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¬Helicobacter pylori CagA and Cag type IV secretion system activity have key roles in triggering gastric transcriptional and proteomic alterations

ABSTRACT: Colonization of the human stomach with cag pathogenicity island (PAI)-positive H. pylori strains is associated with increased gastric cancer risk compared to colonization with cag PAI-negative strains. To evaluate the contributions of the Cag type IV secretion system (T4SS) and CagA (a secreted bacterial oncoprotein) to gastric molecular alterations relevant for carcinogenesis, we infected Mongolian gerbils with a Cag T4SS-positive wild-type (WT) H. pylori strain, one of two Cag T4SS mutant strains (∆cagT or ∆cagY), or a ∆cagA mutant for 12 weeks. Histologic staining revealed a biphasic distribution of gastric inflammation severity (either minimal or severe) in WT-infected animals and minimal inflammation in mutant-infected animals. Atrophic gastritis (a premalignant condition), dysplasia, and gastric adenocarcinoma were only detected in WT-infected animals with high inflammation scores. Transcriptional profiling and analyses of micro-extracted tryptic peptides (LC-MS/MS and imaging mass spectrometry) revealed more than a thousand molecular alterations in gastric tissues from WT-infected animals with high inflammation scores compared to uninfected tissues and few alterations in tissues from other groups of infected animals. Proteins with altered abundance in animals with severe Cag T4SS-induced inflammation mapped to multiple pathways, including the complement/coagulation cascade and proteasome pathway. Proteins exhibiting markedly increased abundance in tissues from H. pylori-infected animals with severe inflammation included calprotectin components, lysozyme, lactoferrin, superoxide dismutase, eosinophil peroxidase, proteins involved in proteasome activation, STAT1, TrpRS, GBP2, and IIGP1. These results demonstrate key roles for CagA and Cag T4SS activity in promoting gastric mucosal inflammation, transcriptional alterations, and proteomic alterations relevant to gastric carcinogenesis.

INSTRUMENT(S): timsTOF HT

ORGANISM(S): Meriones Unguiculatus

TISSUE(S): Stomach

SUBMITTER: Hayes McDonald

LAB HEAD: Timothy L. Cover

PROVIDER: PXD060632 | Pride | 2025-02-28

REPOSITORIES: pride

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Source:

			Action	DRS
	GCF_000196755.1_ASM19675v1_protein_Mongolian_gerbil_fixed_rev.fasta	Fasta
	HT_6328_20230822_60minDDA_Trap_1ul_S10A_Slot2-19_1_288.d.zip	Other
	HT_6328_20230822_60minDDA_Trap_1ul_S10C_Slot2-20_1_290.d.zip	Other
	HT_6328_20230822_60minDDA_Trap_1ul_S11A_Slot2-21_1_295.d.zip	Other
	HT_6328_20230822_60minDDA_Trap_1ul_S11C_Slot2-22_1_299.d.zip	Other

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Project description: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.

Project description:Helicobacter pylori infection promotes chronic inflammation and plethora of DNA damages including strand breaks, base alterations, point mutations, microsatellite instability and epigenetic alterations. The gastric epitheliaI cells, in order to prevent genomic instability, require an integrous DNA damage repair (DDR) machinery which, however, has been reported to be modulated by the infection. CagA is a major Hp virulence factor that deregulates host cell functions such as proliferation, apoptosis and chromosomal integrity. Its pathogenic activity is partly regulated by tyrosine phosphorylation on repeated EPIYA-motifs. Our aim was to identify putative effects of H. pylori infection and CagA protein on DDR machinery, investigating the transcriptome of AGS cells, infected with P12 H. pylori wild-type strain, as well as, its corresponding CagA knock-out and the CagA phosphorylation-defective mutant following tyrosine (EPIYA) substitution by phenylalanine (EPIFA) in the EPIYA-C motifs. Upon RNA-Sequencing on polyA-selected transcripts we performed Differential Expression Analysis, Pathway Enrichment Analysis and visualization on KEGG Pathway Maps per DDR mechanism. Key DDR components that were observed to be downregulated in a CagA-related manner were validated via Western Blot utilizing AGS and the non-cancerous GES1 cell lines. Transcriptome analysis revealed that a notable number of DDR genes were downregulated during H. pylori infection resulting to potential modulation of Base Excision Repair, Mismatch Repair and a more intricate deregulation of Nucleotide Excision Repair, Homologous Recombination and Non-Homologous End-Joining. CagA contributes to MUTYH, FEN1, APE1, POLD1 and LIG1 downregulation and those observations were verified on the protein expression level, with the exception of APE1 that was on contrary observed to be upregulated. Our study accentuates the role of CagA, as a significant contributor of the H. pylori infection-mediated DDR modulation, disrupting the balance between DNA damage introduction and repair thus favoring genomic instability and potentially contributing to gastric carcinogenesis.

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¬Helicobacter pylori CagA and Cag type IV secretion system activity have key roles in triggering gastric transcriptional and proteomic alterations

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