Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR

Couesnon, Aurélie; Raffestin, Stéphanie; Popoff, Michel R.

doi:10.1099/mic.0.28561-0

Cited by 48 publications

(75 citation statements)

References 40 publications

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“…Although data for cntC are not reported, relative expression is anticipated to be similar to that of the other hemagglutinin-encoding genes (cntD and cntE), since all three form an operon regulated by the alternative sigma factor CntR. Indeed, their cotranscription has been reported by Couesnon et al (10). Expression of the regulatory gene, cntR, was consistent with the remainder of the neurotoxin gene cluster but at a lower rate, as observed previously (10).…”

Section: Resultssupporting

confidence: 51%

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Artin

Mason

et al. 2010

Appl Environ Microbiol

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The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO 2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO 2 had little effect on any of these parameters. At all CO 2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.Proteolytic Clostridium botulinum and nonproteolytic C. botulinum are the two neurotoxigenic clostridia most frequently associated with food-borne botulism (22,23,24), an intoxication caused by consumption of contaminated food containing preformed botulinum neurotoxin. The botulinum neurotoxins are the most potent toxins known, with as little as 30 ng being potentially fatal (24, 25). There are seven botulinum neurotoxin subtypes (A to G) and also a number of subtypes, of which subtype A1 has received the most attention to date (25 Carbon dioxide is frequently used as an antimicrobial gas in modified atmosphere packaging (MAP) of food (14,22,23). However, strains of nonproteolytic C. botulinum types B and E, despite having increased lag time and reduced growth rate (13, 15), display an increase in both neurotoxin gene expression and neurotoxin formation with higher CO 2 concentrations (5, 20). The purpose of the present study was to quantify the effect of CO 2 concentration on the growth, relative gene expression, and neurotoxin production by proteolytic C. botulinum type A1 strain ATCC 3502. Relative expression of cntA/A1 and cntR was measured using a new quantitative reverse transcription-PCR (qRT-PCR) method, and a DNA microarray was used to study the transcriptome. MATERIALS AND METHODSCulture conditions and growth parameters. Overnight cultures of proteolytic C. botulinum type A1 strain ATCC 3502 were grown in TPY medium flushed with the appropriate gas mixture (10%, 35%, or 70% CO 2 ; 10% H 2 ; and the balance N 2 [by vol]) and used to inoculate flasks of similarly treated TPY-C broth (5). The initial pH values of the media were 6.8 to 6.4, reflecting the increasing CO 2 concentration. Cultures were grown at 37°C in an anaerobic workstation with the same atmosphere, each in triplicate. Samples for RNA and neurotoxin measurement were withdrawn at four to nine time points during the experiment. Maximum specific growth rates [(OD)] of the change in optical...

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Section: Resultssupporting

confidence: 51%

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Artin

Mason

et al. 2010

Appl Environ Microbiol

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“…TEM micrographs of several type E strains did not consistently show morphologically distinct filaments, suggesting that FlaA1/A2 and FlaB may be found in the same filament. Type E strains possess other unique characteristics, including the mechanism of regulation of the type E BoNT (9).…”

Section: Discussionmentioning

confidence: 99%

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification

Paul

Twine

Tam

et al. 2007

Appl Environ Microbiol

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“…Expression and Purification of Recombinant Proteins-The BoNT isoform A holotoxin was purified as previously described (21). The E. coli strain XL1Blue (Novagen, Madison, WI) was used as the host for recombinant proteins productions.…”

Section: Methodsmentioning

confidence: 99%

Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

Galloux

Vitrac

Montagner

et al. 2008

Journal of Biological Chemistry

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The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

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Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR

Cited by 48 publications

References 40 publications

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification

Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

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