Lee Ellen J, Cowell Brigitte A, Evans David J, Fleiszig Suzanne M J
Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley, California 94720, USA.
Invest Ophthalmol Vis Sci. 2003 Sep;44(9):3892-8. doi: 10.1167/iovs.02-1302.
The exoenzyme S regulatory protein ExsA regulates a type III secretion system in Pseudomonas aeruginosa. In vitro, cytotoxic strains use this system to secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their phagocytosis by epithelial cells. Invasive P. aeruginosa secrete ExoT and ExoS, but exsA mutation has little impact on their short-term interactions with epithelia. In the present study, the contribution of these ExsA-regulated proteins toward corneal infections in vivo was investigated.
After anesthesia, the left cornea of C57BL/6 mice was scratch injured and then inoculated with cytotoxic (PA103) or invasive (PAK) P. aeruginosa or with isogenic mutants in exsA-related genes. Inocula of 10(3) to 10(6) bacteria/5 micro L were used, and at least five animals were assigned to each experimental group. Corneal disease was quantified at regular intervals for 14 days in masked fashion with two different scoring systems.
For the cytotoxic strain, mutation of either exoU or exoT alone had little effect on virulence, whereas simultaneous mutation of both exoT and exoU or of exsA resulted in a significantly reduced capacity to cause corneal disease. Complementation of the double exoUexoT mutant with exoU alone restored bacterial colonization levels (>3-log increase) and disease severity to wild-type levels. Complementation with exoT alone increased colonization ( approximately 3-log increase) and increased virulence to almost the same levels as wild-type or exoU-complemented infections. Virulence of the invasive strain was not reduced by mutation of exsA or of genes encoding the ExsA-regulated secreted proteins.
ExsA contributed to corneal virulence of only cytotoxic P. aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity. This differs from cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.
外毒素S调节蛋白ExsA调控铜绿假单胞菌的III型分泌系统。在体外，具有细胞毒性的菌株利用该系统分泌外毒素（Exo）U和ExoT，从而导致细胞毒性并抑制上皮细胞对它们的吞噬作用。侵袭性铜绿假单胞菌分泌ExoT和ExoS，但exsA突变对其与上皮细胞的短期相互作用影响不大。在本研究中，对这些ExsA调控蛋白在体内角膜感染中的作用进行了研究。
麻醉后，刮伤C57BL/6小鼠的左眼角膜，然后接种具有细胞毒性的（PA103）或侵袭性的（PAK）铜绿假单胞菌，或接种exsA相关基因的同基因突变体。使用每5微升含10³至10⁶个细菌的接种物，每个实验组至少分配五只动物。采用两种不同的评分系统，以盲法定期对角膜疾病进行14天的量化评估。
对于具有细胞毒性的菌株，单独的exoU或exoT突变对毒力影响不大，而exoT和exoU同时突变或exsA突变会导致引起角膜疾病的能力显著降低。用单独的exoU对exoUexoT双突变体进行互补可使细菌定植水平（增加>3个对数）和疾病严重程度恢复到野生型水平。仅用exoT进行互补可增加定植（增加约3个对数）并使毒力增加到与野生型或exoU互补感染几乎相同的水平。侵袭性菌株的毒力不会因exsA或编码ExsA调控的分泌蛋白的基因突变而降低。
ExsA仅对具有细胞毒性的铜绿假单胞菌的角膜毒力有贡献，ExoU和ExoT均对细菌存活和疾病严重程度有贡献。这与肺中具有细胞毒性的铜绿假单胞菌毒力不同，后者依赖于ExoU。
