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SpoT-mediated NapA upregulation promotes oxidative stress-induced Helicobacter pylori biofilm formation and confers multidrug resistance.

ABSTRACT: Recently, there is increased incidence of drug-resistant Helicobacter pylori infection. Biofilm formation confers multidrug resistance to bacteria. Moreover, it has been found that the formation of biofilm on the surface of gastric mucosa is an important reason for the difficulty of eradication of H. pylori The mechanisms underlying H. pylori biofilm formation in vivo have not been elucidated. Reactive oxygen species (ROS) released by the host immune cells in response to H. pylori infection cannot effectively clear the pathogen. Moreover, the extracellular matrix of the biofilm protects the bacteria against ROS-mediated toxicity. This study hypothesized that ROS can promote H. pylori biofilm formation and treatment with low concentrations of hydrogen peroxide (H₂O₂) promoted this process in vitro The comparative transcriptome analysis of planktonic and biofilm-forming cells revealed that the expression of SpoT, a (p)ppGpp (guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bispyrophosphate) synthetase/hydrolase, is upregulated in H₂O₂-induced biofilms and that knockout of spoT inhibited H. pylori biofilm formation. Additionally, this study examined the key target molecules involved in SpoT regulation using weighted gene co-expression network analysis. The analysis revealed that neutrophil-activating protein (NapA; HP0243) promoted H₂O₂-induced biofilm formation and conferred multidrug resistance. Furthermore, vitamin C exhibited anti-H. pylori biofilm activity and downregulated the expression of napA in vitro These findings provide novel insight into the clearance of H. pylori biofilms.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC8092859 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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SpoT-mediated NapA upregulation promotes oxidative stress-induced Helicobacter pylori biofilm formation and confers multidrug resistance.

Zhao Yican Y Cai Yuying Y Chen Zhenghong Z Li Huanjie H Xu Zhengzheng Z Li Wenjuan W Jia Jihui J Sun Yundong Y

Antimicrobial agents and chemotherapy 20210301 5

Recently, there is increased incidence of drug-resistant Helicobacter pylori infection. Biofilm formation confers multidrug resistance to bacteria. Moreover, it has been found that the formation of biofilm on the surface of gastric mucosa is an important reason for the difficulty of eradication of H. pylori The mechanisms underlying H. pylori biofilm formation in vivo have not been elucidated. Reactive oxygen species (ROS) released by the host immune cells in response ...[more]

PMID: 33649116

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Project description:One elusive area in the Helicobacter pylori field is an understanding of why some infections result in gastric cancer, yet others persist asymptomatically for the life-span of the individual. Even before the genomic era, the high level of intraspecies diversity of H. pylori was well recognized and became an intriguing area of investigation with respect to disease progression. Of interest in this regard is the unique repertoire of over 60 outer membrane proteins (OMPs), several of which have been associated with disease outcome. Of these OMPs, the association between HomB and disease outcome varies based on the population being studied. While the molecular roles for some of the disease-associated OMPs have been evaluated, little is known about the role that HomB plays in the H. pylori lifecycle. Thus, herein we investigated homB expression, regulation, and contribution to biofilm formation. We found that in H. pylori strain G27, homB was expressed at a relatively low level until stationary phase. Furthermore, homB expression was suppressed at low pH in an ArsRS-dependent manner; mutation of arsRS resulted in increased homB transcript at all tested time-points. ArsRS regulation of homB appeared to be direct as purified ArsR was able to specifically bind to the homB promoter. This regulation, combined with our previous finding that ArsRS mutations lead to enhanced biofilm formation, led us to test the hypothesis that homB contributes to biofilm formation by H. pylori. Indeed, subsequent biofilm analysis using a crystal-violet quantification assay and scanning electron microscopy (SEM) revealed that loss of homB from hyper-biofilm forming strains resulted in reversion to a biofilm phenotype that mimicked wild-type. Furthermore, expression of homB in trans from a promoter that negated ArsRS regulation led to enhanced biofilm formation even in strains in which the chromosomal copy of homB had been deleted. Thus, homB is necessary for hyper-biofilm formation of ArsRS mutant strains and aberrant regulation of this gene is sufficient to induce a hyper-biofilm phenotype. In summary, these data suggest that the ArsRS-dependent regulation of OMPs such as HomB may be one mechanism by which ArsRS dictates biofilm development in a pH responsive manner.

Project description:With the widespread use and abuse of antibiotics, Helicobacter pylori (H. pylori) has become seriously drug resistant. The development of new antibiotics is an important way to solve H. pylori's drug resistance. Screening antibacterial ingredients from natural products is a convenient way to develop new antibiotics. Phillygenin, an effective antibacterial component, was selected from the natural product, forsythia, in this study. Its minimal inhibitory concentration (MIC) for 18 H. pylori strains was 16-32 μg/ml. The minimum bactericidal concentration (MBC) of H. pylori G27 was 128 μg/ml; the higher the drug concentration and the longer the time, the better the sterilization effect. It was non-toxic to gastric epithelial cell (GES)-1 and BGC823 cells at the concentration of 100 μg/ml. It presented a better antibacterial effect on H. pylori in an acidic environment, and after 24 days of induction on H. pylori with 1/4 MIC of phillygenin, no change was found in the MIC of H. pylori. In the mechanism of action, phillygenin could cause ATP leakage and inhibit the biofilm formation; the latter was associated with the regulation of spoT and Hp1174 genes. In addition, phillygenin could regulate the genes of Nhac, caggamma, MATE, MdoB, flagellinA, and lptB, leading to the weakening of H. pylori's acid resistance and virulence, the diminishing of H. pylori's capacity for drug efflux, H. pylori's DNA methylation, the initiation of human immune response, and the ATP leakage of H. pylori, thus accelerating the death of H. pylori. In conclusion, phillygenin was a main ingredient inhibiting H. pylori in Forsythia suspensa, with a good antibacterial activity, high safety, strong specificity, better antibacterial effect under acidic conditions, and low risk of resistance development by H. pylori. Its mechanism of action was mainly associated with inhibiting the biofilm formation and resulting in ATP leakage. In addition, phillygenin was shown to be able to reduce the acid resistance and virulence of H. pylori.

Project description:UnlabelledHelicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ΔnikR ΔarsS, and Δfur ΔnikR ΔarsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation.ImportanceDespite a paucity of regulatory proteins, adaptation is key to the survival of H. pylori within the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.

Dataset Information

SpoT-mediated NapA upregulation promotes oxidative stress-induced Helicobacter pylori biofilm formation and confers multidrug resistance.

Publications

SpoT-mediated NapA upregulation promotes oxidative stress-induced <i>Helicobacter pylori</i> biofilm formation and confers multidrug resistance.

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