FinO/ProQ家族蛋白调控mcr-1阳性IncI2型质粒适应性的机制研究

The plasmid-mediated colistin resistant gene mcr-1 has been widely spread in the world. IncI2 type plasmid is one of the main vectors carrying mcr-1, which has good stability and does not cause adaptive cost to the host . We found a putative protein-CDS6 with FinO domain in pHNSHP45 plasmid (IncI2 type), and deletion of CDS6 could decrease the adaptability of pHNSHP45 plasmid. By transposon screening, it was found that the growth rate of CDS6 deletion mutants could restore to the wild-type level after the inactivation of mcr-1. Thus, We propose a new adaptive mechanism: FinO/ProQ family protein: CDS6 regulates plasmid IncI2 adaptability, that is, CDS6 can improve plasmid adaptability by down-regulating the expression level of mcr-1. In this project, qPCR, EMSA, RIP and other methods are proposed to further investigate whether CDS6 can improve plasmid adaptability by inhibiting the expression level of mcr-1. This project will clarify the regulatory mechanism of CDS6 on the adaptability of mcr-1 positive IncI2 plasmid, and provide scientific basis for establishing a new target for the prevention and control of mcr-1 diffusion.

质粒介导的黏菌素耐药基因mcr-1已在全球广泛传播，其中IncI2型质粒是携带mcr-1的主要载体之一，其具有良好的稳定性且不会对宿主菌造成适应性代价。我们在pHNSHP45质粒（IncI2型）上发现具有FinO结构域的假定蛋白:CDS6，该蛋白缺失可导致pHNSHP45质粒适应性明显下降；通过转座子筛选实验发现mcr-1被插入失活后，CDS6缺失突变体的生长速率可恢复至野生型水平。由此提出CDS6(FinO/ProQ家族蛋白)调控IncI2型质粒适应性的新机制:CDS6可通过下调mcr-1的表达水平，提高质粒适应性。本项目拟进一步采用qPCR、EMSA和RIP等方法，旨在探讨CDS6是否通过抑制mcr-1的表达水平提高质粒的适应性这一关键科学问题，以阐明CDS6对mcr-1阳性IncI2型质粒适应性的调控机制，为确立防控mcr-1扩散的新靶标提供科学依据。

黏菌素是医学临床抗击多重耐药菌感染的最后一道防线药物。但随着黏菌素在动物生产上的大量使用，出现质粒介导的黏菌素耐药基因mcr-1，其对黏菌素在临床治疗中的有效性构成潜在威胁，引起国内外广泛关注。目前，mcr-1的传播载体主要为IncI2，IncX4和IncHI2质粒，已在全球60多个国家和地区检出，但mcr-1质粒的持留机制仍不清楚。本项目以IncI2型mcr-1质粒（pHNSHP45）为研究对象，寻找到影响该质粒适应性的关键因子PcnR, 明确PcnR调控的靶基因及其调控机制，还探究了PcnR同源蛋白的功能。研究结果表明IncI2质粒编码ProQ/FinO家族蛋白PcnR是确保黏菌素耐药质粒pHNSHP45持留的关键因子，该蛋白通过下调质粒拷贝数，防止高拷贝mcr-1对细菌造成适应性代价；另外，即使是单拷贝质粒编码的MCR-1也会对宿主菌造成适应性代价，这一结果部分解释了黏菌素使用政策调整后mcr-1及其质粒快速下降的原因，同时也提示可以通过影响mcr-1阳性菌株的适应性来减少mcr-1的传播，为防控mcr-1的传播提供理论基础。ProQ/FinO家族蛋白PcnR通过与前导肽RepR mRNA结合抑制复制起始蛋白RepA的表达，从而使质粒拷贝数下降10倍左右; 且PcnR同源蛋白PcnRI也可抑制IncI1质粒的拷贝数，其调控机制与PcnR类似。这一发现不仅拓宽了对ProQ/FinO家族蛋白功能的认识，同时也是对IncI型质粒复制调控机制的补充，为进一步研究IncI家族质粒的复制调控模式和消减耐药质粒的持留奠定基础。