A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

Hutton, Melanie L.; D’Costa, Kimberley; Rossiter, Amanda E.; Wang, Lin; Turner, Lorinda; Steer, David; Masters, Seth L.; Croker, Ben A.; Kaparakis‐Liaskos, Maria; Ferrero, Richard L.

doi:10.3389/fcimb.2017.00219

Cited by 25 publications

(25 citation statements)

References 51 publications

(95 reference statements)

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“…B. burgdorferi and Helicobacter pylori incorporate host cholesterol (Crowley et al ., ; Correia et al ., ) and through the action of a galactose and a glucose transferase, respectively (Lebrun et al ., ; Ostberg et al ., ), make cholesterol glycolipids (Hirai et al ., ). In B. burgdorferi , one of these cholesterol glycolipids, cholesteryl 6‐O‐palmitoyl‐β‐ d ‐galactopyranoside (ACGal), is essential in the formation of lipid rafts (Huang et al ., ), and a recent study detected a flotillin homolog enriched in detergent resistant membranes (DRM), suggesting the presence of lipid rafts in H. pylori (Hutton et al ., ).…”

Section: Introductionmentioning

confidence: 97%

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Toledo

Huang

Coleman

et al. 2018

Molecular Microbiology

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Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.

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Section: Introductionmentioning

confidence: 97%

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Toledo

Huang

Coleman

et al. 2018

Molecular Microbiology

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“…The main limitation of this method is that we were unable to generate mutants in the mouse‐colonizing H pylori SS1 and PMSS1 strains, and although kanamycin‐sensitive and 2‐DOG‐resistant colonies could be isolated, they did not contain the desired mutations (data not shown). It is generally known that SS1 and PMSS1 are not as easily amenable to genetic manipulation as other H pylori strains . Nevertheless, given that antibiotic‐resistant and 2‐DOG‐resistant colonies were isolated, it is likely that, with further optimization, this method may be used to generate mutants in this and other less genetically tractable strains .…”

Section: Discussionmentioning

confidence: 99%

“…Although many H pylori genes have been characterized by cloning and expression in laboratory E coli strains, expression of some H pylori proteins can be deleterious to E coli and so prevents their characterization. Traditional methods for H pylori mutagenesis often involve multiple cloning steps, which precede transformation with donor DNA to generate mutants marked with an antibiotic resistance cassette at the target loci . However, integration of an antibiotic resistance cassette introduces pitfalls to subsequent phenotypic analysis of isolated mutants as antibiotic resistance determinants can introduce polar effects on downstream gene expression .…”

Section: Introductionmentioning

confidence: 99%

Complete genome sequence of Helicobacter pylori B128 7.13 and a single‐step method for the generation of unmarked mutations

et al. 2019

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Background Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction‐modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori , there are relatively few genetic tools for the mutagenesis of this bacterium. Materials and Methods Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter‐selection marker, galactokinase from Escherichia coli . Results We show that this mutagenesis strategy can be used to generate in‐frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori‐ specific DUF874 family protein of unknown function. Conclusions This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.

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“…Indeed, the absence of FLOT reduced the cell scattering output which is mainly attributed to CagA. In vivo experiments showed the absence of FLOT reduced H. pylori colonization in the mice model …”

Section: Virulence Factorsmentioning

confidence: 96%

“…Flolitin‐like protein is described as a membrane raft–associated protein . Protein analysis revealed that FLOT is located in the membrane.…”

Section: Virulence Factorsmentioning

confidence: 99%

Pathogenesis of Helicobacter pylori infection

2018

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In this review, we highlight progress in the last year in characterizing known virulence factors like flagella and the Cag type IV secretion system with sophisticated structural and biochemical approaches to yield new insight on the assembly and functions of these critical virulence determinants. Several aspects of Helicobacter pylori physiology were newly explored this year and evaluated for their functions during stomach colonization, including a fascinating role for the essential protease HtrA in allowing access of H. pylori to the basolateral side of the gastric epithelium through cleavage of the tight junction protein E-cadherin to facilitate CagA delivery. Molecular biology tools standard in model bacteria, including regulated gene expression during animal infection and fluorescent reporter gene fusions, were newly applied to H. pylori to explore functions for urease beyond initial colonization and establish high salt consumption as a mediator of gene expression changes. New sequencing technologies enabled validation of long postulated roles for DNA methylation in regulating H. pylori gene expression. On the cell biology side, elegant work using lineage tracing in the murine model and organoid primary cell culture systems has provided new insights into how H. pylori manipulates gastric tissue functions, locally and at a distance, to promote its survival in the stomach and induce pathologic changes. Finally, new work has bolstered the case for genomic variation as an important mechanism to generate phenotypic diversity during changing environmental conditions in the context of diet manipulation in animal infection models and during human experimental infection after vaccination.

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A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

Cited by 25 publications

References 51 publications

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Complete genome sequence of Helicobacter pylori B128 7.13 and a single‐step method for the generation of unmarked mutations

Pathogenesis of Helicobacter pylori infection

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