Clostridial toxins are responsible for severe diseases in man and animals such as botulism, gangrenes
and necrotic enteritis. Our laboratory investigates the mode of action of certain clostridial toxins and the
regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani.
The passage of botulinum neurotoxin type A (BoNT/A) trough the intestinal barrier was
investigated in polarized intestinal cell monolayers grown on filters. We have found that BoNT/A crosses
intestinal cell monolayers via a receptor-mediated transcytosis and that BoNT/A passage is more efficient
through the intestinal crypt cell line m-ICcl2, We used fluorescent BoNT/A C-terminal part of H chain (Hc) which
mediates toxin binding to cell receptors, to monitor toxin entry into NG108-15 neuronal cells as well as into
Caco-2 and m-ICcl2 intestinal cells. BoNT/A Hc receptors were found to be distributed in membrane structures
closely associated to cholesterol-enriched microdomains but distinct from detergent-resistant microdomains in
both cell types. BoNT/A Hc was trapped into endocytic vesicles, which progressively migrated to a perinuclear
area in NG108-15 cells, and in a more scattered manner in intestinal cells. In both cell types, BoNT/A Hc
entered through a dynamin- and intersectin-dependent pathway, reached an early endosomal compartment
labeled with EEA1. In neuronal cells, BoNT/A Hc entered mainly via a clathrin-dependent pathway, in contrast
to intestinal cells where it followed a Cdc42-dependent pathway (Fig. 1), supporting a differential toxin routing
in both cell types.
Using electronic microscopy, BoNT/A Hc was found to enter neuronal cells via a clathrin pathway and
intestinal cells mainly via non-clathrin vesicles. Upon incubation at 4°C, BoNT/A Hc gold particles were mainly
distributed to cell periphery, including coated pits, and cell extensions of NG108-15 and to a lower extent to mICcl2
cells. A few coated vesicles close from the plasma membrane also contained gold particles in both cell
types. After incubation at 37°C, BoNT/A Hc mostly transited in coated vesicles in NG108-15 cells, whereas in mICcl2
cells toxin gold particles were localized in uncoated vesicles, which support the transcytotic passage of the
toxin, and free in the cytosol probably resulting from an unspecific uptake.
The passage of BoNT/A through the intestinal barrier is investigated using a mouse intestinal loop
model with administration of fluorescent BoNT/A Hc into the loop and then confocal microscopy analysis. We
report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous
muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of
BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of
heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset
of neuroendocrine intestinal crypt cells corresponding to serotonine neuroendocrine cells, which probably
represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the
submucosa and later (90-120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both
submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells
including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by
HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the
consequences of the targeting to non-cholinergic neurons remains to be determined.
Two-component systems are involved in the regulation of botulinum neurotoxin synthesis in Clostridium botulinum type A Hall strain. Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.
Characterization of Botulinum Neurotoxin Type A Neutralizing Monoclonal Antibodies and Influence of Their Half-lives on Therapeutic Activity
Botulinum toxins i.e. BoNT/A to /G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab’)2, Fab’, scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents.
We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab’)2 fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity.
Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination.
These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination.
Characterization of the enzymatic activity of Clostridium perfringens TpeL
TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family with Clostridium difficile toxins A and B, Clostridium sordellii toxins LT and HT, and Clostridium novyi alpha toxin. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We showed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we showed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.
Activation of a JNK pathway by the lethal toxin from Clostridium sordellii, TcsL-82, occurs independently of the toxin intrinsic enzymatic activity and facilitates small GTPase glucosylation
We showed that lethal toxin from Clostridium sordellii (TcsL-82) activates the three MAPK pathways but that only a permeable and specific JNK inhibitor, JNK inhibitor II, prevents toxin-dependent actin depolymerization and cell rounding. We showed that JNK activation is dependent on entry of the toxin N-terminal domain into the cytosol as bafilomycin A1, that prevents acidification of endocytic vesicle and subsequent cytosolic translocation of the toxin N-terminal domain, prevents JNK activation. Inhibition of JNK activity delays small GTPase glucosylation generated by N-terminal domain catalytic activity. Using a cell line mutant deficient in UDP-glucose, we observed that activation of JNK occurs even in the absence of small GTPase glucosylation and, thus, is independent of the toxin intrinsic catalytic activity. Facilitation of target glucosylation by JNK activation appeared to be restricted to TcsL-82 and was not a general feature of large clostridial toxins. Indeed, it was not observed with Toxin B (TcdB) from Clostridium difficile although this toxin also activates JNK.
An atypical Clostridium strain related to the Clostridium botulinum group III isolated from a human case of septic shock
We reported an atypical Clostridium strain related to the Clostridium botulinum group III, which has been identified in a blood culture. Phenotypic and genotypic analysis indicates that the strain AIP981.10 belongs to the physiological group III of C. botulinum, which encompasses C. botulinum type C, C. botulinum type D, C. botulinum mosaic C/D and D/C, and which is closely related to C. novyi and C. haemolyticum. Indeed, the chromosome of the group III C. botulinum isolates is highly conserved and is related to that of C. novyi. However, based on 16s rDNA sequence AIP981.10 is on a phylogenetic sub-branch distinct from those of C. botulinum and C. novyi strains, and multisequence analysis of housekeeping genes shows a close relatedness to C. botulinum D/C mosaic and to a lower extent to C. botulinum C/D mosaic. AIP981.10, which has been isolated from a fatal case of septic shock was found to not produce toxin in vitro and to not contain toxin genes related to those of group III C. botulinum-C. novyi strains except a botulinolysin gene. BoNT genes type C and D as well as C. novyi alpha-toxin gene (tcnA) are localized on phage DNA which are not integrated into the chromosome. These phages can be easily lost upon subcultures and can be interchanged between C. botulinum C or D and C. novyi. Similarly, C2 toxin genes are localized on large plasmids in C. botulinum C and D, which can be lost or acquired. It could be hypothesized that AIP981.10 has lost a phage harboring a bont gene during the isolation and subcultures of the strain, was and is therefore no longer toxigenic. Indeed a C. botulinum type B-like nontoxigenic strain has been isolated from an infant botulism case. However, C. botulinum C or D have been involved only in a few cases of human botulism, and no characteristic symptoms of flaccid paralysis were observed in this patient. Alternatively, AIP981.10 could have lost either a phage harboring tcnA, a plasmid or another mobile genetic element carrying C2 toxin genes or other unknown toxin gene(s), since the group III C. botulinum strains possess various plasmids and mobile elements. An environmental nontoxigenic Clostridium might also be the causative agent in compromised patients as already evidenced with other non-toxic Clostridium species.