Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947

The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30°C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10°C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30°C. The maximum botE expression levels and average neurotoxin levels at 10°C were 45 to 65% of those at 30°C. The relative mRNA levels at 10°C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30°C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30°C, whereas at 10°C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.Botulinum neurotoxins ingested with food and drink can cause a potentially lethal paralysis to humans and animals. The neurotoxins are mainly produced by Clostridium botulinum and appear in seven distinct serotypes (A through G) (7,20,21). Upon secretion, the neurotoxins are associated with nontoxic proteins forming progenitor toxin complexes of differing structures and molecular sizes, depending on serotype and strain (7,8,21).The genes encoding the nontoxic-neurotoxin-associated proteins are located immediately upstream of the neurotoxin gene and vary in structure, composition, and arrangement between different serotypes and different strains (7). All types of the neurotoxin gene clusters share the same organization of bot (also called bont, cnt, and ntx) and ntnh at the 3Ј end but differ at their 5Ј ends (20). Type A1, B, C, D, and G neurotoxin gene clusters are associated with three hemagglutinin-encoding genes (ha70, ha17, and ha33), whereas types A2 to A4 and E and F toxin clusters are associated with p47 and orfx1, orfx2, and orfx3, encoding proteins with unknown function (2,4,6,11,20). In addition, all other types of toxin gene clusters, with the exception of type E, carry botR that encodes a sigma factor responsible for positive regulation of the neurotoxin gene (16).The role of the nontoxic-neurotoxin-associated proteins has not been fully identified. It has been proposed that they play a role in protecting the neurotoxin against the acidity of the gastrointestinal tract (19), and the hemagglutinin components seem to assist in the absorption of ingested neurotoxin from the gastrointestinal tract (17). Nothing is known about the activities of orfx1, orfx2, and orfx...

Section: Vol 74 2008 Qrt-pcr Of Type E Botulinum Neurotoxin Clustersupporting

confidence: 89%

Quantitative Real-Time Reverse Transcription-PCR Analysis Reveals Stable and Prolonged Neurotoxin Cluster Gene Activity in a Clostridium botulinum Type E Strain at Refrigeration Temperature

Chen

Korkeala

Lindén

et al. 2008

“…A large number of studies have focused on the detection of C. botulinum bont/A, bont/B, bont/E, and bont/F genes, which are responsible for toxin production leading to human botulism (1-4, 11, 13, 14, 17, 18, 30, 45). Several studies have also reported on the detection of type C (bont/C) and type D (bont/D) genes by conventional PCR (9,15,19,24,42,48,49), while only a few such genes have been detected by real-time PCR (25,28,29). Real-time PCR technique presents the advantages of being highly specific and sensitive with no need for a post-PCR detection assay, in contrast to conventional PCR.…”

mentioning

confidence: 99%

Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

Woudstra

Skarin

Anniballi

et al. 2012

118

ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

“…M-TC contains BoNT and NTNH but has no HA proteins, whereas LL-TC and L-TC contain different ratios of the BoNT, NTNH, and HA proteins (21,22,29,34). The biological and structural roles of the complex proteins are not completely characterized, although it has been proposed that they serve the role of protecting BoNT from harsh conditions, including pH, salt, temperature, and digestive enzymes, and that they assist BoNT translocation across the intestinal epithelial layer (2,6,17).…”

mentioning

confidence: 99%

Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Lin

Tepp

Pier

et al. 2010

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.