Helicobacter pylori CagA EPIYA Motif Variations Affect Metabolic Activity in B Cells

Background: Helicobacter pylori (Hp) colonizes the human stomach and can induce gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. Clinical observations suggest a role for the Hp virulence factor cytotoxin-associated gene A (CagA) in pathogenesis. The pathogenic activity of CagA is partly regulated by tyrosine phosphorylation of C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in host cells. However, CagA differs considerably in EPIYA motifs, whose functions have been well characterized in epithelial cells. Since CagA is fragmented in immune cells, different CagA variants may exhibit undetected functions in B cells. Methods: B cells were infected with Hp isolates and isogenic mutants expressing different CagA EPIYA variants. CagA translocation and tyrosine phosphorylation were investigated by Western blotting. Apoptosis was analyzed by flow cytometry and metabolic activity was detected by an MTT assay. Results: Isogenic CagA EPIYA variants are equally well translocated into B cells, followed by tyrosine phosphorylation and cleavage. B cell apoptosis was induced in a CagA-independent manner. However, variants containing at least one EPIYA-C motif affected metabolic activity independently of phosphorylation or multiplication of EPIYA-C motifs. Conclusions: The diverse structure of CagA regulates B cell physiology, whereas B cell survival is independent of CagA.

Cortactin Is Required for Efficient FAK, Src and Abl Tyrosine Kinase Activation and Phosphorylation of Helicobacter pylori CagA

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin’s phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.

Diversity of 3′ variable region of cagA gene in Helicobacter pylori strains isolated from Chinese population

Background The cytotoxin-associated gene A (cagA) is one of the most important virulence factors of Helicobacter pylori (H. pylori). There is a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal of CagA protein. This repeat region is thought to play an important role in the pathogenesis of gastrointestinal diseases. The aim of this study was to investigate the diversity of cagA 3′ variable region and the amino acid polymorphisms in the EPIYA segments of the CagA C-terminal region of H. pylori, and their association with gastroduodenal diseases. Methods A total of 515 H. pylori strains from patients in 14 different geographical regions of China were collected. The genomic DNA from each strain was extracted and the cagA 3′ variable region was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analyzed using MEGA 7.0 software. Results A total of 503 (97.7%) H. pylori strains were cagA-positive and 1,587 EPIYA motifs were identified, including 12 types of EPIYA or EPIYA-like sequences. In addition to the four reported major segments, several rare segments (e.g., B′, B″ and D′) were defined and 20 different sequence types (e.g., ABD, ABC) were found in our study. A total of 481 (95.6%) strains carried the East Asian type CagA, and the ABD subtypes were most prevalent (82.1%). Only 22 strains carried the Western type CagA, which included AC, ABC, ABCC and ABCCCC subtypes. The CagA-ABD subtype had statistical difference in different geographical regions (P = 0.006). There were seven amino acid polymorphisms in the sequences surrounding the EPIYA motifs, among which amino acids 893 and 894 had a statistical difference with gastric cancer (P = 0.004). Conclusions In this study, 503 CagA sequences were studied and analyzed in depth. In Chinese population, most H. pylori strains were of the CagA-ABD subtype and its presence was associated with gastroduodenal diseases. Amino acid polymorphisms at residues 893 and 894 flanking the EPIYA motifs had a statistically significant association with gastric cancer.

Diversity of 3’ variable region of cagA gene in Helicobacter pylori strains isolated from Chinese population

Background: The cytotoxin-associated gene A (cagA) is one of the most important virulence factors of Helicobacter pylori (H. pylori). There is a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal of CagA protein. This repeat region is thought to play an important role in the pathogenesis of gastrointestinal diseases. The aim of this study was to investigate the diversity of cagA 3’ variable region and the amino acid polymorphisms in the EPIYA segments of the CagA C-terminal region of H. pylori, and their association with gastroduodenal diseases.Methods: A total of 515 H. pylori strains from patients in 14 different geographical regions of China were collected. The genomic DNA from each strain was extracted and the cagA 3’ variable region was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analyzed using MEGA 7.0 software.Results: A total of 503 (97.7%) H. pylori strains were cagA-positive and 1,587 EPIYA motifs were identified, including 12 types of EPIYA or EPIYA-like sequences. In addition to the four reported major segments, several rare segments (e.g., B’, B’’ and D’) were defined and 20 different sequence types (e.g., ABD, ABC) were found in our study. A total of 481 (95.6%) strains carried the East Asian type CagA, and the ABD subtypes were most prevalent (82.1%). Only 22 strains carried the Western type CagA, which include AC, ABC, ABCC and ABCCCC subtypes. The CagA-ABD subtype had statistical difference in different geographic regions (P = 0.006). There are seven amino acid polymorphisms in the sequences surrounding the EPIYA motifs, among which amino acid residue 893 and 894 had a statistical difference with gastric cancer (P = 0.004).Conclusions: In this study, 503 CagA sequences was studied and analyzed in depth. In Chinese population, most H. pylori strains are of the CagA-ABD subtype and its presence was associated with gastroduodenal diseases. Amino acid polymorphisms at residue 893 and 894 flanking the EPIYA motif had a statistically significant association with gastric cancer.

Diversity of 3’ Variable Region of cagA Gene in Helicobacter Pylori Strains Isolated from Chinese Population

Background: CagA is one of the most important virulence factors of Helicobacter pylori (H. pylori). There is a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the CagA 3’ variable region. This repeat region is thought to play an important role in the pathogenesis of gastrointestinal diseases. The aim of this study was to investigate the diversity of CagA 3’ variable region and the amino acid polymorphisms in the EPIYA segments, and their association with gastroduodenal diseases.Methods: A total of 515 H. pylori isolates from patients in 14 different geographical regions of China were collected and the genomic DNA was extracted. The 3’ variable region of the cagA was amplified by polymerase chain reaction (PCR) and then followed by DNA sequencing, and the amino acid sequences were analyzed with MEGA 7.0 software.Results: A total of 503 (97.7%) H. pylori isolates were cagA-positive and 1,587 EPIYA motifs were obtained, including 12 types of EPIYA or EPIYA-like sequences. In addition to the four reported major segments, several rare segments (e.g., B’, B’’ and D’) were defined and 20 different sequence types (e.g., ABD, ABC) were found in our study. A total of 481 (95.6%) strains were East Asian type, most of them were ABD subtype (82.1%). Only 22 strains were Western type, including types AC, ABC, ABCC and ABCCCC. The CagA-ABD subtype had statistical difference in different geographic regions (P=0.006). There are seven amino acid polymorphisms in the sequences surrounding the EPIYA motifs, among which amino acid residue 893 and 894 had a statistical difference with gastric cancer (P=0.004).Conclusions: In this study, 503 CagA sequences was studied and analyzed in depth. In Chinese population, most H. pylori isolates are of the CagA-ABD subtype and its presence was associated with gastroduodenal disease. Amino acid polymorphisms at residue 893 and 894 flanking the EPIYA motif had a statistically significant association with gastric cancer.

CagA sequence differences and proteome profiles between East Asian and Western H. pylori strains

Background The cytotoxin-associated gene A protein (CagA), an effector protein of Helicobacter pylori (H. pylori), was the first identified bacterium oncoprotein. Based on its sequence characteristics, H. pylori has been classified into East Asian and Western strains. We hypothesized that the differences in structure of CagA and proteomic profiles between East Asian and Western H. pylori strains are the primary cause of the differential clinical outcomes of H. pylori infection. Results In this study, we isolated 27 H. pylori strains from gastric mucosa of Chinese patients with gastric diseases and revealed that the Western CagA has more variation in its EPIYA motifs than East Asian CagA. This result was further confirmed via analyzing the CagA sequences of 150 H. pylori strains from GenBank. More importantly, we detected the deletion or partial deletion of 13 amino acids in CagA in all East Asian strains but not in Western strains. iTRAQ -based proteomic analysis showed that the CagA protein related to cytotoxicity was highly expressed in Western strains, while urease-associated proteins UreA and UreH, flagellin related proteins FlaA, FlgE and FlhA, and cell division proteins FtsZ and FtsI were highly expressed in East Asian strains. These proteins were associated with the colonization, motility, and viability potential of H. pylori in the human stomach and were clustered into an interaction network. Conclusions This study provides significant, unreported differential sequences of CagA and proteomic profiles between East Asian and Western H. pylori, which maybe serves as promising new targets to ascertain the pathogenesis of H. pylori.

The association of Helicobacter pylori CagA EPIYA motifs and vacA genotypes with homologous recombination repair markers during the gastric precancerous cascade

Background: Helicobacter pylori-induced DNA damage and impaired homologous recombination repair are vital molecular mechanisms for gastric cancer, which mainly count on its virulence factors cytotoxic-associated gene A (CagA) and vacuolating cytotoxin A (VacA). However, the relationship between H. pylori CagA EPIYA motifs and vacA genotypes with DNA damage and homologous recombination repair markers is still not clear. Methods: H. pylori positive and negative gastric biopsies were taken from 165 subjects with different gastric precancerous pathologic stages, and DNA damage marker γH2AX and key homologous recombination repair proteins (CtIP and Rad51) were investigated for their association with H. pylori CagA EPIYA motifs and vacAs-, m-, i-, and d-region genotypes and histology (Sydney classification). Results: Out of 165 patients, 78 were identified as H. pylori-positive. CagA EPIYA motifs were identified as AB, ABC, and ABD in 2 (3.3%), 21 (35%), and 37 (61.7%) patients, respectively, while vacA alleles were identified as: s1, s2, m1, m2, i1, i2, d1, and d2 in 50 (89.3%), 6 (10.7%), 24 (42.9%), 32 (57.1%), 45 (80.4%), 11 (19.6%), 40 (71.4%), and 16 (28.6%) patients, respectively. vacAs1m1i1d1, s1m2i1d1, and s1m2i2d2 were the most prevailing genotypes. γH2AX was highly localized in H. pylori-positive tissues with corresponding CagA EPIYA motifs and vacA genotypes, while Rad51 and CtIP signals were weak. Conclusion: H. pylori were positively correlated with the DNA damage marker in precancerous lesions, but were negatively correlated with the key homologous recombination repair proteins, which may be due to the specific CagA EPIYA motifs and vacA genotypes.

Tailor-Made Detection of Individual Phosphorylated and Non-Phosphorylated EPIYA-Motifs of Helicobacter pylori Oncoprotein CagA

The gastric pathogen and carcinogen Helicobacter pylori (H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.