Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.Peer reviewe

Confirmation of tagging <i>C. botulinum</i> BoNT-encoding plasmids pBotCDC-A3-Erm (strain CDC-A3), pCLJ-Erm (strain 657Ba) and pCLL-Erm (strain Eklund 17B) by PFGE and Southern hybridization analysis.

<p>(<b>B</b>) Ethidium bromide stained PFGE of nondigested DNA samples from <i>C. botulinum</i> strains: wild type CDC-A3 (Lane 1), CDC-A3580s1 (Lane 2), wild type 657Ba (Lane 3), 657Ba-CT4 (Lane 4), wild type Eklund 17B (Lane 5) and Eklund17B-CT11 (Lane 6); Lambda PFG Marker (Lane M), (New England Biolabs). The position of BoNT-encoding plasmids is indicated with arrows. (<b>A</b>) Southern hybridization with the <i>bont/A3</i> probe (Lanes 1 and 2); the <i>bont/bvB</i> probe (Lanes 3 and 4) and the <i>bont/npB</i> probe (Lanes 5 and 6); (<b>C</b>) Southern hybridization with the <i>ermB</i> probe. PFGE conditions: 6V/cm, 12°C, 1–20 s pulse time, 24 h.</p

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1–A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy

Comparison of predicted ORFs of pCLL with plasmids of <i>Clostridium perfringens</i>.

<p>Comparison of predicted ORFs of pCLL with plasmids of <i>Clostridium perfringens</i>.</p

Confirmation of plasmid pCLL-Erm transfer from <i>C. botulinum</i> strain Eklund 17BCT11 to strain LNT01.

<p>(<b>A</b>) Ethidium bromide stained PFGE of <i>C. botulinum</i> strains: LNT01 (Lanes 1 and 7), wild type strain Eklund 17B (Lanes 2 and 8); Eklund 17BCT11 (Lanes 3 and 9) and LNT01 transconjugants (pCLL-Erm) (Lanes 4–6 and Lanes10–12). Lanes 1–6, nondigested DNA samples; Lanes 7–12, <i>Nar</i>I digested DNA samples. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pCLL plasmid is indicated with an arrow. Southern hybridization with: (<b>B</b>) the <i>ermB</i> probe, and (<b>C</b>) the <i>bont/npB</i> probe. PFGE conditions: 6V/cm, 12°C, 1–20 s pulse time, 24 h.</p

Confirmation of tagging <i>C. botulinum</i> BoNT-encoded plasmids pBotCDC-A3-Erm (strain CDC-A3), pCLJ-Erm (strain 657Ba) and pCLL-Erm (strain Eklund 17B) by PCR analysis.

<p>PCR products of wild-type <i>C. botulinum</i> strains CDC-A3 (Lane 1), 657Ba (Lane 4) and Eklund 17B (Lane 7) and two erythromycin resistant, thiamphenicol sensitive clones of each of CDC-A3 (Lanes 2 and 3), 657Ba (Lanes 5 and 6) and Eklund 17B (Lanes 8 and 9) strains; 1 kb Plus ladder (Invitrogen) (Lane M).</p