铜绿假单胞菌单PilZ结构域蛋白PA0012和PA4324突变体生物学表型的初步筛选

Preliminary screening of biological phenotypes of single PilZ domain protein PA0012 and PA4324 mutants in Pseudomonas aeruginosa

目的 本研究以铜绿假单胞菌单PilZ结构域的环二鸟苷酸受体蛋白PA0012和PA4324为研究对象,研究这2个蛋白对生物学表型的影响。方法 本研究首先利用同源重组的方法对编码PA0012和PA4324蛋白的基因进行了敲除,得到敲除突变体菌株ΔPA0012和ΔPA4324。然后,将实验分为3组,以野生型菌株PAO1为对照,用这2个突变体进行了表型筛选,包括群集运动能力、感染生菜的能力、对线虫的致病力和蛋白酶活性等,采用单因素方差分析对各组间的差异进行统计分析。随后对这2个突变体和野生型菌株进行了RNA测序,分析2个单PilZ结构域蛋白突变导致的基因差异表达,在转录水平上综合分析2个蛋白对生物学表型的影响。结果 本研究发现这2个单PilZ结构域蛋白的突变均能导致铜绿假单胞菌的群集运动增强,感染生菜的能力减弱,对线虫的毒性和蛋白酶活性的影响则无显著变化。RNA测序结果表明,这2个蛋白突变均可导致铜绿假单胞菌中大量基因的差异表达,且有一些共同的差异基因。结论 铜绿假单胞菌的PA0012和PA4324突变均能增强其群集运动和减轻其对生菜的感染,为后续机制研究奠定了基础。

Objective This research aimed to study the effects of single PilZ domain cyclic diguanosine monophosphate receptor proteins PA0012 and PA4324 on biological phenotypes in Pseudomonas aeruginosa.Methods We first knocked out the genes encoding PA0012 and PA4324 by homologous recombination method,and obtained the mutant strainsΔPA0012 andΔPA4324.Then,the wild-type strain PAO1 was used as the control to screen the phenotypes of the two mutants,including the ability to swarm,the ability to infect lettuce,the pathogenicity to nematodes and proteinase activity etc.One-way analysis of variance(one-way ANOVA)was used to analyze the differences among the groups.Subsequently,we performed RNA sequencing experiment to analyze the differential gene expression caused by the mutation of the two single PilZ domain proteins,and comprehensively analyzed the effects of these two proteins on biological phenotypes at the transcriptional level.Results In this study,we found that the mutation of these two single PilZ domain proteins could enhance the swarming motility,weaken its ability to infect lettuce,and its toxicity to nematodes and protease activity did not change significantly in Pseudomonas aeruginosa.RNA sequencing results showed that mutations in both proteins could lead to the differential expression of a large number of genes in Pseudomonas aeruginosa,and there were some common differential genes.Conclusion Both PA0012 and PA4324 mutations of Pseudomonas aeruginosa can enhance their movement and reduce their infection to lettuce,which lays a foundation for the follow-up mechanism study.