Temporal Control of the Helicobacter pylori Cag Type IV Secretion System in a Mongolian Gerbil Model of Gastric Carcinogenesis

Abstract

The Helicobacter pylori Cag type IV secretion system (T4SS) translocates the effector protein CagA and nonprotein bacterial constituents into host cells. In this study, we infected Mongolian gerbils with an H. pylori strain in which expression of the cagUT operon (required for Cag T4SS activity) is controlled by a TetR/tetO system. Transcript levels of cagU were significantly higher in gastric tissue from H. pylori-infected animals receiving doxycycline-containing chow (to derepress Cag T4SS activity) than in tissue from infected control animals receiving drug-free chow. At 3 months postinfection, infected animals receiving doxycycline had significantly increased gastric inflammation compared to infected control animals. Dysplasia (a premalignant histologic lesion) and/or invasive gastric adenocarcinoma were detected only in infected gerbils receiving doxycycline, not in infected control animals. We then conducted experiments in which Cag T4SS activity was derepressed during defined stages of infection. Continuous Cag T4SS activity throughout a 3-month time period resulted in higher rates of dysplasia and/or gastric cancer than observed when Cag T4SS activity was limited to early or late stages of infection. Cag T4SS activity for the initial 6 weeks of infection was sufficient for the development of gastric inflammation at the 3-month time point, with gastric cancer detected in a small proportion of animals. These experimental results, together with previous studies of cag mutant strains, provide strong evidence that Cag T4SS activity contributes to gastric carcinogenesis and help to define the stages of H. pylori infection during which Cag T4SS activity causes gastric alterations relevant for cancer pathogenesis. IMPORTANCE The “hit-and-run model” of carcinogenesis proposes that an infectious agent triggers carcinogenesis during initial stages of infection and that the ongoing presence of the infectious agent is not required for development of cancer. H. pylori infection and actions of CagA (an effector protein designated a bacterial oncoprotein, secreted by the Cag T4SS) are proposed to constitute a paradigm for hit-and-run carcinogenesis. In this study, we report the development of methods for controlling H. pylori Cag T4SS activity in vivo and demonstrate that Cag T4SS activity contributes to gastric carcinogenesis. We also show that Cag T4SS activity during an early stage of infection is sufficient to initiate a cascade of cellular alterations leading to gastric inflammation and gastric cancer at later time points.

Loss of Corpus-Specific Lipids in Helicobacter pylori-Induced Atrophic Gastritis

“…Male Mongolian gerbils (35 to 45 g weight) were obtained from Charles River Laboratories. Gerbils were fasted overnight and then were infected via oral gavage (day 0 and day 2) with a 0.5-ml suspension of H. pylori strain 7.13 (1 × 10 9 CFU/ml) in Brucella broth ( 13 , 16 , 17 , 19 ). Gerbils in the first cohort were fed an AIN-93M rodent diet (Bio-Serv), beginning 7 days prior to infection and throughout the remainder of the experiment.…”

“…Gerbil stomachs were processed to retain the glandular portion of the stomach (corpus and antrum), and the nonglandular portion (forestomach) was discarded ( 13 , 17 , 19 ). The glandular portion was cut open along the lesser curvature and laid flat ( Fig.…”

“…Inflammation scores (0, 1, 2, and 3, corresponding to absent, mild, moderate, or marked inflammation, respectively) were assigned to evaluate both acute (neutrophils) and chronic (mononuclear leukocytes) inflammation. The inflammation was evaluated in both the corpus and antrum, and these scores were added together to yield a cumulative score of 0 to 12 ( 13 , 17 – 19 , 45 ). The histologic evaluation also included an evaluation of whether gastric ulceration, dysplasia, or gastric adenocarcinoma were present ( 45 ).…”

“…Under most circumstances, H. pylori -infected wild-type mice do not develop gastric ulcers or gastric cancer ( 11 ). In contrast, H. pylori -infected Mongolian gerbils can develop gastric ulcers, hyperplasia, premalignant pathology (parietal and chief cell loss and dysplasia), or gastric cancer ( 13 – 19 ).…”

An infection-activated redox switch promotes tumor growth

“…Gerbil infection protocols were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee. Experiments were performed as previously described 55 . H. pylori strain 7.13 was cultured in Brucella broth with 10% (v/v) FBS for ~16 h under microaerophilic conditions.…”