Régulation de la toxinogenèse chez Clostridium botulinum et Clostridium tetani

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Correction: Two-Component Systems Are Involved in the Regulation of Botulinum Neurotoxin Synthesis in Clostridium botulinum Type A Strain Hall.

International audienceClostridium 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

Growth kinetics and BoNT/A production in representative Hall isogenic antisense strains.

<p>(A) Hall/707, Hall/714, and Hall/1146 showed a similar growth curve monitored at OD₆₀₀ than the Hall/pAT19 strain. (B) Hall/1147 and Hall/1148 grew more rapidly than the control Hall/pAT19 strain, whereas Hall/1001 displayed a slower growth curve. Hall/652, Hall716, and Hall/723 show a similar kinetics compare to Hall/pAT19. (C) Monitoring of growth kinetics of Hall/pAT19, Hall/707 and Hall/1147 by bacterial counting on TGY agarose and expressed as unit forming colony (UFC)/ml. (D) BoNT/A production assayed by ELISA was significantly reduced in the culture supernatants of Hall/707, Hall/714, Hall/1146, Hall/1147, and Hall/748, versus the control Hall/pAT19 strain. BoNT/A production was significantly reduced in Hall/306 at 24 h culture. Data are mean values +/− SD from 3 independent experiments, *P<0.05.</p

Two-component system and orphan response regulator genes in <i>C. botulinum</i> strain Hall, homology with regulatory genes in other bacterial species, Hall isogenic antisense strains, and primers used for the construction of recombinant plasmids generating antisense mRNA.

a<p>In bold the accession number of the gene which has been targeted in the isogenic antisense strains and in bracket the associated gene of the corresponding TCS.</p>b<p>Protein identity >60% (<i>C. sporogenes</i> is omitted due to its high similarity to <i>C. botulinum</i>).</p

Kinetics of transcript levels of CLC_1093, CLC_1914, and CLC_0661 in Hall strain.

<p>Transcript levels were quantified by qRT-PCR and normalized versus <i>rpoB</i> gene. Data are mean values +/− SD, from 3 independent experiments.</p

Genetic environment of the TCS genes involved in the regulation of toxin synthesis in strain Hall.

<p>Genetic environment of the TCS genes involved in the regulation of toxin synthesis in strain Hall.</p