Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E.

Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control.

Differential effects of sporulation temperature on the high pressure resistance of Clostridium botulinum type E spores and the interconnection with sporulation medium cation contents.

High pressure thermal (HPT) processing can be used to improve traditional preservation methods and increase food safety and durability, whereas quality related characteristics can be largely maintained. Clostridium (C.) botulinum type E is a non-proteolytic, psychrotrophic, toxin-producing spore former, commonly associated with aquatic environments in temperate regions of the northern hemisphere. Sporulation in nature is likely to occur under varying conditions including temperature and nutrient availability, which might affect resistance properties of resulting spores. In our study, we determined the effect of sporulation temperature (13-38 °C) on the resistance of three Clostridium botulinum type E strains to differently intense HPT treatments (200 MPa at 40 and 80 °C, and 800 MPa at 40 and 80 °C). Furthermore, the effect of cations on sporulation temperature-mediated alterations in HHP resistance was investigated. Results indicate that low and high sporulation temperatures can increase and decrease sporal HPT resistance, respectively, in a treatment-dependent (pressure level, treatment temperature) manner, whereas the trends observed are largely unaffected by pressure dwells (1 s-10 min). Furthermore, results show that the cation content of the sporulation medium (Ca(2+), Mg(2+), Mn(2+)) marginally influences and partially counteracts effects on the HPT resistance of spores grown at low and elevated temperatures, respectively. This suggests that sporulation temperature and medium cations provoke changes in some common spore resistance structures. Sporulation conditions can markedly affect spore resistance properties and, thus, should be considered for the experimental setup of worst case studies aiming to evaluate the effectiveness of food processes in terms of the inactivation of C. botulinum type E spores.

Effect of sporulation medium and its divalent cation content on the heat and high pressure resistance of Clostridium botulinum type E spores.

Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II and produces the highly potent Botulinum neurotoxin E (BoNT/E) even at refrigerated temperatures. As C. botulinum type E spores are highly prevalent in aquatic environments, seafood and fishery products are commonly associated with this organism. Hydrostatic high pressure (HHP) treatments, or treatments combining HHP with elevated temperatures (HHPT), can be used to improve traditional preservation methods and increase food safety, quality and durability. In this study, we assessed the effect of different sporulation media and cation concentration on the heat resistance, HHP resistance, and HHPT resistance of spores from three C. botulinum type E strains. SFE (sediment fish extract) sporulation media yielded the most resistant spores, whereas, in M140 media, the least resistant spores were produced. Furthermore our results indicate that the divalent cation content (Ca(2+), Mg(2+) and Mn(2+)) plays a role in the differential development of C. botulinum type E spore resistance to heat, HHP and HHPT in different media. Calcium cations confer heat and HPPT resistance to spores, while high amounts of magnesium cations appear to have a negative effect. Manganese cations in low concentrations are important for the development resistance to HPP and HPPT treatments, but not heat alone. This study provides valuable information on the nature of non-proteolytic C. botulinum type E spores grown in different media. The data provided here can be useful to the food industry and to researchers when considering spore properties in food safety risk assessment and the experimental design of future inactivation studies.

SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E.

Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.

Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum Type E.

Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.

Persistence of Clostridium botulinum neurotoxin type E in tissues from selected freshwater fish species: implications to public health.

Rainbow trout (Oncorhynchus mykiss), round gobies (Neogobius melanostomas), yellow walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) were given Clostridium botulinum neurotoxin type E (BoNT/E) at four doses (0, 800, 1500, and 4000 mouse lethal doses). BoNT/E was sought in the fish tissues at death or at the conclusion of the experiment (10 days after treatment). Fish were divided into a "fillet" (axial musculature) and a "nonfillet" sample before testing for BoNT/E toxicity with a mouse bioassay. BoNT/E was detected in all species. The percentage of positive BoNT samples ranged across the species and doses from 0 (trout, perch, and walleye) to 17% (round goby) in fillet tissues and from 0 (perch) to 92% (round goby) in nonfillet tissues. The lack of positive fillet samples in three key commercial fish species suggests that the public health implications of eating these fish are minimal. However, the presence of toxin in the nonfillet compartment of a high proportion of fish supports the hypothesis that live intoxicated fish are a vehicle for the transfer of BoNT/E to fish-eating birds, which are then in turn, intoxicated.

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.

Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 degrees C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 degrees C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 degrees C. However, incubation at 44 degrees C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 degrees C was evident.

Development and application of Real-Time PCR assays to detect fragments of the Clostridium botulinum types A, B, and E neurotoxin genes for investigation of human foodborne and infant botulism.

Real-time PCR assays for detection of Clostridium botulinum neurotoxin (BoNT) gene fragments specific to BoNTA, B, and E were developed as alternatives to the mouse bioassay. The expected specificities of the PCR assays were demonstrated by in silico analysis as well as empirical testing of target DNA extracted from 83 pure cultures of C. botulinum, and 44 bacteria from other species. The sensitivities of the assays were found to be equivalent to 16, 10, and 141 genomes for BoNT A, B, and E, respectively. The assays were shown to be applicable to both purified DNA, as well as crude DNA extracted from cultures and enrichment broths. The assays were evaluated using DNA extracted directly from clinical and food specimens as well as from inoculated broths using material collected from seven confirmed and one suspected case of botulism. The appropriate BoNT genes were detected in material from seven of the eight cases of botulism and provided a supportive diagnosis faster than the conventional bioassay. These assays have already proven useful for pubic health microbiological investigation of suspected cases of human botulism by substantially improving the diagnostic process.