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Unité des Bactéries anaérobies et Toxines, Institut Pasteur, Paris, France
Correspondence
Michel R. Popoff
mpopoff{at}pasteur.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Meunier et al., 2002; Schiavo et al., 2000).
BoNTs and associated non-toxic proteins (ANTPs) form complexes of various sizes (300–900 kDa). ANTPs consist of a non-toxic non-haemagglutinin (NTNH) component, the size of which is similar to BoNT, and haemagglutinin (HA) components. ANTPs spontaneously associate with BoNT at low pH by non-covalent bonds and form stable complexes that dissociate at high pH (pH 8 and above) (reviewed by Popoff & Marvaud, 1999). The precise role of ANTPs and botulinum complexes remains unclear, but they possibly protect BoNT against acidic pH and proteolytic degradation during passage through the stomach and intestine. ANTPs might also be involved in transporting BoNT across the intestinal barrier (Fujinaga et al., 1997, 2000).
In C. botulinum A, bont and antp genes are clustered in a chromosomal DNA segment called the botulinum locus (reviewed by Henderson et al., 1997; Popoff & Marvaud, 1999). These genes are organized in two operons. The first operon (ntnh/A-bont/A), which is located at the 3' end of the botulinum locus, encompasses the bont gene immediately preceded by the ntnh/A gene. Both genes are transcribed in the same orientation, and the organization of this operon is highly conserved in all C. botulinum types. The second operon contains the ha genes and differs slightly between various toxinotypes. In C. botulinum A, the has operon contains successive genes for the 34 kDa (ha34), 17 kDa (ha17), and 70 kDa (ha70) haemagglutinins (Henderson et al., 1996). Genes of the ha operon are transcribed in an opposite orientation versus the ntnh/A-bont/A genes. The botR gene, which has features of a DNA-binding protein (i.e. highly basic isoelectric point and helix–turn–helix motif), is localized at the 5' end of the botulinum locus in C. botulinum types C and D and between the two operons in C. botulinum types A, B and F (Henderson et al., 1997; Popoff & Marvaud, 1999). botR/A from C. botulinum type A has been characterized as a transcriptional activator of bont and ha genes based on botR/A overexpression or partial inhibition by antisense mRNA in C. botulinum (Marvaud et al., 1998b). Recently, BotR/A was found to be an alternative sigma factor of RNA polymerase, which forms, with Clostridium tetani TetR, Clostridium difficile TxeR and Clostridium perfringens UviA, a new subgroup of the sigma 70 family of RNA polymerase involved in controlling clostridial toxin genes (Raffestin et al., 2005).
Genetic organization of the botulinum locus is slightly different in C. botulinum E strains, which are non-proteolytic bacteria unable to activate BoNT/E. The BoNT/E locus contains no gene related to botR, but immediately upstream of ntnh/E-bont/E and in a position equivalent to that of botR/A lies a gene encoding a 47 kDa protein (p47). The second operon encompasses orfX1 and orfX2 instead of ha genes (Hatheway, 1993; Popoff & Marvaud, 1999).
In this study, we report that the expression of botR/A is concomitant with that of bont/A and antp. This expression occurs during the transition from the exponential to the stationary growth phase, as assessed by quantitative reverse transcription-PCR (RT-PCR). In C. botulinum E, bont/E and p47 are also expressed during the transition phase. In addition, high temperature (44 °C) does not act as a BotR/A-mediated regulation signal, but activates a protease which degrades BoNT/A and NTNH/A.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Kinetic experiments were performed by incubating 500 ml TGY inoculated with either 10 ml of an overnight preculture (non-sporulated cultures) or 10 ml of a culture containing 0·5 ml heat-activated (10 min, 95 °C) spore suspension (about 2x107 spores ml–1). Bacterial growth was monitored as OD600. Culture samples (5–10 ml) were removed at 3, 6, 9, 10, 11, 12, 24 and 48 h. Bacteria were collected by centrifugation and stored at –20 °C until RNA extraction, and the supernatants were stored at 4 °C for mouse bioassays. Total DNA was prepared as previously described (Marvaud et al., 1998b), and at least three experiments were independently performed for each strain and condition.
Total RNA extraction.
Bacterial pellets were washed and suspended in 1 ml distilled water treated with diethyl pyrocarbonate (DEPC, Sigma). For total RNA extraction, 400 µl bacterial suspension was used when the culture OD600 was below 0·4, 200 µl when the OD600 ranged between 0·4 and 2, and 100 µl otherwise. Bacteria were centrifuged at 4 °C (7500 g, 10 min) and total RNA was extracted using a RNeasy Mini kit (Qiagen) according to the manufacturer's guidelines. Contaminating genomic DNA was degraded by treating the 60 µl RNA sample with 2 U DNA-free DNase (Ambion) and 1x reaction buffer. The reaction mixture was incubated at 37 °C for 30 min and the DNase was removed by adding DNase Inactivation Reagent (DNase-free, 0·1x) (Ambion) to the mixture. Total RNA concentrations were determined by measuring A260 and the formula 1 A260 unit=40 µg RNA. RNA purity was monitored using the ratio A260/A280 (between 1·8 and 2·2) and real-time PCR was then done on the RNA samples, which were immediately used as a template in cDNA synthesis.
Reverse transcription.
First-strand cDNA synthesis was performed in triplicate with random hexamers PDN6 and the M-MLV kit (Invitrogen) in a 20 µl final volume according to the handbook protocol. The reaction mixture contained 1 µg total RNA, 25 ng PDN6 µl–1 (Roche Boehringer), 10 mM each nucleotide dATP, dTTP, dCTP and dGTP (Amersham Biosciences), 40 U RNA guard RNase inhibitor (Amersham Biosciences), 0·01 M DTT (Invitrogen), 1x first-strand buffer (Invitrogen), and 200 U M-MLV reverse transcriptase (Invitrogen). Before RT enzyme was added, the reaction mixture was heated to 65 °C for 5 min and thereafter chilled on ice. It was then incubated for 10 min at 25 °C and 50 min at 37 °C. The reaction was inactivated by heating at 70 °C for 15 min. The three replicates were pooled and aliquoted. An aliquot was diluted 10-fold in distilled DEPC-treated water. cDNA solutions were stored at –20 °C before PCR amplification.
Quantitative real-time PCR assay.
The real-time PCR assay was based on the double-stranded DNA-specific dye SYBR Green I methodology. The primers used are listed in Table 1. A primer pair specific for each of six genes in the botulinum gene locus (bont/A, ntnh/A, ha17, ha34, ha70 and botR/A) or the 16S rRNA gene was designed from the Hall A genome sequence (http://www.ncbi.nlm.nih.com) with Beacon Designer version 2.0 (Bio-Rad). Primers were also designed for bont/E, p47 and the 16 rRNA gene from C. botulinum E (X62089 for bont/E, D88418 for p47 and L37592 for 16 rRNA).
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Quantitative analysis was performed using the iCycler iQ Real-Time PCR Detection System Software Version 3.0 (Bio-Rad). Amplification plots displaying fluorescence for each well at every cycle were obtained. Each sample was amplified in triplicate. The background fluorescence was automatically removed by setting a noise band. The threshold cycle (cT) for each transcript was determined as the PCR cycle at which the fluorescence rose above background. Because the cT is inversely proportional to the logarithm of the initial number of template molecules, the threshold cycle value was used in computation for quantification of gene expression. A standard curve consisting of the cT versus log chromosomal DNA dilutions (corresponding to the log copy numbers) was generated by amplifying serial dilutions of chromosomal DNA corresponding to an unknown amount of amplicon. For each primer set, PCR was performed in parallel reactions using different amounts of Hall A chromosomal DNA (150 ng to 1·5 pg) and each cDNA sample in the same 96-well analysis. For each target gene, the software determined the unknown concentration in the cDNA samples by interpolating the cT of the sample against the standard curve of known concentrations.
Expression levels of cDNA from the six botulinum locus genes were standardized against 16S rRNA gene expression. The 16S rRNA gene primer set was run concurrently with each set of experimental primers. At different points during the growth kinetics, relative expression of the six genes (X1, X2,..., Xi,..., X6) compared to 16S rRNA gene expression (Y) was determined by the formula Xi=Xi'/Y. Xi' is the arbitrary genomic DNA dilution given by cT values on a standard curve and corresponds to the amount of gene i transcripts in DNA samples.
The identity and specificity of PCR products were verified by high-resolution agarose gel electrophoresis, and a single PCR product migrating at the expected size was observed for each primer pair. In addition, an iQ iCycler melting curve confirmed the presence of a unique peak for the negative first derivative of the temperature versus fluorescence plotted against temperature (melt curve) with single product-specific melting temperatures (Tm). For this, fluorescence was continuously monitored in samples heated from 55 to 95 °C at a transition rate of 0·1 °C. In order to check for amplification of any contaminating genomic DNA in the RNA sample after extraction or in the PCR mixture, DNase-treated RNA and sterile water were added to the PCR.
Mouse bioassay.
Serial twofold dilutions of supernatant samples (0·5 ml) in 50 mM sodium phosphate buffer (pH 6·3) containing 0·2 % (w/v) gelatin were injected intraperitoneally into mice weighing 18–20 g. For type E supernatant, the toxin was activated with trypsin (200 µg ml–1, Sigma) for 15 min at room temperature prior to dilution. Three mice were used for each dilution. The mice were observed over 4 days, and the number that died was recorded, with results expressed as mouse lethal dose (MLD) per millilitre.
Toxin stability assay.
One-millilitre aliquots of supernatant from a 24 h Hall culture were incubated at 44 °C for different time periods: 30 min, 3 h, 6 h and 24 h. The supernatant was also incubated with protease inhibitors: EDTA (0·5 and 1 mM), EGTA (5 and 1 mM), o-phenanthroline (5 mM), PMSF (1 mM), pepstatin (10 µg ml–1) and benzamidine (1 mM) for 6 h at 44 °C. Toxin was then precipitated at pH 3·0 with 10 M H2SO4 and centrifuged 10 min at 7500 g and 4 °C. The precipitates were washed with distilled water and resuspended in 0·2 M phosphate buffer, pH 6·0. Protein concentration was determined by the Bradford method (Bio-Rad) using BSA as standard.
PAGE and Western blotting.
Each precipitate (5 µg total protein) was separated by electrophoresis on SDS-polyacrylamide gels (10 %). Proteins were transferred to PVDF membranes (Hybond-C, Amersham), which were blocked with 5 % skimmed milk (w/v) in PBS (pH 7·2) and incubated 1 h at room temperature with rabbit antibodies (1/1000) against purified recombinant BoNT/A (Hc or Lc), NTNH/A, HA34 or HA52 (processed form of HA70) (Tavallaie et al., 2004). DNAs encoding BoNT/A Hc (amino acids 872–1296), Lc (amino acids 1–438), NTNH/A (amino acids 783–1206), HA34 (amino acids 1–293) and HA52 (amino acids 203–626) were cloned in pET28 vector in fusion with a N-terminal His6-tag, and the recombinant proteins were purified on a cobalt column (Clontech) as previously described (Tavallaie et al., 2004). After three washes in PBS/0·1 % Tween 20, the blots were treated with horseradish peroxidase-conjugated protein A (Bio-Rad) and analysed by the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The lag phase was slightly longer at 5–6 h versus 2–3 h in non-sporulated cultures, but the growth curve was similar, although strain Hall showed a slower decrease in OD during the stationary phase in sporulated cultures compared to strain NCTC 2916 (Fig. 1b). Spores were determined by phase-contrast microscopy and expressed as a percentage of the total bacterial population. C. botulinum strain NCTC 2916 was selected since it yielded a high percentage of sporulation in our culture conditions. Spores of strain NCTC 2916 were detected within 10 h after inoculation, corresponding to the transition phase, and increased to 65 % at 24 h and almost 100 % at 48 h. In contrast, spores of strain Hall were not apparent until 32 h after inoculation of the culture and then increased to 28 % at 48 h and reached only 85 % at 156 h.
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). These results were in agreement with previous reports (Bradshaw et al., 2004; Siegel & Metzger, 1979).
Relative expression of the type A botulinum locus genes by real-time PCR
Gene expression was assayed by mRNA reverse transcription and cDNA amplification with real-time PCR. The amplified products were quantified by measuring SYBR Green fluorescence incorporated into double-stranded DNA resulting from a single specific gene product as determined by melting temperature curves. For this purpose, a primer pair was designed for each target gene in order to define a DNA fragment having a high Tm. Since the C. botulinum genome has a low G+C content, the Tm of the PCR products of the botulinum toxin locus genes ranged from 76·5 to 81 °C (Table 1). However, the Tm of the PCR product the of 16S rRNA gene, which has a greater G+C content (52 mol% versus 21–28 mol% for the botulinum toxin locus genes), was higher (88 °C). Despite the different Tm values, calibration assays of PCR amplification of each gene with C. botulinum Hall or NCTC 2916 genomic DNA showed a correlation coefficient above 0·995 over 40 amplification cycles, indicating a linear measurement of 15 pg to 150 ng DNA (data not shown). All culture samples submitted to RT and cDNA real-time PCR yielded DNA amplification within this linear range. In addition, RNA samples prior to reverse transcription treatment did not yield any significant PCR amplification, indicating that DNA contamination was negligible.
Quantitative expression of the 16 rRNA gene as calculated by fluorescence intensity per microgram of RNA was constant over bacterial growth, at least until 48 h, in sporulated and non-sporulated cultures. Therefore, the 16S rRNA gene was used as a calibrator gene. An equal amount of total mRNA from each sample was used for relative quantification of gene expression. The levels of each gene transcript were then normalized to the 16S rRNA gene. Transcript of each botulinum toxin locus gene was detectable in all stages of growth in both strains and in sporulated and non-sporulated cultures. However, the relative expression of botR/A in C. botulinum strains Hall and NCTC 2916 was approximately 100-fold less than that of the other botulinum toxin locus genes.
Relative quantification of gene expression within the botulinum toxin locus, versus that of the 16 rRNA gene, revealed a concomitant expression of the botR/A, bont/A and antp genes in sporulated and non-sporulated cultures (Fig. 2). The expression pattern over time was similar for all these genes in both strains. A low expression level was detected at the beginning of exponential growth, followed by a rapid increase in expression during late exponential growth. A maximum expression value was reached at the transition phase (10 h incubation) and then expression decreased during the stationary phase. Relative expression of each gene within the botulinum toxin locus was increased 100-fold in the transition phase versus levels in the early exponential phase. After 24 h incubation, relative gene expression decreased to levels found in the early growth phase. Thereafter, the expression remained at basal levels during stationary phase, with a slight transient increase at 48 h.
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shows quantification of the botulinum toxin locus gene expression versus the 16 rRNA gene obtained from three independent sporulated and non-sporulated cultures. In both strains and culture conditions, the relative expression of each botulinum toxin locus gene predominated at the transition phase. In the non-sporulated Hall strain, the relative levels of bont/A and antp transcripts at the transition phase were similar, whereas in sporulated cultures each ha gene was expressed in higher amounts (about twofold) compared to the bont/A and ntnh/A genes. In contrast, C. botulinum NCTC 2916 showed similar values of relative expression in both sporulated and non-sporulated cultures. However, the botulinum toxin locus genes were expressed in different amounts: the relative expression of bont/A was approximately twice that of the ntnh/A gene, and ha34 transcript levels were about twofold higher than those of ha17 and ha70 (Fig. 3).
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Stability of BoNT/A and ANTPs at 44 °C
To further explore the temperature-dependent stability of BoNT/A and ANTPs, supernatant from a 37 °C Hall culture was incubated at 44 °C. As shown in Fig. 4(a), a progressive decrease in lethal activity was observed, similar to that found in a 44 °C culture during the 24–48 h incubation period. BoNT/A and ANTPs were monitored over time by Western blotting of culture supernatant incubated at 44 °C. BoNT/A significantly decreased after 0·5 h incubation at 44 °C and was not detected at 3 h or longer incubation times as monitored with antibodies specific for BoNT/A L or H chains (Fig. 4b). A similar pattern was observed with NTNH/A. After 0·5 h incubation at 44 °C, only a small amount of uncleaved NTNH/A (119 kDa) was detected, whereas the processed form (106 kDa) was not evident. In contrast, HA protein levels did not change during 24 h incubation at 44 °C (Fig. 4b). This suggests that BoNT/A and NTNH/A were sensitive to a proteolysis at 44 °C, in contrast to HA proteins. Among the inhibitors for various proteases tested, only EDTA or EGTA (1 mM minimal concentration) blocked degradation of BoNT/A and NTNH/A (Fig. 4c, d). This indicated that a metalloprotease requiring Ca2+ but not Zn2+, as evidenced by o-phenanthroline results, was active at 44 °C (not 37 °C) and degraded BoNT/A and NTNH/A in crude culture supernatant from C. botulinum A strain Hall.
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). The sporulation pattern was the same as for NCTC 2916 in non-sporulated cultures (data not shown). BoNT/E production was monitored in trypsin-treated culture supernatant of C. botulinum E strain HV grown at 37 °C. As in C. botulinum A strains, BoNT/E progressively increased during the exponential growth phase and reached its maximum value at the entry into stationary phase at about 12 h (Fig. 5a). The toxicity level of the culture supernatant was then stable until at least 48 h.
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).
BoNT/E and NTNH/E are stable in culture supernatant incubated at 44 °C
We tested whether a 44 °C incubation of supernatant from a 24 h culture of strain HV at 37 °C induced a proteolytic degradation of BoNT/E and NTNH/E as found in C. botulinum A strain Hall. No decrease in lethal activity was observed in the HV culture supernatant incubated at 44 °C until at least 24 h (Fig. 5c). In addition, BoNT/E and NTNH/E degradation was not evidenced by SDS-PAGE and Western blotting after incubation at 44 °C (Fig. 5d). This suggests that either no protease activated at 44 °C is produced by C. botulinum E or BoNT/E is more protease resistant than BoNT/A. To test this last hypothesis, we mixed equal volumes of 37 °C culture supernatant from Hall (type A) and HV (type E) strains. The mixtures were incubated at 37 °C or 44 °C for 24 h and the mouse lethal activity of BoNT/E was monitored after addition of a specific neutralizing anti-BoNT/A serum. After incubation at 37 °C, the level of mouse lethal activity was the same as in untrypsinized HV culture, indicating that BoNT/E was not activated by C. botulinum A proteases, in agreement with previous findings (DasGupta & Sugiyama, 1972; Dekleva & DasGupta, 1989). However, a 44 °C incubation induced a total loss of lethal activity, likely resulting from protease degradation (data not shown). This further argues that BoNT/E is sensitive to protease degradation induced by C. botulinum A incubated at 44 °C and that no such protease is produced by C. botulinum E.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), acting as an RNA polymerase alternative sigma factor specific for the two promoters controlling the two operons (ntnh/A-bont/A and ha-34-ha17-ha70) of the botulinum toxin locus (Raffestin et al., 2005). BotR/A is functionally related to TetR, which controls the expression of the tent gene in C. tetani in vivo (Marvaud et al., 1998a). Moreover, BotR and TetR are also related to the alternative sigma factors TxeR and UviA, which control expression of toxin genes in C. difficile and a bacteriocin gene in C. perfringens, respectively (Dupuy et al., 2005; Garnier & Cole, 1988; Mani & Dupuy, 2001). In this study, we analysed by real-time RT-PCR the relative expression of the botulinum toxin locus genes versus the 16 rRNA gene as calibrator gene (Broussolle et al., 2002), in C. botulinum A and C. botulinum E strains, which in contrast to C. botulinum A contain no gene related to botR. Real-time RT-PCR is a very sensitive method to quantify gene expression and has been used to investigate various bacterial virulence genes and in particular to detect C. botulinum in food and clinical samples (Kimura et al., 2001) and to monitor expression of neurotoxin genes in C. botulinum B and C. botulinum E (Lövenklev et al., 2004a; McGrath et al., 2000).
The patterns of relative expression for all botulinum toxin locus genes were identical in strains Hall and NCTC 2916 as well as in non-sporulated and sporulated cultures, although strain NCTC 2916 possesses a slightly different genetic organization, with two botulinum toxin locus genes. One is similar to that from type A1 strains, including ntnh, botR and has genes, but its bont/A gene is replaced with a silent bont/B gene, while the other locus is typical of type A2 strains but has a bont/A type A1 gene and is organized in two operons, p47-ntnh/A-bont/A1 and orfx1-orfx2-orfx3 (Dineen et al., 2004; Henderson et al., 1996; Jovita et al., 1998). The botulinum toxin locus genes were concomitantly expressed and their kinetics showed a bell-shaped curve (Fig. 2). A low expression level was measured during entry into exponential growth, and maximal expression (50- to 200-fold increase compared to the baseline) occurred during the transition phase. Expression of the botulinum toxin locus genes then decreased to baseline at about 24 h of culture. A small increase in gene expression was observed late (48 h) in sporulated strain Hall cultures. Synchronous expression of botR/A, bont/A and antp genes, as well as botR/A being expressed about 100-fold less than the other botulinum toxin locus genes, further supports the hypothesis that BotR/A is a regulatory factor controlling bont/A and antp gene expression. In addition, the RT-PCR results clearly show that botR/A, bont/A and antp gene expression was mainly restricted to late exponential growth/early stationary phase, indicating that BotR/A is an alternative sigma factor specific to the transition phase and independent of sporulation. The time correlation between the expression of BotR/A and botulinum toxin locus genes suggests that BotR/A is the main regulatory factor. However, this does not rule out that another regulatory or sigma factor is also involved. These results are in agreement with a recent report showing by Northern hybridizations that bontA, ntnhA and ha genes are temporally expressed during late exponential and early stationary phase in C. botulinum type A strains 62A, Hall A-hyper and NCTC 2916 (Bradshaw et al., 2004). It is noteworthy that TxeR from C. difficile is also predominantly expressed during the transition phase, as evidenced by a fusion between a 
-glucuronidase reporter gene and TxeR in C. perfringens (Karlsson et al., 2003).
Analysis of mRNA by Northern blotting and transcriptional study with purified BotR/A-RNA polymerase core enzyme has indicated that the bont/A and antp genes from C. botulinum type A1 strains such as Hall strain are transcribed in two divergent operons (ntnh/A-bont/A1 and ha34-ha17-ha70) and that bont/A1 can also be expressed separately (Bradshaw et al., 2004; Henderson et al., 1996; Raffestin et al., 2005). RT-PCR results showing equivalent expression levels of each gene of the botulinum toxin locus in the Hall strain are in agreement with the genetic organization in two operons. However, strain NCTC 2916 yielded twofold more expression of ha34 versus ha17 and ha70. It is noteworthy that relative amounts of HA proteins vary in the different-sized botulinum complexes. Indeed, the ratio of HA34 versus HA17 or HA70 is about twice in 19S compared to 12S complexes (Inoue et al., 1996; Oguma et al., 1999; Sharma et al., 2003). Regulation of HA production at the transcription and/or translation level in C. botulinum strains such as in NCTC 2916 remains to be defined.
BoNT/A was detected in the culture supernatant at low levels during early exponential growth and reached a maximum level at 24 h of culture, which remained stable until at least 72 h. No obvious bacterial lysis was observed during the transition and early stationary phases in either strain. This indicates that BoNT/A is esentially synthesized during the transition into early stationary phase and is transported in a delayed manner into the external medium. Similar patterns of growth and toxin production without apparent cell lysis were previously reported (Bradshaw et al., 2004; Siegel & Metzger, 1979). An exfoliation process without cell lysis has been reported (Call et al., 1995). It could be argued that BoNT degradation and resynthesis take place during stationary phase; however, BoNT/A is activated by an endogenous protease from C. botulinum and is stable in acidic cultures at 37 °C (DasGupta, 1989).
An important question is which are the environmental factors controlling neurotoxin gene production in C. botulinum. Although nitrogen and carbon sources are important for the toxinogenesis, specific external signals controlling toxinogenesis have not been clearly identified in C. botulinum or C. tetani. In contrast to C. difficile (Dupuy & Sonenshein, 1998), C. botulinum requires glucose for high toxin production. CO2 seems to stimulate bont/B gene expression and subsequent neurotoxin production in C. botulinum type B (Lövenklev et al., 2004b). Moreover, short peptides from media containing pancreatic digest of casein are essential for toxin production by C. tetani (Porfirio et al., 1997). Growth temperature is a common signal involved in the control of alternative sigma factors and subsequent regulated gene expression. Thereby a temperature of 42 °C prevents transcription of txeR and the subsequent expression of toxA and toxB genes in C. difficile (Karlsson et al., 2003). Cultures of C. botulinum Hall incubated at 44 °C showed an initial toxin production similar to that found in cultures at 37 °C and then a dramatic decrease in toxin activity after 24–48 h of growth (data not shown), which is in agreement with previous observations (Siegel & Metzger, 1979). However, no decrease in botR/A, bont/A and antp gene expression was observed in cultures at 44 versus 37 °C, indicating that declining toxin activity resulted not from gene regulation but rather from a post-translational event. Our data show that a calcium-dependent protease degraded BoNT/A in culture supernatant incubated at 44 °C. Interestingly, the same type of protease is responsible for BoNT/A activation into the di-chain form (Dekleva & DasGupta, 1990). Characteristics of the BoNT/A-activating protease from C. botulinum A include: (1) a thiol-reducing agent and Ca2+ requirement; (2) maximum activity in the pH range 6·2–6·4; and (3) a temperature optimum of 70 °C (Dekleva & DasGupta, 1990). Although weakly active at 37 °C, this protease efficiently activates BoNT/A by a single proteolytic nicking and excision of 10 residues between the light and heavy chains (Krieglstein et al., 1994), whereas increased activity at 44 °C caused neurotoxin degradation (Fig. 4) by cleaving multiple sites. This is further supported by the fact that prolonged exposure of BoNT/A to purified protease led to considerable toxin degradation in multiple fragments (Dekleva & DasGupta, 1990). Seemingly, C. botulinum A uses a temperature-dependent protease to control BoNT/A activation and degradation. We can not rule out that an increase in temperature also induces a conformational change in BoNT/A, leading to exposure of cleavage sites on the surface of the molecule. One of the functions for ANTPs may be to protect BoNT/A against protease degradation. As shown in Fig. 4, NTNH/A was as sensitive as BoNT/A to proteolytic degradation at 44 °C, whereas HA34 and HA52 were resistant. Trypsin or pepsin resistance of HA34 versus NTNH/A at 25 °C has already been observed (Chen et al., 1998; Fu et al., 1997; Sharma et al., 1999). Since BoNT/A from culture supernatant with increased temperature was degraded, it is excluded that HA proteins have a protective effect for the neurotoxin, at least against this type of protease.
In contrast to the other C. botulinum types, C. botulinum E strains do not contain an equivalent of the botR gene in their botulinum toxin locus, and no related gene has been identified in their genome (Quinn & Minton, 2001). However, p47, which encodes a 47 kDa protein, is located immediately upstream of ntnh/E and bont/E, similarly to botR/A in C. botulinum A (Popoff & Marvaud, 1999). In contrast to botR/A, p47 was expressed at a level similar to that of bont/E. This is further supported by the fact that C. botulinum E transformed with a recombinant plasmid expressing p47 antisense mRNA did not decrease BoNT/E synthesis (data not shown), in contrast to botR/A antisense mRNA in C. botulinum A (Marvaud et al., 1998b), indicating that P47 is probably not a regulator of the botulinum toxin E locus genes. Interestingly, bont/E and p47 were selectively expressed at the transition phase, as the botulinum toxin A locus genes, but by an as yet unknown regulator.
A possible regulation of the toxinogenesis in C. botulinum E by high temperature was investigated. However, C. botulinum E strains did not grow at 44 °C and only weakly at 40 °C. This group of bacteria is adapted to grow at low temperature and the optimum temperature is around 25–30 °C (Hatheway, 1989). Toxinogenesis was the same at 37 versus 30 °C (data not shown), suggesting that high temperature is probably not involved in toxin gene regulation by C. botulinum E. C. botulinum E strains are non-proteolytic and trypsin treatment of the culture supernatant is required to obtain fully active BoNT/E (Hatheway, 1989). In contrast to C. botulinum A strain Hall, no decrease in BoNT/E activity and no proteolytic degradation of BoNT/E and NTNH/E were apparent in culture supernatant incubated at 44 or 37 °C. This argues that a unique protease is involved in activation/degradation of BoNTs in proteolytic C. botulinum strains such as C. botulinum A.
In conclusion, the botR/A, bont/A and antp genes of C. botulinum A are mainly expressed during the transition from exponential into stationary phase, the level of botR/A expression being about 100-fold less than that for the bont/A and antp genes. This further argues that BotR/A is an alternative sigma factor controlling botulinum toxin A locus genes during the transition phase. In C. botulinum E, expression of bont/E and p47 is also positively regulated to an equivalent extent during the transition phase but by an unknown factor. Although high temperature is not an inhibitory signal for toxin gene regulation, it stimulates a calcium-dependent protease, which activates BoNT/A at 37 °C but degrades BoNT/A and NTNH/A at 44 °C. No such activating or degradative protease was found in C. botulinum E.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Broussolle, V., Alberto, F., Shearman, C. A., Mason, D. R., Botella, L., Nguyen-The, C., Peck, M. W. & Carlin, F. (2002). Molecular and physiological characterization of spore germination in Clostridium botulinum and Clostridium sporogenes. Anaerobe 8, 89–100.[CrossRef]
Call, J. E., Cooke, P. H. & Miller, A. J. (1995). In situ characterization of Clostridium botulinum neurotoxin synthesis and export. J Appl Bacteriol 79, 257–263.[Medline]
Chen, F., Kuziemko, G. M. & Stevens, R. C. (1998). Biophysical characterization of the stability of the 150-kilodalton botulinum toxin, the nontoxic component, and the 900-kilodalton botulinum toxin complex species. Infect Immun 66, 2420–2425.
DasGupta, B. R. (1989). The structure of the botulinum neurotoxins. In Botulinum Neurotoxin and Tetanus Toxin, pp. 53–67. Edited by L. L. Simpson. San Diego: Academic Press.
DasGupta, B. R. & Sugiyama, H. (1972). Role of protease in natural activation of Clostridium botulinum neurotoxin. Infect Immun 6, 587–590.
Dekleva, M. L. & DasGupta, B. R. (1989). Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease. Biochem Biophys Res Commun 162, 767–772.[CrossRef][Medline]
Dekleva, M. L. & DasGupta, B. R. (1990). Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into di-chain form. J Bacteriol 172, 2498–2503.
Dineen, S. S., Bradshaw, M., Karasek, C. E. & Johnson, E. A. (2004). Nucleotide sequence and transcriptional analysis of the type A2 neurotoxin gene cluster in Clostridium botulinum. FEMS Microbiol Lett 235, 9–16.[CrossRef][Medline]
Dupuy, B. & Sonenshein, A. L. (1998). Regulated transcription of Clostridium difficile toxin genes. Mol Microbiol 27, 107–120.[CrossRef][Medline]
Dupuy, B., Mani, N., Katayama, S. & Sonenshein, A. L. (2005). Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor. Mol Microbiol 55, 1196–1206.[CrossRef][Medline]
Fu, F. N., Sharma, S. K. & Singh, B. R. (1997). A protease-resistant novel hemagglutinin purified from type A Clostridium botulinum. J Protein Chem 17, 53–60.
Fujinaga, Y., Inoue, K., Watanabe, S., Yokota, K., Hirai, Y., Nagamachi, E. & Oguma, K. (1997). The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig intestine, leading to the efficient absorption of the toxin. Microbiology 143, 3841–3847.[Abstract]
Fujinaga, Y., Inoue, K., Nomura, T., Sasaki, J., Marvaud, J. C., Popoff, M. R., Kozaki, S. & Oguma, K. (2000). Identification and characterization of functional subunits of Clostridium botulinum A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Lett 467, 179–183.[CrossRef][Medline]
Garnier, T. & Cole, S. T. (1988). Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro. Mol Microbiol 2, 607–614.[CrossRef][Medline]
Hatheway, C. L. (1989). Bacterial sources of clostridial neurotoxins. In Botulinum Neurotoxin and Tetanus Toxin, pp. 3–24. Edited by L. L. Simpson. San Diego: Academic Press.
Hatheway, C. L. (1993). Clostridium botulinum and other clostridia that produce botulinum neurotoxin. In Clostridium Botulinum: Ecology and Control in Foods, pp. 3–20. Edited by A. H. W. Hauschild & K. L. Dodds. New York: Marcel Dekker.
Henderson, I., Whelan, S. M., Davis, T. O. & Minton, N. P. (1996). Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC2916. FEMS Microbiol Lett 140, 151–158.[CrossRef][Medline]
Henderson, I., Davis, T., Elmore, M. & Minton, N. (1997). The genetic basis of toxin production in Clostridium botulinum and Clostridium tetani. In The Clostridia: Molecular Biology and Pathogenesis, pp. 261–294. Edited by I. Rood. New York: Academic Press.
Humeau, Y., Doussau, F., Grant, N. J. & Poulain, B. (2000). How botulinum and tetanus neurotoxins block neurotransmitter. Biochimie 82, 427–446.[Medline]
Inoue, K., Fujinaga, Y., Watanabe, T., Ohyama, T., Takeshi, K., Moriishi, K., Nakajima, H., Inoue, K. & Oguma, K. (1996). Molecular composition of Clostridium botulinum type A progenitor toxins. Infect Immun 64, 1589–1594.[Abstract]
Jovita, M. R., Collins, M. D. & East, A. K. (1998). Gene organization and sequence determination of the two botulinum neurotoxin gene clusters in Clostridium botulinum. Curr Microbiol 36, 226–231.[CrossRef][Medline]
Karlsson, S., Dupuy, B., Mukherjee, K., Norin, E., Burman, L. G. & Akerlund, T. (2003). Expression of Clostridium difficile toxins A and B and their sigma factor TcdD is controlled by temperature. Infect Immun 71, 1784–1793.
Kimura, B., Kawasaki, S., Nakano, H. & Hujii, T. (2001). Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish. Appl Environ Microbiol 67, 206–218.
Krieglstein, K. G., DasGupta, B. R. & Henschen, A. H. (1994). Covalent structure of botulinum neurotoxin A: location of sulfhydryl bridges and identification of C-termini of light and heavy chains. J Protein Chem 13, 49–57.[CrossRef][Medline]
Lövenklev, M., Holst, E., Borch, E. & Radstrom, P. (2004a). Relative neurotoxin gene expression in clostridium botulinum type B, determined using quantitative reverse transcription-PCR. Appl Environ Microbiol 70, 2919–2927.
Lövenklev, M., Artin, I., Hagberg, O., Borch, E., Holst, E. & Radström, P. (2004b). Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B. Appl Environ Microbiol 70, 2928–2934.
Mani, N. & Dupuy, B. (2001). Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98, 5844–5849.
Marvaud, J. C., Eisel, U., Binz, T., Niemann, H. & Popoff, M. R. (1998a). tetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR. Infect Immun 66, 5698–5702.
Marvaud, J. C., Gibert, M., Inoue, K., Fujinaga, V., Oguma, K. & Popoff, M. R. (1998b). botR/A is a positive regulator of botulinum neurotoxin and associated non-toxin protein genes in Clostridium botulinum A. Mol Microbiol 29, 1009–1018.[CrossRef][Medline]
McGrath, S., Dooley, J. S. & Haylock, R. W. (2000). Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription-PCR. Appl Environ Microbiol 66, 1423–1428.
Meunier, F. A., Herreros, J., Schiavo, G., Poulain, B. & Molgo, J. (2002). Molecular mechanism of action of botulinal neurotoxins and the synaptic remodeling they induce in vivo at the skeletal neuromuscular junction. In Handbook of Neurotoxicology, pp. 305–347. Edited by J. Massaro. Totowa, NJ: Humana Press.
Oguma, K., Inoue, K., Fujinaga, Y., Yokota, K., Watanabe, T., Ohyama, T., Takeshi, K. & Inoue, K. (1999). Structure and function of Clostridium botulinum progenitor toxin. J Toxicol 18, 17–34.[CrossRef]
Popoff, M. R. & Marvaud, J. C. (1999). Structural and genomic features of clostridial neurotoxins. In The Comprehensive Sourcebook of Bacterial Protein Toxins, pp. 174–201. Edited by J. E. Alouf & J. H. Freer. London: Academic Press.
Porfirio, Z., Prado, S. M., Vancetto, M. D. C., Fratelli, F., Alves, E. W., Raw, I., Fernandes, B. L., Camargo, A. C. M. & Lebrun, I. (1997). Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin. J Appl Microbiol 83, 678–684.[CrossRef][Medline]
Quinn, C. P. & Minton, N. P. (2001). Clostridial neurotoxins. In Clostridia, pp. 211–250. Edited by H. Bahl & P. Dürre. Weinheim: Wiley-VCH.
Raffestin, S., Dupuy, B., Marvaud, J. C. & Popoff, M. R. (2005). BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. Mol Microbiol 55, 235–249.[CrossRef][Medline]
Schiavo, G., Matteoli, M. & Montecucco, C. (2000). Neurotoxins affecting neuroexocytosis. Physiol Rev 80, 717–766.
Sharma, S. K., Fu, F. N. & Singh, B. R. (1999). Molecular properties of a hemagglutinin purified from type A Clostridium botulinum. J Protein Chem 18, 29–38.[CrossRef][Medline]
Sharma, S. K., Ramzan, M. A. & Singh, B. R. (2003). Separation of the components of type A botulinum neurotoxin complex by electrophoresis. Toxicon 41, 321–331.[Medline]
Siegel, L. S. & Metzger, J. F. (1979). Toxin production by Clostridium botulinum type A under various fermentation conditions. Appl Environ Microbiol 38, 606–611.
Tavallaie, M., Chenal, A., Gillet, D., Raffestin, S., Popoff, M. R. & Marvaud, J. C. (2004). Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies. FEBS Lett 572, 299–306.[CrossRef][Medline]
Received 4 October 2005;
revised 30 November 2005;
accepted 2 December 2005.
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) was determined by measuring OD600. BoNT/A production was assayed with the mouse test (MLD ml–1, 
). It was confirmed that lethal activity corresponded to BoNT/A by using specific neutralizing antibodies against BoNT/A. The lag phase of cultures in sporulated cultures is not reported. Note that BoNT/A was detected at a low level during entry into exponential growth, increased to a maximal value at 24 h and then remained constant, independent of sporulation. Data are mean values from two independent experiments for each strain and condition.
, botR/A (x100); 
, bont/A; 
, NTNH/A; 
, ha34; 
) were determined by measurement of OD600. Lag phase of the sporulated cultures is not reported. Data are mean values from three independent experiments for each strain and condition.
