The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse

Abstract

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Binding of YY1/CREB to an Enhancer Region Triggers Claudin 6 Expression in H. pylori LPS-Stimulated AGS Cells

“…YY1 function is regulated through the IL-4/STAT6 signaling pathway in tumor-associated immune cells [ 54 ]. LPS is not the only pathogenic factor in H. pylori capable of inducing gastric carcinogenesis; cytotoxin-associated gene A (CagA) protein, another major and substantial component of H. pylori , is known to provoke genomic hypermutations, DNA double strand breaks, subverted DNA damage responses that include the down-regulation of DNA repair genes, and transcriptomic and proteomic alterations that increase the chance of generating a “hit-and-run mechanism” for gastric carcinogenesis [ 55 , 56 , 57 ].…”

Potential Antigenic Candidates for the Development of Peptide-Based Vaccines to Induce Immunization against Helicobacter pylori Infection in BALB/c Mice

“…The selection of proteins that match the required criteria for vaccine development is crucial. That said, we have included the CagA oncoprotein that is associated with risk of cancer [ 36 , 37 , 38 ]. The distinct effects of the VacA toxin and its effects on various cells have been reported in several studies [ 39 , 40 ].…”

The Helicobacter pylori cag pathogenicity island as a determinant of gastric cancer risk

“… 131 CagA is also implicated in the downregulation of several genes involved in DNA repair. 133 , 135 …”