Zolfaghar Irandokht, Evans David J, Ronaghi Reza, Fleiszig Suzanne M J
School of Optometry, University of California, Berkeley, CA 94720-2020, USA.
Infect Immun. 2006 Jul;74(7):3880-9. doi: 10.1128/IAI.01891-05.
Mutation of retS (rtsM) of Pseudomonas aeruginosa strain PA103 reduces its virulence in both ocular and respiratory murine models of infection. In vitro, retS mutants exhibit loss of the ExsA-regulated type III secretion system (TTSS), reduced twitching motility, and a decrease in association with, invasion of, and survival within corneal epithelial cells. In addition, transcription of multiple other virulence genes is positively and negatively affected by retS mutation. Since our published data show that ExoU and ExoT, the two TTSS effectors encoded by strain PA103, each confer virulence in this corneal model, we hypothesized that loss of virulence of retS mutants follows loss of type III secretion. Corneal pathology, bacterial colonization, and phagocyte infiltration were compared for wild-type PA103, retS mutants, and various TTSS mutants after infection with approximately 10(6) CFU bacteria. Results showed that either a retS or an exsA (TTSS) mutation delayed disease progression, as illustrated by reduced severity scores and colonization levels during the first 48 h postinfection. Surprisingly, retS mutant infections then became more severe than those involving exsA mutants. By day 7, colonization levels of retS mutants even surpassed those of wild-type bacteria (more than twofold, P = 0.028). Although retS mutants caused more severe opacification of central corneas than both the wild type and the exsA mutants, neither mutant caused the peripheral ring opacity commonly associated with wild-type infection, suggesting that the TTSS was involved. Histological experiments with retS and various TTSS mutants showed that ring opacification required ExoU but not ExoT and that it consisted of dense polymorphonuclear phagocyte infiltration at the corneal periphery and the absence of any cell type in the central cornea. These data suggest that these P. aeruginosa TTSS effectors have different effects on innate immunity and that RetS influences virulence beyond its effects on the TTSS.
铜绿假单胞菌PA103菌株的retS（rtsM）突变降低了其在眼部和呼吸道小鼠感染模型中的毒力。在体外，retS突变体表现出ExsA调控的III型分泌系统（TTSS）缺失、颤动运动能力降低，以及与角膜上皮细胞的黏附、侵袭和在其中存活能力的下降。此外，retS突变对多个其他毒力基因的转录有正向和负向影响。由于我们已发表的数据表明，PA103菌株编码的两种TTSS效应蛋白ExoU和ExoT在该角膜模型中各自赋予毒力，我们推测retS突变体毒力的丧失是由于III型分泌的丧失。在用约10⁶CFU细菌感染后，比较了野生型PA103、retS突变体和各种TTSS突变体的角膜病理学、细菌定植和吞噬细胞浸润情况。结果表明，retS或exsA（TTSS）突变均延迟了疾病进展，如感染后最初48小时严重程度评分和定植水平降低所示。令人惊讶的是，retS突变体感染随后比exsA突变体感染更严重。到第7天，retS突变体的定植水平甚至超过了野生型细菌（超过两倍，P = 0.028）。尽管retS突变体导致中央角膜的浑浊比野生型和exsA突变体更严重，但两种突变体均未引起通常与野生型感染相关的周边环形浑浊，这表明TTSS参与其中。对retS和各种TTSS突变体进行的组织学实验表明，环形浑浊需要ExoU而非ExoT，并且它由角膜周边密集的多形核吞噬细胞浸润以及中央角膜中缺乏任何细胞类型组成。这些数据表明，这些铜绿假单胞菌TTSS效应蛋白对先天免疫有不同影响，并且RetS对毒力的影响超出了其对TTSS的作用。
