Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Invest Ophthalmol Vis Sci. 2013 Apr 17;54(4):2821-8. doi: 10.1167/iovs.13-11788.
The virulence contribution of Pseudomonas aeruginosa small protease (PASP) during experimental keratitis was studied by comparing a PASP-deficient mutant with its parent and rescue strains.
The pasP gene of P. aeruginosa was replaced with the tetracycline resistance gene via allelic exchange. A plasmid carrying the pasP gene was introduced into the PASP-deficient mutant to construct a rescue strain. The PASP protein in the culture supernatants was determined by Western blot analysis. Corneal virulence was evaluated in rabbit and mouse keratitis models by slit lamp examination (SLE), bacterial enumeration, and/or histopathological analysis. Various host proteins and the rabbit tear film were analyzed for their susceptibility to PASP degradation.
The PASP-deficient mutant produced a significantly lower mean SLE score when compared with the parent or rescue strain (P ≤ 0.03) at 29 hours postinfection (PI). All of the strains grew equally in the rabbit cornea (P = 0.971). Corneas infected with the PASP-deficient mutant showed moderate histopathology compared with those infected with the parent or rescue strain, which produced severe pathology inclusive of epithelial erosions, corneal edema, and neutrophil infiltration. In the mouse model, eyes inoculated with the PASP-deficient mutant had a significantly lower mean SLE score at 24 hours PI than the eyes inoculated with the parent or rescue strain (P ≤ 0.007). PASP was found to degrade complement C3, fibrinogen, antimicrobial peptide LL-37, and constituents of the tear film.
PASP is a commonly secreted protease of P. aeruginosa that contributes significantly to the pathogenesis of keratitis.
通过比较铜绿假单胞菌小蛋白酶（PASP）缺陷突变株与其亲本和拯救株，研究其在实验性角膜炎中的毒力贡献。
通过等位基因交换将铜绿假单胞菌的 pasP 基因替换为四环素抗性基因。将携带 pasP 基因的质粒导入 PASP 缺陷突变株中，构建拯救株。通过 Western blot 分析测定培养上清液中的 PASP 蛋白。通过裂隙灯检查（SLE）、细菌计数和/或组织病理学分析，在兔和鼠角膜炎模型中评估 PASP 的角膜毒力。分析各种宿主蛋白和兔泪膜对 PASP 降解的敏感性。
与亲本或拯救株相比，PASP 缺陷突变株在感染后 29 小时（PI）时产生的平均 SLE 评分显著降低（P≤0.03）。所有菌株在兔角膜中的生长情况相同（P=0.971）。与感染亲本或拯救株的角膜相比，感染 PASP 缺陷突变株的角膜显示出中度组织病理学改变，而感染亲本或拯救株的角膜则产生严重的病理学改变，包括上皮糜烂、角膜水肿和中性粒细胞浸润。在小鼠模型中，感染 PASP 缺陷突变株的眼睛在 24 小时 PI 时的平均 SLE 评分明显低于感染亲本或拯救株的眼睛（P≤0.007）。发现 PASP 可降解补体 C3、纤维蛋白原、抗菌肽 LL-37 和泪膜成分。
PASP 是铜绿假单胞菌普遍分泌的一种蛋白酶，对角膜炎的发病机制有重要贡献。
