Mehdizadeh Gohari Iman, Li Jihong, Rood Julian I, McClane Bruce A
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh Pennsylvania, USA.
Infection Program, Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Victoria, Australia.
mBio. 2022 Apr 26;13(2):e0049622. doi: 10.1128/mbio.00496-22. Epub 2022 Mar 23.
Clostridium perfringens type B and D strains produce epsilon-toxin (ETX). Our 2011 study (mBio 2:e00275-11, 2011, https://doi.org/10.1128/mBio.00275-11) reported that the Agr quorum-sensing (QS) system regulates ETX production by type D strain CN3718. However, subsequent studies have brought that conclusion into question. For example, we reported in 2012 (Infect Immun 80:3008-3017, 2012, https://doi.org/10.1128/IAI.00438-12) that the Agr-like QS system is not required for wild-type ETX production levels by two type B strains. Consequently, we reexamined whether the Agr-like QS system regulates ETX production in type D strains by using Targetron insertional mutagenesis to construct new null mutants of two type D strains, CN3718 and CN2068. Western blotting showed that both mutants still produce wild-type ETX levels. However, the newly constructed mutants of both type D strains produced reduced amounts of alpha-toxin, and this effect was reversible by complementation, which confirms loss of functional AgrB production by these mutants since alpha-toxin production is known to be regulated by AgrB. Coupled with the previously published results for type B strains, these new findings indicate the Agr-like QS system is not usually necessary for C. perfringens to produce wild-type ETX levels. Since epsilon-toxin (ETX) is necessary for the virulence of C. perfringens type D and, likely, type B strains, understanding the regulation of ETX production is important. In 2011, we reported that an null mutant of type D strain CN3718 produces less ETX than its wild-type parent. However, when new mutants were constructed in type D strains CN3718 and C2068, ETX production was unaffected. Those newly constructed mutants produced less alpha-toxin, and this phenotype was reversible by complementation, confirming construction of null mutants since alpha-toxin production is regulated by AgrB. Coupled with previous results for type B strains, these new type D results support the conclusion that the Agr QS is not usually necessary for wild-type ETX production levels.
B型和D型产气荚膜梭菌菌株可产生ε毒素（ETX）。我们2011年的研究（《mBio》2:e00275 - 11, 2011, https://doi.org/10.1128/mBio.00275 - 11）报道，Agr群体感应（QS）系统调节D型菌株CN3718的ETX产生。然而，随后的研究对这一结论提出了质疑。例如，我们在2012年报道（《感染与免疫》80:3008 - 3017, 2012, https://doi.org/10.1128/IAI.00438 - 12），两种B型菌株产生野生型水平的ETX并不需要Agr样QS系统。因此，我们通过使用靶基因插入诱变构建D型菌株CN3718和CN2068的新缺失突变体，重新研究Agr样QS系统是否调节D型菌株的ETX产生。蛋白质免疫印迹分析表明，这两种突变体仍能产生野生型水平的ETX。然而，新构建的两种D型菌株突变体产生的α毒素量减少，并且这种效应通过互补作用是可逆的，这证实了这些突变体中功能性AgrB的产生缺失，因为已知α毒素的产生受AgrB调节。结合之前发表的关于B型菌株的结果，这些新发现表明，产气荚膜梭菌产生野生型水平的ETX通常不需要Agr样QS系统。由于ε毒素（ETX）对于D型产气荚膜梭菌以及可能对于B型菌株的毒力是必需的，了解ETX产生的调控很重要。2011年，我们报道D型菌株CN3718的一个缺失突变体产生的ETX比其野生型亲本少。然而，当在D型菌株CN3718和C2068中构建新的突变体时，ETX的产生未受影响。那些新构建的突变体产生的α毒素较少，并且这种表型通过互补作用是可逆的，证实了缺失突变体的构建，因为α毒素的产生受AgrB调节。结合之前关于B型菌株的结果，这些关于D型菌株的新结果支持了以下结论：野生型ETX产生水平通常不需要Agr QS。
