) in order to be processed in the laboratory. DNA extraction began using about 14 mg of tissue. The QIAamp DNA Mini® kit was used, following the manufacturer's instructions. The sample was previously fragmented to obtain better results. The DNA concentration was determined by spectrophotometry using a NanoDrop® (Thermo Scientific). The presence of H. pylori was detected in 39.4% (n = 91/231) samples, of which 50 corresponded to AG, and 41 to NAG. Figure 1A. The vacA gene was identified in 72.5% (66/91) of all biopsies for positive H. pylori; of these, 29 were related to NAG and 37 to AG, with a p-value>0.05. The s1/m1 genotype ( Figures 1C and 1D) was more prevalent in AG, 17 cases in MGA and 14 in IM.
The cagA gene was detected in 62.6% (57/91) of all samples ( Figure 1B). The EPIYA motifs were found in patients with both AG and NAG, the most frequent being the ABC motif with 54.4% (31/57) - Figure   2A (Table 2). However, it could not be concluded that there is a significant statistical relationship between the EPIYA motifs and the severity of gastric injury. It was observed a variation of A by T in the EPIYT-B motif (44%) - Figure   2F.
The combination of genotypes was found more frequent for cagA+vacA+ s1/m1+/EPIYA-ABC with

Discussion
Although the association between H. pylori and diseases such as chronic atrophic gastritis, peptic ulcer, or gastric cancer are not completely clear, there is strong evidence that the presence of the vacA and cagA virulence genes or its variants increase this relationship (25).
The general prevalence of H. pylori was of 39.4% in dyspeptic patients diagnosed with AG and NAG.
Similar prevalence have been reported in Mexico (26) with 30.5%; and Brazil (2) with 57%. In Ecuador (Reyes et al.), with a similar population, 45% was reported by using conventional culture techniques to identify them (27). Although there are reports that in Latin America the prevalence is higher (70 and 80%) (28) due to sociodemographic factors. Burucoa et al suggests that the current prevalence has decreased due to health improvements (29).
The association between AG and the development of gastric cancer has been widely described (30).
About two thirds of patients with AG have infection due to H. pylori; in many cases its progression towards a carcinogenic disease is irreversible (31). In our study it was found that H. pylori was present in 42.4% (50/118) of patients with atrophic gastritis; results similar to those reported by Vilar and Silva who reported 50.5% (32). In Asian countries such as Japan, this relationship is increasing; it has been reported between 54% and 70%, possibly influenced by its high population density (33,34).
In our study, 72.5% of positive samples for H. pylori presented the vacA gene; of these, 81.8% corresponded to the s1/m1 alleles, being the most frequent genotype (54/66) associated to patients with gastric atrophy and intestinal metaplasia. In order to ensure its permanence in the gastric mucosa, the genetic information contained in the vacA gene allows H. pylori to evade the immune response. Its immunomodulatory role (35) prevents the maturation of phagosomes or prevents interaction with T lymphocytes; additionally, they have direct cytopathic effects on gastric cells, inducing cytotoxic vacuolization (36) , causing gastric inflammation and consequently atrophy or intestinal metaplasia (37). However, the activation of oncogenic pathways is the most worrisome event; the evidence suggests that the presence of the s1/m1 genotype corresponds to the most virulent ones and is associated with the development of AG and later that of gastric cancer (38,39), thus becoming risk markers (6,40).
The cagA gene is one of the virulence factors with a prevalence between 60 and 70% of circulating strains (41); in our study it was 62.6%. The presence of EPIYA ABC and ABCC motifs associated with the vacA s1m1 genotype was found in patients with precancerous histopathological alterations such as multifocal gastric atrophy (14 patients) and intestinal metaplasia (11 patients). Gao suggests that these two virulence mechanisms are associated (42) and therefore their virulent genotypes too.
The EPIYA motifs activate multiple signaling pathways in the host cell, depending on phosphorylation, such as: a) the family of Src kinases (SFK) that control the processes of mobility, differentiation and cell proliferation; they are also key players in the genesis of tumors; b) the family of Abl, c-Abl and Arg kinases that can directly phosphorylate EPIYA CagA motifs in late infection (10,15,43,44). It also inhibits the pathways of the family of PAR1/MARK kinases that, together with apoptosis-stimulating protein p53-2 (ASPP2), prevents cellular apoptosis (45).
Apparently, the EPIYA motifs are the most important factors associated with the development of malignant neoplasms (16), showing that the higher the number of EPIYA-C repeats, the higher the probability of gastric atrophy, intestinal metaplasia and gastric cancer; thus, EPIYA-ABCCC has the capacity to activate in a higher proportion the growth factors, phosphorylation pathways, and inflammatory processes that contribute to intestinal metaplasia (46,49). EPIYA-A, B and D have been described with higher prevalence in Asian regions (Japan, Korea and China), while EPIYA-A, B, C and their several repetitions are in the western region (Europe, North America, Latin America and Australia) (44,47). In our study only EPIYA-C motifs were identified, corroborating the close relationship between the EPIYA motifs and the geographical area.
It is interesting to note that we identified the EPIYT-B motif, a mutation that suggests an EPIYA/EPIYT change, being the only mutation present in our study, with 44% of the analyzed samples. Zhang et al.
(48) found EPIYT-B with 32.9%, proposing that this mutation has the ability to regulate the activity of CagA, thus interfering with the signaling pathways of the host related to the sequential process of cancer; therefore, it was found less associated with gastric pathologies such as cancer.
Despite the wide variability in the distribution of H. pylori in the gastric mucosa, the use of molecular tests allows us to make a more accurate identification of the pathogenicity factors, by means of molecular sequencing techniques. Karlsson et al, for example, showed that the culture introduces a bias in the number of EPIYA motifs versus molecular determination directly done from gastric biopsy.

Conclusion
This study suggests that the sum of the pathogenicity factors such as the cagA+/vacA s1/m1+/EPIYA ABCC genotype, increases the virulence potential of H. pylori, suggesting that the coexistence of these genes increases the severity of inflammation progression, which leads to pre-cancerous lesions such as intestinal metaplasia, which is a point of no return in carcinogenic progression.