[铜绿假单胞菌Ⅲ型分泌系统毒力相关蛋白差异表达研究]

Li Xiao-qiang, Zhuo Chao, Liao Dong-jiang, Xiao Shu-nian, Jin Guang-yao, Zhong Nan-shan

Department of Infectious Diseases, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.

Zhonghua Yi Xue Za Zhi. 2010 Mar 30;90(12):849-52.

OBJECTIVE

To analyze the difference of virulence-related protein concerned with type III secretion system (T3SS) in Pseudomonas aeruginosa during the changes of antibiotic sensitivity and interpret the clinical patient data to explore the relationship between the changes in resistance and variance of virulence.

METHODS

The isolates of Pseudomonas aeruginosa was isolated from the respiratory tract of a same patient with an altered sensitivity of antibiotics. It turned out to be one clone. The homolog of isolates was determined by ERIC-PCR. The Kirby-Bauer antibiotic testing was employed to detect the sensitivity of antibiotics of isolates. PCR was used to detect the gene of T3SS and virulence of isolates and two-dimensional gel electrophoresis to compare the whole-cell proteins. The mass spectrometry was employed to analyze a variety of protein spots. The relevant information was retrieved from protein databases. Clinical record was collected to study the relationship of clinical features, bacteria resistance and virulence-associated protein.

RESULTS

One subject was diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with pneumonia. Pseudomonas aeruginosa was isolated from his respiratory tract three times in one year. Sensitivity spectrum of isolates were as follows: sensitivity, multi-drug resistant (MDR) and pan-drug resistance (PDR). Virulence gene was exo U+/exo S-. Twenty-one differentially expressed proteins were revealed by two-dimensional gel electrophoresis in three isolates. The functions of 11 proteins were definite. Only 9 proteins were associated with basal bacterial metabolism. Disulfide oxidoreductase A (DsbA) corresponded to the variation of virulence and sensitivity spectrum. Clinical record revealed that the severe lung infection was caused by the PDR strain and the patient died within one month.

CONCLUSIONS

The sensitivity spectrum and virulence of Pseudomonas aeruginosa may undergo changes when there is an alteration of eco-environment during colonization and infection over a long period of time. DsbA plays a key role in the variety of virulence.

目的

分析铜绿假单胞菌在抗生素敏感性变化过程中与Ⅲ型分泌系统（T3SS）相关的毒力蛋白差异，并解读临床患者数据，以探讨耐药性变化与毒力变异之间的关系。

方法

从一名抗生素敏感性发生改变的同一患者呼吸道分离出铜绿假单胞菌菌株，结果显示为一个克隆株。通过ERIC-PCR确定分离株的同源性。采用 Kirby-Bauer 抗生素检测法检测分离株的抗生素敏感性。用 PCR 检测分离株的 T3SS 基因和毒力，并用二维凝胶电泳比较全细胞蛋白。采用质谱分析法分析各种蛋白斑点。从蛋白质数据库中检索相关信息。收集临床记录以研究临床特征、细菌耐药性和毒力相关蛋白之间的关系。

结果

一名患者被诊断为慢性阻塞性肺疾病急性加重期（AECOPD）合并肺炎。一年内从其呼吸道三次分离出铜绿假单胞菌。分离株的药敏谱如下：敏感、多重耐药（MDR）和泛耐药（PDR）。毒力基因为外毒素 U+/外毒素 S-。二维凝胶电泳显示三个分离株中有 21 种差异表达蛋白。11 种蛋白的功能明确。只有 9 种蛋白与基础细菌代谢相关。二硫键氧化还原酶 A（DsbA）与毒力和药敏谱的变化相对应。临床记录显示，严重肺部感染由 PDR 菌株引起，患者在一个月内死亡。