More than 50% of the world population is infected with Helicobacter pylori. The bacterium is highly linked to peptic ulcer diseases (PUD). At least 10% of infected individuals develop PUD, and 1% - 3% develop gastric cancer [1]. The gastric cancer risk in H. pylori infected people is 2 to 7 times that of the uninfected. Ninety percent of distal gastric cancers are now considered to be the consequence of H. pylori infection. The WHO classified H. pylori as a group I carcinogen in 1994 (ref IARC) which they reconfirmed later [2].

H. pylori exerts its pathogenicity through/via several virulence factors, some of which influence colonization and the severity of the disease. One of the best characterized virulence factors is the CagA protein encoded by the cagA gene present in the cag pathogenicity island (cagPAI). The cagPAI also encodes a type IV secretion system (T4SS), representing a needle-like pilus, which is induced upon contact with host cells [3]. CagA is translocated by this T4SS across both the bacterial and host cell membranes into the cytoplasm of target cells. CagA represents a prime example of a tyrosine-phosphorylatable bacterial virulence factor [4] [5]. Upon delivery, members of the c-Src [3] and c-Abl host tyrosine kinase families were identified as having phosphorylated CagA. Mass spectrometry and site-directed mutagenesis of CagA identified a set of Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat motifs as phosphorylation sites [5] [6].

CagA positive H. pylori strains are associated with increased inflammation and increased risk of PUD and gastric carcinoma in humans and experimental animals [7]. The presence of the protein CagA generally coincides with the presence of other virulence factors, including VacA, BabA and OipA [8]. Thus, the pathogenesis of H. pylori is multifactorial and cannot be reduced to a gene. The CagA protein is responsible for alterations of many cell signaling systems which profoundly influence the physiology of the host cell. When H. pylori is in contact with the host cells, the CagA protein is directly injected into the cytoplasm of the host cell where it is phosphorylated and binds to the host’s SHP2 domain [9]. SHP-2 is a phosphatase involved in signal transduction for the tyrosine kinase receptor [10]. CagA also causes the passage/transformation/evolution of an epithelial cell to a mesenchymal cell phenotype [11]. All of these phenotypes are associated with gastric carcinogenesis [12]. The vacuolating toxin VacA has been named for its ability to induce many large vacuoles in cultured cells. Unlike CagA, the VacA protein forms an autotransporter structure and secretes itself without the need for contact with the host cell. VacA proteins then oligomerize to form pore-like structures. VacA is transported to the receptor tyrosine phosphatase (RPTPα and RPTPβ) and other transmembrane glycosylated proteins on the surface of the host cell [13]. VacA then enters by endocytosis and forms selective anion channels in the vacuole membrane. The channels allow the accumulation of chloride anions and weak bases, resulting in osmotic swelling [14]. VacA also inserts into mitochondrial membranes, causing mitochondrial dysfunction and apoptotic cell death [15]. Vacuolization is not the only effect of VacA intoxication. VacA disrupts the barrier function of epithelial cells, allowing leakage of essential nutrients such as iron, nickel, and amino acids. All H. pylori strains contain vacA genes, but not all strains produce a functional VacA protein. This is due to polymorphisms in the vacA gene, particularly at the amino-terminal (s region), in the middle of the gene (m region), and in the intermediate region (region i). The s2 polymorphism gives/results in an inactive toxin [16]. Thus, strains with the s2 allele are often called “VacA negative”. Polymorphisms have been discovered more recently and influence vacuolating activity; vacA containing the allele i1 produces the most active toxin. Strains harboring the s1m1 allele have been most commonly associated with PUD and gastric carcinomas, but it now appears that the i1 allele is more strongly associated with these diseases than the presence of the s1m1 genotype [16].

The aim of this work was to identify cagA and vacA polymorphism genotypes corresponding to H. pylori pathogenicity factors in Brazzaville, Congo.

This study reports a high prevalence of H. pylori infected individuals in the Congo (75.5%). These results are similar to those in the study by Ankouane Andoulo et al. in Cameroun, who reported a prevalence of 72.5% [25].

Indeed, most patients harbour strains with the cagA gene (93.9%) and all cagA gene have phosphorylatable motifs with 92.2% of cagAP1, P2, P3. Some studies reported that cagA is present in approximately 70% of strains worldwide, but this rate varies geographically, from between 90% - 95% in East Asian countries (South Korea, China, Japan) to only about 40% in Western countries [26] [27]. In Africa, Kidd, Lastovica, Atherton, et al. found the presence of cagA in all South African strains [28]. Our results indicate that our patients, are at risk for severe gastroduodenal diseases due to the CagA proteins. Indeed, H pylori could directly deliver the CagA protein into the host epithelial cell cytoplasm via the cagPAI-coded type IV export system [29]. Inside the epithelial cells, the CagA protein undergoes tyrosine phosphorylation by the host Src family protein tyrosine kinases, and the CagA protein binds an Src homology 2 (SH-2) domain-containing tyrosine phosphatase SHP-2, and stimulates the division and proliferation of gastric epithelial cells [9]. The CagA-Csk interaction activates Csk and inactivates the Src family kinases, thereby bringing about a decrease in CagA tyrosine-phosphorylation as well as in CagA-SHP2 interactions as a feedback mechanism [30]. Through/Via this mechanism, chronic infection with CagA-positive strains persists, thus causing the host damage. A typical characteristic of AGS gastric epithelial cells infected with cagPAI-positive H. pylori is their “hummingbird” phenotype [4] [6]. This in vitro phenotype likely mirrors numerous in vivo signaling activities that control host cell motility, invasive growth and metastasis of cancer cells [31] [32]. Otherwise the oncogenic role of CagA is further supported by in vivo experiments in mice, where transgenic cagA expression in the stomach leads to gastric epithelial hyperplasia, adenocarcinoma, myeloid leukemia and B-cell lymphoma [33] [34].

In addition, patients with chronic H. pylori infection in Brazzaville also have the risk of developing gastritis and ulcer pathologies. Indeed, this demonstrates that the CagA protein is a multiple effector via phosphorylation independ/via T4SS to activate the NF-κB-inducing kinase (NIK) and IκB kinase α/β (IKKα/β) resulting in subunit IκBα of NF-κB (trimer IκBα/p50/p60) phosphorylation and then degradation [35]. Active NF-κB (dimer p50/p60) translocates into the nucleus to transcribe the inflammatory factor genes [cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS)], proinflammatory cytokine genes [interleukin-6 (IL-6), interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α)], and the chemokine IL-8 gene [35]. This is called the NF-κB pathway. All of these related proteins can result in severe inflammation of the gastric mucosa for infected cells [26] [35] [36] [37] (Figure 4).

The vacA gene represents another locus involved in the disease. Concerning the vacA gene polymorphisms in this study, variations of the vacA alleles (s1a, s1b, s1c, or s2), (m1 or m2), or (i1 or i2) also exist. This study noted that 81.9% of patients have the allelic combination s1m1 and all of those tested (100%) have the i1 allele. H. pylori strains with vacA alleles s1/m1/i1 are associated with an increased risk of developing severe disease, compared to positive s2/m2/i2 vacA strains [24] [38]. In fact, among the possible allelic combinations, the vacAs1/m1 alleles are the most virulent combination, while the s1/m2 and s2/m2 genotypes display virtually no cytotoxicity [39].

Together, while each of the vacA polymorphisms has been used as a predictor for VacA-induced disease severity, it is clear that other factors, including the presence of CagA (discussed below), contribute to disease. For this reason, individually typing vacA alone may not provide sufficient information to understand the virulence potential of a strain and multiple virulence factor typing appears to be required to understand strain dependent disease contributions [39].

As shown in Figure 5, VacA oligomer p88 forms anionselective channels in the cytoplasmic membrane, which can further react with early and late endosomal compartments (EE/LE) to form anion-selective channels in the vacuole membrane. Such channels increase permeability to small organic molecules and cations Fe³⁺/Ni²⁺ which can further interact with from H. pylori generating an osmotic force for the driving water influx and vesicle swelling, finally leading to vacuolation [26] [35]. On the other hand, the p88/EE/LE complex could be activated by Bax and Bak, resulting in mitochondrial transmembrane

potential (ΔΨm) disruption, followed by the release of cytochrome c from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and finally proceeding to apoptosis. However, apoptosis is inhibited by CagA [26] [35].

In our study, there was nostatistically significant difference regarding the cagA gene or vacA alleles and age group, gastritis and another pathology in the study population. These results are on the contrary of those obtained in some studies. El Khadir noted a difference in the vacA and cagA combination of H. pylori in PUD and gastric cancer cases [20] [40]. Lehours did not find a difference between gastritis and gastric MALT lymphoma patients regarding H. pylori

pathogenicity factors [20]. Our results can be explained by the fact that H. pylori is acquired early in childhood, as there is no statistically significant difference between the age groups and the strains exist in many parts of the population, even if the results of the endoscopy are normal [41].

The results of this study can explain the frequency of gastritis, PUD and gastric cancers in Brazzaville, Congo. Indeed, Ibara et al. reported 62.02% gastritis (first cause of gastric pathologies), 11.29% PUD and 3.60% gastric adenocarcinomas, despite the fact that a causal relationship with H. pylori has not been established [42]. H. pylori infection and gastric carcinogenesis processes in Congo must be fought using strategies based on the recommendation for H. pylori diagnosis and antimicrobial susceptibility testing in order to eradicate H. pylori and prevent gastritis, PUD and carcinoma.

Despite the evidence that cagA positivity, CagA and VacA seropositivity, and/or vacA polymorphism contribute to disease severity, numerous studies have not found this association [26] [43].

For example, in Tunisia, the vacA type is significantly different between patients with peptic ulceration and gastritis, while CagA status is not [26] [44].

In China, no association between cagA status and peptic ulceration orchronic gastritis was established, due likely to the high presence of CagA in both patient populations. The differences observed between disease severity and toxin type/presence in these epidemiological studies may be due to differences that exist in H. pylori strains well beyond the described cagA and vacA polymorphisms. As noted at the outset of this review, environmental, geographic, and host influences could contribute to the differences observed in disease severity between these studies. As such, while individual evaluation of cagA and vacA genotypes show that both contribute to disease, the lack of evaluation of both genotypes in combination with other factors is problematic in determining how both toxins contribute to disease [26].

The presence of genes associated with severe gastric diseases indicates the importance of H. pylori eradication in the prevention of these diseases in Congo.

The authors declare no conflicts of interest regarding the publication of this paper.

Ontsira Ngoyi, E.N., Guilloteau, C., Benejat, L., Mongo Onkouo, A., Buissonnière, A., Sifre, E., Aloumba, A., Mieret, T., Bossali, F., Yala, F., Abena, A.A., Vadivelu, J., Goh, K.L., Ibara, J.R., AtipoIbara, B.I., Menard, A., Lehours, P. and Megraud, F. (2019) cagA and vacA Helicobacter pylori Pathogenicity Factors in Brazzaville, Congo. Open Journal of Medical Microbiology, 9, 186-200. https://doi.org/10.4236/ojmm.2019.94018