PDF(Pubmed)
Abstract：
The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.
摘要：
多药耐药（MDR）细菌感染的患病率持续上升，因为对抗这些感染所需的抗生素的开发仍然停滞不前。MDR肠球菌是这场危机的主要原因。对抗MDR肠球菌的潜在治疗方法是噬菌体（噬菌体）疗法，使用裂解病毒来感染和杀死致病菌。虽然已经确定了裂解某些MDR肠球菌菌株的噬菌体，其他菌株显示出高水平的抗性，并且这种抗性的机制尚不明确。这里,我们使用CRISPR干扰(CRISPRi)筛选来鉴定在粪肠球菌可动员质粒上发现的与噬菌体抗性有关的基因位点.该基因座编码推定的丝氨酸重组酶，然后编码IV型限制酶（TIV-RE），我们显示该酶限制了耐万古霉素的粪肠球菌中噬菌体phi47的复制。我们进一步发现，phi47通过在TIV-RE抑制剂蛋白中获得错义突变而进化以克服限制。我们证明了这种抑制剂，称为IV型限制抑制因子A(TIA)，结合和灭活不同的TIV-RE。总的来说,我们的发现促进了我们对耐药粪肠球菌中噬菌体防御的理解,并提供了对噬菌体如何进化以克服抗噬菌体防御系统的机制见解.
