铜绿假单胞菌Ⅲ型分泌系统毒力相关蛋白的差异表达

Research of differential expression of virulence-related proteins of T3SS in Pseudomonas aeruginosa

目的分析铜绿假单胞菌在耐药性变化时菌株Ⅲ型分泌系统（T3SS）毒力相关蛋白的表达差异，结合患者的临床资料，探讨耐药性变化与细菌毒力变化的相关性。方法筛选源于同一患者呼吸道、多次分离、药敏谱发生动态改变的铜绿假单胞菌，用肠杆菌基因间重复一致序列PCR（ERIC．PCR）方法确定菌株的同源性，以同一克隆的菌株体系为研究对象，Kirby—Bauer纸片法检测菌株对药物的敏感性，PCR方法检测T3SS系统和菌株的毒力基因型，双向蛋白电泳比较菌株的全菌蛋白表达谱，质谱分析差异蛋白点，并在蛋白质数据库进行检索。收集患者临床信息，了解临床特征、细菌耐药性、毒力相关蛋白三者问的关系。结果筛选出1例慢性阻塞性肺疾病急性加重期（AECOPD）合并肺炎的患者符合入选条件，其1年内3次呼吸道分离出铜绿假单胞菌，药敏谱依次为：敏感一多重耐药（MDR）-泛耐药（PDR）。3株菌株毒力基因属exoU＋／exoS-型，经ERIC-PCR证实为同一克隆。双向凝胶电泳显示3株菌有21个差异表达蛋白。质谱分析发现其中11个蛋白功能明确（9个与细菌基础代谢有关）。仅毒力相关蛋白二硫化物氧化还原酶A（DsbA）在PDR株时表达明显上调，在敏感株表达最低。临床信息显示该患者在分离出PDR时，肺部感染最重，并于1个月后死亡。结论在长期定植、感染过程中，生存环境的变化可能导致铜绿假单胞菌的耐药表型发生变化，同时菌株毒力加强，共同导致感染难于控制。DsbA在铜绿假单胞菌毒力变化中可能发挥了重要的作用。

Objective To analyze the difference of virulence-related protein concerned with type Ⅲsecretion 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 eleetrophoresis 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.