CagA-dependent Manner Research Articles

Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Helicobacter pylori CagA protein induces gastric cancer stem cell-like properties through the Akt/FOXO3a axis.

The presence of Helicobacter pylori (H. pylori) infection poses a substantial risk for the development of gastric adenocarcinoma. The primary mechanism through which H. pylori exerts its bacterial virulence is the cytotoxin CagA. This cytotoxin has the potential to induce inter-epithelial mesenchymal transition, proliferation, metastasis, and the acquisition of stem cell-like properties in gastric cancer (GC) cells infected with CagA-positive H. pylori. Cancer stem cells (CSCs) represent a distinct population of cells capable of self-renewal and generating heterogeneous tumor cells. Despite evidence showing that CagA can induce CSCs-like characteristics in GC cells, the precise mechanism through which CagA triggers the development of GC stem cells (GCSCs) remains uncertain. This study reveals that CagA-positive GC cells infected with H. pylori exhibit CSCs-like properties, such as heightened expression of CD44, a specific surface marker for CSCs, and increased ability to form tumor spheroids. Furthermore, we have observed that H. pylori activates the PI3K/Akt signaling pathway in a CagA-dependent manner, and our findings suggest that this activation is associated with the CSCs-like characteristics induced by H. pylori. The cytotoxin CagA, which is released during H. pylori infection, triggers the activation of the PI3K/Akt signaling pathway in a CagA-dependent manner. Additionally, CagA inhibits the transcription of FOXO3a and relocates it from the nucleus to the cytoplasm by activating the PI3K/Akt pathway. Furthermore, the regulatory function of the Akt/FOXO3a axis in the transformation of GC cells into a stemness state was successfully demonstrated.

Helicobacter pylori-Induced Angiopoietin-Like 4 Promotes Gastric Bacterial Colonization and Gastritis.

Helicobacter pylori infection is characterized as progressive processes of bacterial persistence and chronic gastritis with features of infiltration of mononuclear cells more than granulocytes in gastric mucosa. Angiopoietin-like 4 (ANGPTL4) is considered a double-edged sword in inflammation-associated diseases, but its function and clinical relevance in H. pylori-associated pathology are unknown. Here, we demonstrate both pro-colonization and pro-inflammation roles of ANGPTL4 in H. pylori infection. Increased ANGPTL4 in the infected gastric mucosa was produced from gastric epithelial cells (GECs) synergistically induced by H. pylori and IL-17A in a cagA-dependent manner. Human gastric ANGPTL4 correlated with H. pylori colonization and the severity of gastritis, and mouse ANGPTL4 from non-bone marrow-derived cells promoted bacteria colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Il17a -/-, Angptl4 -/-, and Il17a -/- Angptl4 -/- mice. Mechanistically, ANGPTL4 bound to integrin αV (ITGAV) on GECs to suppress CXCL1 production by inhibiting ERK, leading to decreased gastric influx of neutrophils, thereby promoting H. pylori colonization; ANGPTL4 also bound to ITGAV on monocytes to promote CCL5 production by activating PI3K-AKT-NF-κB, resulting in increased gastric influx of regulatory CD4+ T cells (Tregs) via CCL5-CCR4-dependent migration. In turn, ANGPTL4 induced Treg proliferation by binding to ITGAV to activate PI3K-AKT-NF-κB, promoting H. pylori-associated gastritis. Overall, we propose a model in which ANGPTL4 collectively ensures H. pylori persistence and promotes gastritis. Efforts to inhibit ANGPTL4-associated pathway may prove valuable strategies in treating H. pylori infection.

Tubulointerstitial nephritis antigen-like 1 is a novel matricellular protein that promotes gastric bacterial colonization and gastritis in the setting of Helicobacter pylori infection.

The interaction between the gastric epithelium and immune cells plays key roles in H. pylori-associated pathology. Here, we demonstrate a procolonization and proinflammatory role of tubulointerstitial nephritis antigen-like 1 (TINAGL1), a newly discovered matricellular protein, in H. pylori infection. Increased TINAGL1 production by gastric epithelial cells (GECs) in the infected gastric mucosa was synergistically induced by H. pylori and IL-1β via the ERK-SP1 pathway in a cagA-dependent manner. Elevated human gastric TINAGL1 correlated with H. pylori colonization and the severity of gastritis, and mouse TINAGL1 derived from non-bone marrow-derived cells promoted bacterial colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Tinagl1-/- and Tinagl1ΔGEC mice and were increased in mice injected with mouse TINAGL1. Mechanistically, TINAGL1 suppressed CCL21 expression and promoted CCL2 production in GECs by directly binding to integrin α5β1 to inhibit ERK and activate the NF-κB pathway, respectively, which not only led to decreased gastric influx of moDCs via CCL21-CCR7-dependent migration and, as a direct consequence, reduced the bacterial clearance capacity of the H. pylori-specific Th1 response, thereby promoting H. pylori colonization, but also resulted in increased gastric influx of Ly6Chigh monocytes via CCL2-CCR2-dependent migration. In turn, TINAGL1 induced the production of the proinflammatory protein S100A11 by Ly6Chigh monocytes, promoting H. pylori-associated gastritis. In summary, we identified a model in which TINAGL1 collectively ensures H. pylori persistence and promotes gastritis.

Helicobacter pylori promotes epithelial-to-mesenchymal transition by downregulating CK2β in gastric cancer cells

Strains of Helicobacter pylori that are positive for the oncoprotein CagA (cytotoxin-associated gene A) are associated with gastric cancer and might be related to the epithelial-to-mesenchymal transition (EMT). Casein kinase 2 (CK2) is a serine/threonine protein kinase that plays a major role in tumorigenesis through signaling pathways related to the EMT. However, the role played by the interaction between CagA and CK2 in gastric carcinogenesis is poorly understood. Although CK2α protein expression remained unchanged during H. pylori infection, we found that CK2α kinase activity was increased in gastric epithelial cells. We also found that the CK2β protein level decreased in H. pylori-infected gastric cancer cells in CagA-dependent manner and demonstrated that CagA induced CK2β degradation via HDM2 (human double minute 2; its murine equivalent is MDM2). We observed that CagA induced HDM2 protein phosphorylation and that p53 levels were decreased in H. pylori-infected gastric cancer cells. In addition, downregulation of CK2β induced AKT Ser473 phosphorylation and decreased the AKT Ser129 phosphorylation level in gastric cancer cells. We also found that the downregulation of CK2β triggered the upregulation of Snail levels in gastric cancer cells. Furthermore, our in vivo experiments and functional assays of migration and colony formation suggest that CK2β downregulation is a major factor responsible for the EMT in gastric cancer. Therefore, CK2 could be a key mediator of the EMT in H. pylori-infected gastric cancer and could serve as a molecular target for gastric cancer treatment.

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

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.

Activation of Aquaporin 5 by carcinogenic Helicobacter pylori infection promotes epithelial-mesenchymal transition via the MEK/ERK pathway.

Helicobacter pylori (H.pylori) is a major risk factor for gastric cancer. The water channel protein Aquaporin 5 (AQP5) is involved in the tumorigenesis and progression of various cancers. In this study, we aimed to explore the role of AQP5 in H.pylori-induced gastric carcinogenesis. We collected 160 samples which inculded CNAG, IM, Dys and gastric cancer from patients who underwent endoscopy and detected the expression of AQP5. In vivo and vitro H.pylori infection models, we explored the relationship between AQP5 and H.pylori. Plasmid, siRNA and inhibitors were used to investigated the relationship between AQP5 and EMT and the role of AQP5 in H.pylori-induced gastric carcinogenesis. AQP5 expression was gradually increased in human gastric tissues with the progression of chronic nonatrophic gastritis to gastric cancer and associated with the H.pylori infection status. In vivo and in vitro studies showed that H.pylori infection induced AQP5 expression in gastric epithelial cells in a CagA-dependent manner. Knockdown of AQP5 reversed H.pylori-induced cell proliferation and invasion, and -suppressed cell apoptosis. Additionally, knockdown of AQP5 suppressed H.pylori-induced Epithelial-mesenchymal transition (EMT) phenotypes by regulating transcriptional factors, mesenchymal markers, and epithelial markers. We explored the underlying mechanism and our results indicated that knockdown of AQP5 significantly suppressed H.pylori infection-induced phosphorylation of ERK1/2, MEK and the expression levels of downstream genes. Treatment with an ERK inhibitor suppressed the EMT induced by H.pylori infection. Taken together, this study suggest that pathogenic H.pylori infection promotes AQP5 expression to induce the EMT via the MEK/ERK signaling pathway.

H. pylori infection confers resistance to apoptosis via Brd4‐dependent BIRC3 eRNA synthesis

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance. While H. pylori infection-induced expression of pro-inflammatory cytokines for chronic inflammation is well studied, the molecular mechanism underlying the apoptosis resistance in infected cells is not well understood. In this study, we demonstrated that H. pylori infection induced apoptosis resistance in gastric epithelial cells triggered by Raptinal, a drug that directly activates caspase-3. This resistance resulted from the induction of cIAP2 (encoded by BIRC3) since depletion of BIRC3 by siRNA or inhibition of cIAP2 via BV6 reversed H. pylori-suppressed caspase-3 activation. The induction of cIAP2 was regulated by H. pylori induced BIRC3 eRNA synthesis. Depletion of BIRC3 eRNA decreased H. pylori-induced cIAP2 and reversed H. pylori suppressed caspase-3 activation. Mechanistically, H. pylori stimulated the recruitment of bromodomain-containing factor Brd4 to the enhancer of BIRC3 and promoted BIRC3 eRNA and mRNA synthesis. Inhibition of Brd4 diminished the expression of BIRC3 eRNA and the anti-apoptotic response to H. pylori infection. Importantly, H. pylori isogenic cagA deficient mutant failed to activate the synthesis of BIRC3 eRNA and the associated apoptosis resistance. Finally, in primary human gastric epithelial cells, H. pylori also induced resistance to Raptinal-triggered caspase-3 activation by activating the Brd4-dependent BIRC3 eRNA synthesis in a CagA-dependent manner. These results identify a novel function of Brd4 in H. pylori-mediated apoptosis resistance via activating BIRC3 eRNA synthesis, suggesting that Brd4 could be a potential therapeutic target for H. pylori-induced gastric cancer.

Arrestin domain containing 3 promotes Helicobacter pylori-associated gastritis by regulating protease-activated receptor 1.

Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori–associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM–derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C–Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3–/– mice but increased in protease-activated receptor 1–/– (Par1–/–) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori–associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori–associated gastritis.

H. pylori infection confers resistance to apoptosis via Brd4-dependent BIRC3 eRNA synthesis

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance. While H. pylori infection-induced expression of pro-inflammatory cytokines for chronic inflammation is well studied, the molecular mechanism underlying the apoptosis resistance in infected cells is not well understood. In this study, we demonstrated that H. pylori infection-induced apoptosis resistance in gastric epithelial cells triggered by Raptinal, a drug that directly activates caspase-3. This resistance resulted from the induction of cIAP2 (encoded by BIRC3) since depletion of BIRC3 by siRNA or inhibition of cIAP2 via BV6 reversed H. pylori-suppressed caspase-3 activation. The induction of cIAP2 was regulated by H. pylori-induced BIRC3 eRNA synthesis. Depletion of BIRC3 eRNA decreased H. pylori-induced cIAP2 and reversed H. pylori-suppressed caspase-3 activation. Mechanistically, H. pylori stimulated the recruitment of bromodomain-containing factor Brd4 to the enhancer of BIRC3 and promoted BIRC3 eRNA and mRNA synthesis. Inhibition of Brd4 diminished the expression of BIRC3 eRNA and the anti-apoptotic response to H. pylori infection. Importantly, H. pylori isogenic cagA-deficient mutant failed to activate the synthesis of BIRC3 eRNA and the associated apoptosis resistance. Finally, in primary human gastric epithelial cells, H. pylori also induced resistance to Raptinal-triggered caspase-3 activation by activating the Brd4-dependent BIRC3 eRNA synthesis in a CagA-dependent manner. These results identify a novel function of Brd4 in H. pylori-mediated apoptosis resistance via activating BIRC3 eRNA synthesis, suggesting that Brd4 could be a potential therapeutic target for H. pylori-induced gastric cancer.

A Summary of the 2020 Gastric Cancer Summit at Stanford University

There exists no coherent national strategy for the early detection or prevention of gastric cancer in the United States (US), even among identified high-risk groups such as Asian Americans, African Americans, Hispanic Americans, and Alaska Native/American Indian peoples. As a result, patients with gastric cancer in the US are diagnosed at later stages and demonstrate worse overall survival compared to nations of East Asia with established screening programs (Table 1). The under-recognition of gastric cancer risk within minority communities is a significant unaddressed healthcare disparity.

Inhibition of autophagy aggravates DNA damage response and gastric tumorigenesis via Rad51 ubiquitination in response to H. pylori infection

ABSTRACT Helicobacter pylori (H. pylori) infection is the strongest known risk factor for the development of gastric cancer. DNA damage response (DDR) and autophagy play key roles in tumorigenic transformation. However, it remains unclear how H. pylori modulate DDR and autophagy in gastric carcinogenesis. Here we report that H. pylori infection promotes DNA damage via suppression of Rad51 expression through inhibition of autophagy and accumulation of p62 in gastric carcinogenesis. We find that H. pylori activated DNA damage pathway in concert with downregulation of repair protein Rad51 in gastric cells, C57BL/6 mice and Mongolian gerbils. In addition, autophagy was increased early and then decreased gradually during the duration of H. pylori infection in vitro in a CagA-dependent manner. Moreover, loss of autophagy led to promotion of DNA damage in H. pylori-infected cells. Furthermore, knockdown of autophagic substrate p62 upregulated Rad51 expression, and p62 promoted Rad51 ubiquitination via the direct interaction of its UBA domain. Finally, H. pylori infection was associated with elevated levels of p62 in gastric intestinal metaplasia and decreased levels of Rad51 in dysplasia compared to their H. pylori- counterparts. Our findings provide a novel mechanism into the linkage of H. pylori infection, autophagy, DNA damage and gastric tumorigenesis.

SHP2-Independent Tyrosine Dephosphorylation of Cortactin and Vinculin during Infection with Helicobacter pylori.

The gastric pathogen Helicobacter pylori colonizes approximately half of the human world population. The bacterium injects the effector protein cytotoxin associated gene A (CagA) via a type-IV secretion system into host epithelial cells, where the protein becomes phosphorylated at specific EPIYA-motifs by cellular kinases. Inside the host cell, CagA can interact with over 25 different proteins in both phosphorylation-dependent and phosphorylation-independent manners, resulting in manipulation of host-cell signaling pathways. During the course of an H. pylori infection, certain host-cell proteins undergo tyrosine dephosphorylation in a CagA-dependent manner, including the actin-binding proteins cortactin and vinculin. A predominant response of intracellular CagA is the binding and activation of tyrosine phosphatase, the human Src-homology-region-2-domain-containing-phosphatase-2 (SHP2). Here, we considered the possibility that activated SHP2 might be responsible for the dephosphorylation of cortactin and vinculin. To investigate this, phosphatase inhibitor studies were performed. Additionally, a complete knockout mutant of SHP2 in AGS cells was created by CRISPR/Cas9 technology, and these cells were infected with H. pylori. However, neither the presence of an inhibitor nor the inactivation of SHP2 prevented the dephosphorylation of cortactin and vinculin upon CagA delivery. Tyrosine dephosphorylation of these proteins is therefore independent of SHP2 and instead must be caused by another, as yet unidentified, protein tyrosine phosphatase.

Helicobacter pylori-induced adrenomedullin modulates IFN-\u03b3-producing T-cell responses and contributes to gastritis

Adrenomedullin (ADM) is a multifunctional peptide that is expressed by many surface epithelial cells, but its relevance to Helicobacter pylori (H. pylori)-induced gastritis is unknown. Here, we found that gastric ADM expression was elevated in gastric mucosa of H. pylori-infected patients and mice. In H. pylori-infected human gastric mucosa, ADM expression was positively correlated with the degree of gastritis; accordingly, blockade of ADM resulted in decreased inflammation within the gastric mucosa of H. pylori-infected mice. During H. pylori infection, ADM production was promoted via PI3K–AKT signaling pathway activation by gastric epithelial cells in a cagA-dependent manner, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterized by the increased IFN-γ-producing T cells, whose differentiation was induced via the phosphorylation of AKT and STAT3 by ADM derived from gastric epithelial cells. ADM also induced macrophages to produce IL-12, which promoted the IFN-γ-producing T-cell responses, thereby contributing to the development of H. pylori-associated gastritis. Accordingly, blockade of IFN-γ or knockout of IFN-γ decreased inflammation within the gastric mucosa of H. pylori-infected mice. This study identifies a novel regulatory network involving H. pylori, gastric epithelial cells, ADM, macrophages, T cells, and IFN-γ, which collectively exert a pro-inflammatory effect within the gastric microenvironment.

Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes

ABSTRACT RNautophagy and DNautophagy (RDA) are unconventional autophagic pathways where nucleic acids are directly transported through the lysosomal membrane, then degraded inside lysosomes. We have previously shown that bitopic protein LAMP2C and putative RNA transporter SIDT2, both lysosomal membrane proteins, mediate the direct transport of nucleic acids into lysosomes and that LAMP2C interacts with the nucleic acids and functions as a receptor during RDA. Because SIDT2-mediated RDA occurs in isolated lysosomes that lack LAMP2C, in this study, we tested the hypothesis that SIDT2 itself could also interact with the nucleic acids. Our results show that SIDT2 directly binds RNA and DNA through an arginine-rich motif (ARM) located within its main cytosolic domain, and disruption of this motif dramatically impairs SIDT2-mediated RNautophagic activity. We also found that SIDT2 interacts with exon 1 of HTT (huntingtin) transcript through the ARM in a CAG-dependent manner. Moreover, overexpression of SIDT2 promoted degradation of HTT mRNA and reduced the levels of polyglutamine-expanded HTT aggregates, hallmarks of Huntington disease. In addition, a comparative analysis of LAMP2C and SIDT2 functions at the cellular level revealed that the two proteins exert a synergistic effect on RNautophagic activity and that the ARMs which mediate the interactions of SIDT2 and LAMP2C with RNA are essential for the synergy. Together, our results point out the importance of nucleic acid-binding capacity of SIDT2 for its function in translocating nucleic acids through the lipid bilayer and suggests a potential application of RNautophagy activation to reduce the expression levels of disease-causing toxic proteins. Abbreviations: ACTB/β-actin: actin beta; ARM: arginine-rich motif; CBB: Coomassie Brilliant Blue; CD: cytosolic domain; COX4I1/COX4: cytochrome c oxidase subunit 4I1; E. coli: Escherichia coli; EGFP: enhanced green fluorescent protein; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GST: glutathione S-transferase; HRP: horseradish peroxidase; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; HTTex1: exon 1 of the HTT gene; LAMP2: lysosomal associated membrane protein 2; LMNA: lamin A/C; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; PEI: polyethyleneimine; polyQ: polyglutamine; qPCR: quantitative PCR; RAB5A: RAB5A, member RAS oncogene family; RDA: RNautophagy and DNautophagy; SCARB2/LIMP2: scavenger receptor class B member 2; SDS: sodium dodecyl sulfate; SID-1: systemic RNA interference deficient-1; SIDT2: SID1 transmembrane family member 2; WT: wild type.

Targeted mobilization of Lrig1+ gastric epithelial stem cell populations by a carcinogenic Helicobacter pylori type IV secretion system

Helicobacter pylori-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the cag type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within H. pylori-infected gastric mucosa. Lineage tracing was induced in Lrig1-CreERT2/+;R26R-YFP/+ (Lrig1/YFP) mice that were uninfected or subsequently infected with cag+H. pylori or an isogenic cagE- mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) H. pylori for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the cagE- mutant. WT H. pylori-infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT H. pylori In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic H. pylori infection stimulates Lrig1-expressing progenitor cells in a cag-dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.

Modification of the Gastric Mucosal Microbiota by a Strain-Specific Helicobacter pylori Oncoprotein and Carcinogenic Histologic Phenotype.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma; however, most infected individuals never develop this malignancy. Strain-specific microbial factors, such as the oncoprotein CagA, as well as environmental conditions, such as iron deficiency, augment cancer risk. Importantly, dysbiosis of the gastric microbiota is also associated with gastric cancer. To investigate the combinatorial effects of these determinants in an in vivo model of gastric cancer, Mongolian gerbils were infected with the carcinogenic cag+H. pylori strain 7.13 or a 7.13 cagA isogenic mutant, and microbial DNA extracted from gastric tissue was analyzed by 16S rRNA sequencing. Infection with H. pylori significantly increased gastric inflammation and injury, decreased α-diversity, and altered microbial community structure in a cagA-dependent manner. The effect of iron deficiency on gastric microbial communities was also investigated within the context of infection. H. pylori-induced injury was augmented under conditions of iron deficiency, but despite differences in gastric pathology, there were no significant differences in α- or β-diversity, phyla, or operational taxonomic unit (OTU) abundance among infected gerbils maintained on iron-replete or iron-depleted diets. However, when microbial composition was stratified based solely on the severity of histologic injury, significant differences in α- and β-diversity were present among gerbils harboring premalignant or malignant lesions compared to gerbils with gastritis alone. This study demonstrates that H. pylori decreases gastric microbial diversity and community structure in a cagA-dependent manner and that as carcinogenesis progresses, there are corresponding alterations in community structure that parallel the severity of disease.IMPORTANCE Microbial communities are essential for the maintenance of human health, and when these communities are altered, hosts can become susceptible to inflammation and disease. Dysbiosis contributes to gastrointestinal cancers, and specific bacterial species are associated with this phenotype. This study uses a robust and reproducible animal model to demonstrate that H. pylori infection induces gastric dysbiosis in a cagA-dependent manner and further that dysbiosis and altered microbial community structure parallel the severity of H. pylori-induced gastric injury. Ultimately, such models of H. pylori infection and cancer that can effectively evaluate multiple determinants simultaneously may yield effective strategies for manipulating the gastric microbiota to prevent the development of gastric cancer.

Carcinogenic Helicobacter pylori Strains Selectively Dysregulate the In Vivo Gastric Proteome, Which May Be Associated with Stomach Cancer Progression*[S

Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.

Helicobacter pylori-induced IL-33 modulates mast cell responses, benefits bacterial growth, and contributes to gastritis

Interleukin (IL)-induced inflammatory responses are critical for the pathogenesis of Helicobacter pylori (H. pylori)-induced gastritis. IL-33 represents a recently discovered proinflammatory cytokine involved in inflammatory diseases, but its relevance to H. pylori-induced gastritis is unknown. Here, we found that gastric IL-33 mRNA and protein expression were elevated in gastric mucosa of both patients and mice infected with H. pylori, which is positively correlated with bacterial load and the degree of gastritis. IL-33 production was promoted via extracellular regulated protein kinases (ERK) signaling pathway activation by gastric epithelial cells in a cagA-dependent manner during H. pylori infection, and resulted in increased inflammation and bacteria burden within the gastric mucosa. Gastric epithelial cell-derived IL-33 promoted TNF-α production from mast cells in vitro, and IL-33 increased TNF-α production in vivo. Increased TNF-α inhibited gastric epithelial cell proliferation, conducing to the progress of H. pylori-associated gastritis and bacteria colonization. This study defined a patent regulatory networks involving H. pylori, gastric epithelial cell, IL-33, mast cell, and TNF-α, which jointly play a pathological effect within the gastric circumstances. It may be a valuable strategy to restrain this IL-33-dependent pathway in the treatment of H. pylori-associated gastritis.

Glutamine Codon Usage and polyQ Evolution in Primates Depend on the Q Stretch Length.

Amino acid usage in a proteome depends mostly on its taxonomy, as it does the codon usage in transcriptomes. Here, we explore the level of variation in the codon usage of a specific amino acid, glutamine, in relation to the number of consecutive glutamine residues. We show that CAG triplets are consistently more abundant in short glutamine homorepeats (polyQ, four to eight residues) than in shorter glutamine stretches (one to three residues), leading to the evolutionary growth of the repeat region in a CAG-dependent manner. The length of orthologous polyQ regions is mostly stable in primates, particularly the short ones. Interestingly, given a short polyQ the CAG usage is higher in unstable-in-length orthologous polyQ regions. This indicates that CAG triplets produce the necessary instability for a glutamine stretch to grow. Proteins related to polyQ-associated diseases behave in a more extreme way, with longer glutamine stretches in human and evolutionarily closer nonhuman primates, and an overall higher CAG usage. In the light of our results, we suggest an evolutionary model to explain the glutamine codon usage in polyQ regions.