Proteomic analysis of the function of spot in Helicobacter pylori anti‐oxidative stress in vitro and colonization in vivo

Abstract

As a microaerobe, Helicobacter pylori employs the global regulator SpoT for defending against oxidative stress in vitro. However, the mechanisms how SpoT affects bacterial gene expression is still unknown. Moreover, the function of SpoT in H. pylori colonization in the host is remaining undetermined. To explore the functions of the SpoT in H. pylori pathogenesis, we constructed H. pylori 26695 spoT-deficient mutant (ΔspoT). While grown in ambient atmosphere, protein expression profile of the ΔspoT was analyzed with 2D gel electrophoresis and real-time PCR. Compared to the wild type, the spoT-deficient strain downregulated its transcription of the oxidative-induced genes, as well as the genes responsible for protein degradation and that related to energy metabolism. Meanwhile, the colonization ability of ΔspoT strains in Mongolian gerbil was tested, the results demonstrated a decayed colonization in the mouse stomach with ΔspoT than the wild type. As a matter of facts, the AGS cells infected with the ΔspoT strains excreted increased level of the gastric inflammation cytokines IL-8, and the ΔspoT strains showed poor survival ability when treated with reactive oxygen stress (sodium nitroprusside). The elevated capacity of stimulating cytokines and fragility to reactive oxygen stress may be contribute to decreased colonization of the spoT-deficient mutant in the mouse stomach. Conclusively, we speculate that spoT is a key regulator of the genes for H. pylori spreading in the air and colonization in host stomach.

Bifunctional Enzyme SpoT Is Involved in Biofilm Formation of Helicobacter pylori with Multidrug Resistance by Upregulating Efflux Pump Hp1174 ( gluP )

“…In our previous studies, we successfully constructed an spoT mutant strain (the Δ spoT strain) ( 62 ) and an spoT -complemented strain (the spoT* strain) ( 44 ). The plasmids (pILL570 and pUC18K2) used to construct the mutant strain were kindly provided by Agnès Labigne (Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur).…”

“…The plasmids (pILL570 and pUC18K2) used to construct the mutant strain were kindly provided by Agnès Labigne (Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur). The construction of the gluP mutant strain was identical to the construction of the Δ spoT strain, as described in a previous study ( 62 ). Briefly, the gluP gene from the genome of H. pylori 26695 was destroyed with the insertion of the nonpolar aphA-3 gene, encoding a kanamycin resistance cassette.…”

Potential Role of Biofilm Formation in the Development of Digestive Tract Cancer With Special Reference to Helicobacter pylori Infection

“…Co-culture of a gastric non-cancer epithelial cell line with coccoid or helical forms of Hp revealed that helical Hp led to more severe inflammatory and apoptotic responses, while coccoid Hp maintained host cell in a high proliferation rate (Liu et al, 2006). Interestingly, in animal models, colonization efficiency of Hp mutants in coccoid associated genes, aimA and spoT , was markedly reduced (Sun et al, 2012; Chaput et al, 2016). Despite substantial contrasting findings in the observations, overall, accumulated data suggest that coccoid Hp retains virulence, can survive better in vivo and may contribute to carcinogenesis, while helical Hp can be more easily eliminated by acute host immunoinflammatory responses.…”

The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471

“…pylori strains used in this study were reference strain 26695, which was kindly provided by Zhang Jianzhong (Chinese Disease Control and Prevention Center), and its isogenic spoT mutant, which was used in our previous report and comes from the collection of our laboratory (52). All E.…”

“…The construction of hp0939 , hp1017 , hp0497 , and hp0471 mutant strains was identical to the construction of the Δ spoT strain, as described in the literature (52). Briefly, these four genes from the genome of H.…”