Dissemination of cagA and cagE Virulence Genes Among H. Pylori Strains From Sudanese Patients With Gastric Discomfort

Helicobacter pylori cytotoxin-associated gene pathogenicity island (cag-PAI) is one of the strain-specic genes (they do not exist in all strains). cag-PAI is involved in inducing inammation, ulceration, and carcinogenesis. This study aimed to detect and characterize cagA and cagE virulence genes among H. pylori strains from Sudanese patients with gastric discomfort. Result Out of 288 gastric biopsies screened for the presence of H. pylori, 34% (98/288) were positive, cagA gene was present in 41% (40/98) of specimens, mostly in patients with gastritis 72.5% (29/40), followed by duodenal ulcer 15% (6/40), esophagitis 5% (2/40), and 7.5% (3/40) in patients diagnosed normal by endoscopy. cagE gene was present in 39% (38/98) of specimens, the majority 73.7% (28/38) were from patients with gastritis, 10.5% (4/38) duodenal ulcer, 5.3% (2/38) esophagitis, 2.6% (1/38) gastric ulcer, and 7.9% (3/38) were diagnosed as normal. The cagA and cagE protein sequences have synonymous amino acid variations.


Introduction
Helicobacter pylori (H. pylori) is a gram-negative bacterium, grows in a microaerophilic environment at 37 ∘ C. It is one of the causative agents of gastric diseases [1], like chronic gastritis, ulcers (duodenal or gastric), gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma [2].
Previous studies showed that soil, water, animal manure, and human stool are the primary resources for H. pylori [3]. There is an opportunity for transmission from person to person [4]. Some factors affect the incidence of H. pylori infection; race, gender, social and socioeconomic status of the population [5].
The pathogenicity of H. pylori infection is mediated by environmental, bacterial virulence factors, and internal interaction with the host [6].
The virulence genes of H. pylori are classi ed into 3 categories; strain-speci c genes, phase variable genes, and genes with variable genotypes or structures depending on the strain [7]. Cytotoxin-associated gene pathogenicity island (cag-PAI) and plasticity island genes are strain-speci c genes, and they do not exist in all strains [7].
The cag-PAI is a DNA fragment that contains 31 genes, including; cagA and cagE in addition to other genes [8]. cag-PAI genes involved in inducing in ammation, ulceration, and carcinogenesis [3]. The presence of cag-PAI is strongly associated with the outcomes of H. pylori infection [9].
The cagA protein behaves as a bacterial oncoprotein, affects the expression of vital proteins or its function in oncogenic or tumor suppressor signaling pathways [10].
The cagE gene encodes a secretory protein that induces secretion of IL-8 from infected epithelial cells; also this gene is important for translocation and phosphorylation of the cagA protein, some studies have suggested that it is a more accurate marker of an intact pathogenicity island and can be used with cagA as a marker for cag-PAI [11,12,13].
To date there are limited published studies about the detection and characterization of cagA and cagE genes of H. pylori in Sudan, this information is of great importance in determining the virulence and clinical outcomes of H. pylori strains. Thus, this study was conducted to detect and characterize cagA and cagE genes of H. pylori strains from gastric biopsies of patients with gastric discomfort in Khartoum State.

Materials And Methods
This was a descriptive cross-sectional study conducted during the period from August 2018 to October 2019. The sampling technique was convenience (Non-probability).

Collection of clinical specimens
A total of 288 gastric biopsies were collected by gastric endoscope by the gastroenterologist in different Khartoum hospitals: Omdurman Medical Military Hospital, Police Hospital, Al-Buqa'a Specialized Hospital, Ibn Sina Hospital, and Fedail Hospital. The specimens were collected from patients suffering from gastric discomfort and indicated for endoscopy, in which both antrum and corpus were sampled.
The specimens were collected using endoscopes, using 1.0 ml brain heart infusion broth with 15% glycerol as transport media.
DNA extraction and polymerase chain reaction (PCR) DNA was extracted using the guanidine chloride extraction method [14]. The extracted DNA was stored at -70˚C until use.
The PCR was performed using a thermocycler (Convergys, Germany) and speci c primer (Table 1) according to the following protocols: For glmM gene: initial activation at 94°C for 3 minutes, followed by 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30seconds, then a nal extension at 72°C for 3 minutes [15].
For 16S rRNA gene: initial activation at 94°C for 3 minutes, followed by 35 cycles at 94°C for 30 seconds, 53°C for 30 seconds, and 72°C for 45 seconds, then a nal extension at 72°C for 5 minutes [16].
For cagA gene: initial activation at 94°C for 3 minutes, followed by 35 cycles at 94°C for 30 seconds, 53°C for 30 seconds, and 72°C for 45 seconds, then a nal extension at 72°C for 5 minutes [15].
For cagE genes: initial activation at 94°C for 3 minutes, followed by 35 cycles at 94°C for 30 seconds, 53°C for 30 seconds, and 72°C for 45 seconds, then a nal extension at 72°C for 5 minutes [17].
There was no statistically signi cant association between the presence of cagA and cagE virulence genes and clinical diagnosis (p-value= 0.305).

DNA sequencing
The comparison of 5 cagA protein sequences with the JF798705.1 reference from GenBank revealed substitution of proline (P) with alanine (A), with 100% identity to MK074991.1 reference sequence ( Figure   3).
The comparison of 21 cagE protein sequences with the AY153111.1 reference sequence from GenBank, revealed a substitution mutation in one sequence, the sequence has valine (V) instead of isoleucine (I), with 100% identity to AB191082.1 reference sequence ( Figure S1 and Figure S2).

Discussion
In the present study, out of 288 gastric biopsies, 98 (34%) were positive for H. pylori, using PCR targeting 16S rRNA and glmM genes. These genes are more sensitive than other genes in detecting H. pylori infection from gastric biopsies [18]. So, we have used them for the detection of H. pylori in biopsy samples.
H. pylori infection prevalence was 79.1% in Africa, 54.7% in Asia, and 37.1% in Northern America [19]. The geographic variation in the prevalence may be due to the uneven distribution of H. pylori in the stomach in the different clinical settings, gender, race, social and socioeconomic status of the population [5,20].
In Sudan, there is scanty information about the prevalence of H. pylori infection [14]. The prevalence in this study may indicate a low rate of H. pylori infection. Most of the previous studies investigating H. pylori seroprevalence using tests for the detection of H. pylori IgM and IgG antibodies. They reported a seroprevalence ranging from 20% up to 70% [21]. While PCR was used in this study which has signi cantly higher accuracy than stool antigen test and antibody-based tests used in the mentioned Sudanese studies.
In this study 50/98 (51%) of the infected patients were females, this may attributed to the possibility of transmission from animal and their products, as women may deal with foods with animal origins when preparing food. This result in contrasts with other reports, noting a high existence of infection among males [22]. The majority of the infected patients were adults 96/98 (98%), the infected adolescents were 2/98 (2%), this result is in harmony with El-Shenawy [23] and Salih [24] studies, they mentioned that; the prevalence of H. pylori infection increases with age.
In the present study, the cagA gene was more frequent than cagE, and this result in agreement with Lima [28] study in which 28% of specimens were positive for both genes (cagA and cagE), which might be attributed to the importance of cagE for cagA translocation, as mention in Yong [10] study.
There was no statistically signi cant association between the presence of these virulence genes and the clinical outcomes in this study. This result is similar to Moaddeb [29] study, which found that; there is no signi cant association between cagA-positive strains and gastroduodenal diseases. In contrast with Yong [10] and Skoog [9] studies, they mentioned that the presence of cag-PAI is strongly associated with the outcomes of H. pylori infection.
The differences in H. pylori virulence may be attributed to geographic variations in the strain-speci c (non-conserved) genes and reduced virulence of African H. pylori strains, as in Bullock [30] study, who also mentioned that cagA proteins produced by East Asian strains cause more extensive alterations in gastric epithelial cells than that produced by strains from other parts of the world.
The analysis of 5 cagA protein sequences in this study revealed a substitution mutation in two cagA protein sequences. This variation may be due to the high level of geographic variability in H. pylori protein sequences, which may lead to differences in H. pylori virulence.  Figure 1 Ampli ed DNA of the glmM gene (A) (Lanes 7, 8& 9 contain gene does not included in this manuscript) and 16S rRNA gene (B) on 1.5% agarose gel electrophoresis.

Figure 2
Ampli ed DNA of the cagA gene (A) and cagE gene (B) on 1.5% agarose gel electrophoresis.

Supplementary Files
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