Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions.
Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions.
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DOI：
10.1167/iovs.04-1483
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发表时间：
2005-10
影响因子：
4.4
通讯作者：
M. Marquart;A. Caballero;M. Chomnawang;B. Thibodeaux;S. Twining;R. O'Callaghan
中科院分区：
文献类型：
--
作者：
M. Marquart;A. Caballero;M. Chomnawang;B. Thibodeaux;S. Twining;R. O'Callaghan
目的鉴定一种新的假单胞菌蛋白酶，并探讨其在角膜炎中的作用。方法对铜绿假单胞菌PA 103和ATCC 19660的浓缩培养上清进行酶谱分析。从菌株PA 103纯化铜绿假单胞菌小蛋白酶（PASP），并且从菌株ATCC 19660纯化修饰的弹性蛋白酶B（LasB）。对纯化的PASP和修饰的LasB进行SDS-PAGE和Western印迹分析。通过质谱和氨基端测序进一步分析PASP。Pasp基因的克隆和表达，亲和纯化的变性形式从包涵体，并通过去除变性剂重折叠。通过酶谱法分析纯化的重组PASP的蛋白酶活性。将PASP和热灭活的PASP注射到兔角膜中，并监测角膜由蛋白酶活性引起的糜烂。结果各菌株均能产生一种蛋白酶，酶谱分析表明其分子量约为80 kDa。LasB抗血清将ATCC 19660蛋白酶鉴定为修饰的LasB。质谱法将PA 103蛋白酶定义为具有18.5kDa的分子量。铜绿假单胞菌基因组序列的氨基末端测序和分析确定PA 103 Pasp基因序列与菌株PAO 1的PA 0423序列具有>99%的同一性。重组PASP具有蛋白水解性，酶谱质量为50 kDa。从PA 103纯化的PASP产生广泛的角膜上皮糜烂，而热灭活的PASP不产生糜烂。结论PASP是一种以前未被鉴定的蛋白酶。它引起角膜上皮糜烂，表明其可能作为假单胞菌角膜炎的促毒因子。
PURPOSE The purpose of this study was to identify a new Pseudomonas protease and determine its possible role in keratitis. METHODS Concentrated culture supernatants of the Pseudomonas aeruginosa strains PA103 and ATCC 19660 were analyzed by zymography. P. aeruginosa small protease (PASP) was purified from strain PA103, and modified elastase B (LasB) was purified from strain ATCC 19660. SDS-PAGE and Western blot analysis were performed on purified PASP and modified LasB. PASP was further analyzed by mass spectrometry and amino-terminal sequencing. The Pasp gene was cloned and expressed, affinity-purified in denatured form from inclusion bodies, and refolded by removal of the denaturant. Purified recombinant PASP was analyzed by zymography for protease activity. PASP and heat-inactivated PASP were injected into rabbit corneas, and the corneas were monitored for erosions caused by protease activity. RESULTS Each strain produced a protease with a molecular mass of 80 kDa on zymograms. LasB antiserum identified the ATCC 19660 protease as modified LasB. Mass spectrometry defined the PA103 protease as having a molecular mass of 18.5 kDa. Amino-terminal sequencing and analysis of the P. aeruginosa genome sequence determined that the PA103 Pasp gene sequence was >99% identical with the PA0423 sequence of strain PAO1. Recombinant PASP was proteolytic, with a zymogram mass of 50 kDa. PASP purified from PA103 produced extensive corneal epithelial erosions, whereas heat-inactivated PASP produced no erosions. CONCLUSIONS PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.