Hollingshead Sarah, McVicker Gareth, Nielsen Maria R, Zhang YuGeng, Pilla Giulia, Jones Rebekah A, Thomas Jonathan C, Johansen Sarah E H, Exley Rachel M, Brodersen Ditlev E, Tang Christoph M
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Department of Biosciences, Nottingham Trent University, Nottingham, United Kingdom.
mBio. 2025 Feb 5;16(2):e0261624. doi: 10.1128/mbio.02616-24. Epub 2024 Dec 20.
Toxin:antitoxin (TA) systems are widespread in bacteria and were first identified as plasmid addiction systems that kill bacteria lacking a TA-encoding plasmid following cell division. TA systems have also been implicated in bacterial persistence and antibiotic tolerance, which can be precursors of antibiotic resistance. Here, we identified a clinical isolate of (CS14) with a remarkably stable pINV virulence plasmid; pINV is usually frequently lost from , but plasmid loss was not detected from CS14. We found that the plasmid in CS14 is stabilized by a single nucleotide polymorphism (SNP) in its TA system. VapBC TA systems are the most common Type II TA system in bacteria, and consist of a VapB antitoxin and VapC PIN domain-containing toxin. The plasmid stabilizing SNP leads to a Q12L substitution in the DNA-binding domain of VapB, which reduces VapBC binding to its own promoter, impairing autorepression. However, VapBC mediates high-level plasmid stabilization because VapB is more prone to degradation by Lon than wild-type VapB; this liberates VapC to efficiently kill bacteria that no longer contain a plasmid. Of note, mutations that confer tolerance to antibiotics in also map to the DNA-binding domain of VapBC encoded by the chromosomally integrated F plasmid. We demonstrate that the tolerance mutations also enhance plasmid stabilization by the same mechanism as VapB. Our findings highlight the links between plasmid maintenance and antibiotic tolerance, both of which can promote the development of antimicrobial resistance.
Our work addresses two processes, the maintenance of plasmids and antibiotic tolerance; both contribute to the development of antimicrobial resistance in bacteria that cause human disease. Here, we found a single nucleotide change in the vapBC toxin:antitoxin system that stabilizes the large virulence plasmid of . The mutation is in the vapB antitoxin gene and makes the antitoxin more likely to be degraded, releasing the VapC toxin to efficiently kill cells without the plasmid (and thus unable to produce more antitoxin as an antidote). We found that vapBC mutations in that lead to antibiotic tolerance (a precursor to resistance) also operate by the same mechanism (, generating VapB that is prone to cleavage); free VapC during tolerance will arrest bacterial growth and prevent susceptibility to antibiotics. This work shows the mechanistic links between plasmid maintenance and tolerance, and has applications in biotech and in the design and evaluation of vaccines against shigellosis.
毒素-抗毒素（TA）系统在细菌中广泛存在，最初被鉴定为质粒成瘾系统，可在细胞分裂后杀死缺乏编码TA质粒的细菌。TA系统也与细菌的持留性和抗生素耐受性有关，而这可能是抗生素耐药性的先兆。在此，我们鉴定出一株临床分离株（CS14），其携带的pINV毒力质粒异常稳定；pINV通常在福氏志贺菌中频繁丢失，但在CS14中未检测到质粒丢失。我们发现CS14中的质粒通过其TA系统中的一个单核苷酸多态性（SNP）得以稳定。VapBC TA系统是细菌中最常见的II型TA系统，由VapB抗毒素和含VapC PIN结构域的毒素组成。该质粒稳定SNP导致VapB的DNA结合结构域发生Q12L替换，这减少了VapBC与其自身启动子的结合，削弱了自身抑制作用。然而，VapBC介导高水平的质粒稳定作用，因为与野生型VapB相比，VapB更容易被Lon降解；这释放出VapC以有效杀死不再含有质粒的细菌。值得注意的是，福氏志贺菌中赋予抗生素耐受性的突变也定位于染色体整合的F质粒编码的VapBC的DNA结合结构域。我们证明这些耐受性突变也通过与VapB相同的机制增强质粒稳定作用。我们的发现突出了质粒维持与抗生素耐受性之间的联系，这两者均可促进抗菌药物耐药性的发展。
我们的工作涉及两个过程，即质粒维持和抗生素耐受性；两者都有助于导致人类疾病的细菌中抗菌药物耐药性的发展。在此，我们在vapBC毒素-抗毒素系统中发现了一个单核苷酸变化，该变化稳定了福氏志贺菌的大毒力质粒。该突变位于vapB抗毒素基因中，使抗毒素更有可能被降解，释放出VapC毒素以有效杀死没有质粒的细胞（因此无法产生更多抗毒素作为解毒剂）。我们发现福氏志贺菌中导致抗生素耐受性（耐药性的先兆）的vapBC突变也通过相同机制起作用（即产生易于裂解的VapB）；耐受性期间游离的VapC将阻止细菌生长并防止对抗生素敏感。这项工作揭示了质粒维持与耐受性之间的机制联系，并在生物技术以及针对志贺菌病的疫苗设计和评估中具有应用价值。
