Dataset Information

Uptake of clostridium botulinum C3 exoenzyme into intact HT22 and J774A.1 cells.

ABSTRACT: The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-?-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae.

SUBMITTER: Rohrbeck A

PROVIDER: S-EPMC4344630 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Uptake of clostridium botulinum C3 exoenzyme into intact HT22 and J774A.1 cells.

Rohrbeck Astrid A von Elsner Leonie L Hagemann Sandra S Just Ingo I

Toxins 20150202 2

The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, sug ...[more]

PMID: 25648844

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Project description:The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships. Clostridium botulinum produces botulinum neurotoxin (BoNT)and is classified as a “Category A” select agent. BoNT can be classified into seven serotypes designated A-G. There is considerable genetic variation within these serotypes, as demonstrated by the recognition of at least 47 subtypes. The most studied serotype, BoNT/A, has been found in a large and diverse group of clostridia, most of which express the subtype BoNT/A1. The BoNT/A1 producing C. botulinum strain ATCC 3502, used to obtain an initial annotated genome sequence, is not representative of the diverse clostridia group producing BoNT. Nearly 50% of C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed variants of BoNT/B with a distinct cluster arrangement. This nucleotide cluster is completely absent from the published genome sequence. In addition, a recently identified novel BoNT/A1 strain lacks the gene cluster seen in the genome sequence of ATCC 3502. Furthermore, a strain designated Hall A Hyper differs greatly from the sequenced strain as indicated by its ability to produce higher quantities of BoNT/A1. The genetic and phenotypic basis for this difference in BoNT expression is currently unknown, and the sequences of the BoNT gene and the cluster are identical in both strains. This observation supports the hypothesis that genes outside the toxin cluster are involved in the regulation and maturation of BoNT. The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a “species” DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 20 diverse C. botulinum strains were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for C. botulinum group members. The availability of the C. botulinum species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of C. botulinum group.

Dataset Information

Uptake of clostridium botulinum C3 exoenzyme into intact HT22 and J774A.1 cells.

Publications

Uptake of clostridium botulinum C3 exoenzyme into intact HT22 and J774A.1 cells.

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