Tang Aihua, Marquart Mary E, Fratkin Jonathan D, McCormick Clare C, Caballero Armando R, Gatlin Hattie P, O'Callaghan Richard J
Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3794-801. doi: 10.1167/iovs.08-3107. Epub 2009 Feb 28.
To analyze PASP in terms of its gene distribution and expression, its corneal pathologic effects, its enzymatic properties, and the protectiveness of the immune response to this protease.
Twenty-five strains of P. aeruginosa were analyzed for the PASP gene and secreted protein by PCR and Western blot analysis, respectively. Active recombinant (r)PASP (10 microg/20 microL) or heat-inactivated rPASP was intrastromally injected into rabbit corneas. Pathologic changes were monitored by slit lamp examination (SLE) and histopathology. Purified rPASP was assayed for cleavage of collagens and susceptibility to TLCK. Rabbit antibody to rPASP was produced and tested for enzyme inactivation, and actively immunized rabbits were challenged by intrastromal injection of active rPASP (5 microg).
All 25 strains of P. aeruginosa analyzed were positive for the PASP gene and protein. SLE scores of eyes injected with active rPASP were significantly higher than control eyes at all postinjection times (PI; P <or= 0.004). Histopathologic studies documented the destruction of the corneal epithelial layer and portions of the corneal stroma at 9 hours PI, and polymorphonuclear (PMN) leukocyte infiltration into the cornea by 24 hours after active rPASP injection. PASP cleaved type I and IV collagens and was susceptible to TLCK inhibition. PASP was present in the cytoplasm and periplasm, but only secreted PASP was enzymatically active. A high antibody titer (ELISA titer >or= 10,000) was produced, but this antibody did not protect against active rPASP challenge.
PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.
从基因分布与表达、角膜病理效应、酶学特性以及免疫反应对该蛋白酶的保护作用等方面分析铜绿假单胞菌碱性丝氨酸蛋白酶（PASP）。
分别采用聚合酶链反应（PCR）和蛋白质免疫印迹分析对25株铜绿假单胞菌的PASP基因和分泌蛋白进行分析。将活性重组（r）PASP（10微克/20微升）或热灭活的rPASP基质内注射到兔角膜中。通过裂隙灯检查（SLE）和组织病理学监测病理变化。检测纯化的rPASP对胶原蛋白的裂解作用以及对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）的敏感性。制备兔抗rPASP抗体并检测其对酶的灭活作用，对主动免疫的兔子进行基质内注射活性rPASP（5微克）攻击。
所分析的25株铜绿假单胞菌的PASP基因和蛋白均呈阳性。在所有注射后时间点（PI），注射活性rPASP的眼睛的SLE评分均显著高于对照眼睛（P≤0.004）。组织病理学研究表明，在PI 9小时时角膜上皮层和部分角膜基质被破坏，在活性rPASP注射后24小时多形核（PMN）白细胞浸润到角膜中。PASP可裂解I型和IV型胶原蛋白，且对TLCK抑制敏感。PASP存在于细胞质和周质中，但只有分泌型PASP具有酶活性。产生了高抗体滴度（酶联免疫吸附测定滴度≥10,000），但该抗体不能保护免受活性rPASP攻击。
PASP是一种常见的铜绿假单胞菌蛋白酶，可裂解胶原蛋白并导致角膜糜烂。
