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Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

ABSTRACT: The concept of Helicobacter pylori biofilm formation is relatively new. To help provide a foundation for future biofilm studies, we characterized the biofilm formation ability of a common H. pylori lab strain, G27. The goal of this study was to evaluate biofilm formation by G27 in response to common culture conditions and to explore the biofilm matrix. Our results indicate that while various types of growth media did not dramatically affect biofilm formation, surface selection had a significant effect on the final biofilm mass. Furthermore, enzymatic assays and confocal microscopy revealed that proteins appear to be the primary structural component of the H. pylori extracellular matrix; extracellular DNA (eDNA) and polysaccharides were also present but appear to play a secondary role. Finally, we found that two well-characterized antibiofilm cationic peptides differentially affected early and late-stage biofilms. Together these results provide interesting avenues for future investigations that will seek to understand H. pylori biofilm formation.IMPORTANCE The study of H. pylori biofilm formation is still in its infancy. As such, there is great variability in how biofilm assays are performed across labs. While several groups have begun to investigate factors that influence H. pylori biofilm formation, it is not yet understood how H. pylori biofilm formation may vary based on commonly used conditions. These inconsistencies lead to difficulties in interpretation and comparison between studies. Here, we set out to characterize biofilm formation by a commonly available lab strain, G27. Our findings provide novel insight into optimal biofilm conditions, the biofilm matrix, and possible mechanisms to block or disrupt biofilm formation.

SUBMITTER: Windham IH

PROVIDER: S-EPMC6029101 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

Windham Ian H IH Servetas Stephanie L SL Whitmire Jeannette M JM Pletzer Daniel D Hancock Robert E W REW Merrell D Scott DS

Applied and environmental microbiology 20180702 14

The concept of <i>Helicobacter pylori</i> biofilm formation is relatively new. To help provide a foundation for future biofilm studies, we characterized the biofilm formation ability of a common <i>H. pylori</i> lab strain, G27. The goal of this study was to evaluate biofilm formation by G27 in response to common culture conditions and to explore the biofilm matrix. Our results indicate that while various types of growth media did not dramatically affect biofilm formation, surface selection had ...[more]

PMID: 29752266

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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:UNLABELLED:The gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (?luxS strains) or are defective for chemotaxis (?cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxS(OP)) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ?luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal. IMPORTANCE:Bacterial biofilms are ubiquitous in nature, but the mechanisms governing their assembly and spatial organization are not fully understood. Bacterial communication through quorum sensing has been shown to influence biofilm growth through the regulation of biofilm genes. Our study revealed a new role for quorum sensing in biofilms through rapid chemotactic responses to quorum signals. Specifically, we studied how chemorepulsion of Helicobacter pylori from the universal quorum signal autoinducer-2 (AI-2) shapes the spatial organization of its biofilms. We demonstrate that the chemorepulsive response of H. pylori to AI-2 is necessary to promote its dispersal from biofilms grown on both abiotic and biotic surfaces and is sufficient to promote dispersal in a chemotaxis-dependent manner. This work has broad implications for understanding the mechanisms by which endogenously produced microbial compounds shape the assembly and spatial organization of microbial communities in their environments.

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Dataset Information

Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

Publications

Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

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