Table_1_Role of cpxA Mutations in the Resistance to Aminoglycosides and β-Lactams in Salmonella enterica serovar Typhimurium.DOCX

Although it has been reported that deletion of the response regulator, CpxR, in the CpxRA system confers sensitivity to aminoglycosides (AGAs) and β-lactams in Salmonella enterica serovar Typhimurium, the regulatory effects of CpxA on multidrug resistance (MDR) are yet to be fully investigated in this organism. Here, to explore the role of CpxA in MDR, various cpxA mutants including a null mutant (JSΔcpxA), a site-directed mutant (JSΔcpxA38) and an internal in-frame deletion mutant (JSΔcpxA92–104) of the S. enterica serovar Typhimurium strain JS, were constructed. It was revealed that cpxA and cpxR deletion mutants have opposing roles in the regulation of resistance to AGAs and β-lactams. Amikacin and cefuroxime can activate the CpxRA system, which results in increased resistance of the wild-type compared with the cpxR deletion mutant. All the cpxA mutations significantly increased resistance to AGAs and β-lactams due to CpxRA system activation via the phosphorylation of CpxR. Moreover, AckA-Pta-dependent activation of CpxR increased the antibiotic resistance of cpxA deletion mutants. Further research revealed that the AcrAB-TolC conferred resistance to some AGAs and β-lactams but does not influence the regulation of resistance by CpxRA against these antibiotics. The detection of candidate MDR-related CpxR regulons revealed that the mRNA expression levels of spy, ycca, ppia, htpX, stm3031, and acrD were upregulated and that of ompW was downregulated in various cpxA mutants. Furthermore, the expression levels of nuoA and sdhC mRNAs were downregulated only in JSΔcpxA92–104. These results suggested that cpxA mutations contribute to AGAs and β-lactams resistance, which is dependent on CpxR.

尽管已有研究报道，肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium）中CpxRA双组分调控系统（CpxRA system）的应答调节子CpxR缺失会导致菌株对氨基糖苷类抗生素（AGAs）和β-内酰胺类抗生素（β-lactams）敏感，但CpxA对该菌多重耐药性（MDR）的调控作用尚未得到充分阐释。本研究为探究CpxA在多重耐药性中的功能，以肠炎沙门氏菌鼠伤寒血清型菌株JS为出发菌株，构建了多种cpxA突变株，包括敲除突变株（null mutant）JSΔcpxA、定点突变株（site-directed mutant）JSΔcpxA38以及内部框内缺失突变株（internal in-frame deletion mutant）JSΔcpxA92–104。研究发现，cpxA与cpxR缺失突变株在调控菌株对氨基糖苷类与β-内酰胺类抗生素的耐药性方面发挥相反的作用。阿米卡星与头孢呋辛可激活CpxRA系统，使得野生型菌株相较于cpxR缺失突变株的耐药性显著提升。所有cpxA突变均可通过CpxR的磷酸化激活CpxRA系统，进而显著增强菌株对氨基糖苷类与β-内酰胺类抗生素的耐药性。此外，依赖AckA-Pta途径的CpxR磷酸化可进一步提升cpxA缺失突变株的抗生素耐药性。进一步研究表明，AcrAB-TolC外排泵系统可赋予菌株对部分氨基糖苷类与β-内酰胺类抗生素的耐药性，但并不影响CpxRA系统对上述抗生素的耐药性调控。对候选多重耐药性相关CpxR调控子的检测分析显示，在各类cpxA突变株中，spy、ycca、ppia、htpX、stm3031以及acrD的mRNA表达水平均上调，而ompW的mRNA表达水平则下调。此外，仅在JSΔcpxA92–104突变株中，nuoA与sdhC的mRNA表达水平出现下调。上述研究结果表明，cpxA突变可通过依赖CpxR的途径增强菌株对氨基糖苷类与β-内酰胺类抗生素的耐药性。