Yahr T L, Vallis A J, Hancock M K, Barbieri J T, Frank D W
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13899-904. doi: 10.1073/pnas.95.23.13899.
The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.
外毒素S调节子是铜绿假单胞菌一组协调调控的毒力基因。该调节子编码的蛋白质包括III型分泌和转运装置、基因表达调节因子以及效应蛋白。效应蛋白包括两种具有ADP核糖基转移酶活性的酶（ExoS和ExoT）以及一种急性细胞毒素（ExoU）。在本研究中，我们鉴定出ExoY是该调节子的第四种效应蛋白。ExoY与百日咳博德特氏菌（CyaA）和炭疽芽孢杆菌（EF）的细胞外腺苷酸环化酶同源。这三种腺苷酸环化酶之间的同源性仅限于两个短区域，其中一个区域具有ATP结合基序。在腺苷酸环化酶活性测定中，重组ExoY（rExoY）催化形成cAMP，其比活性与CyaA的基础活性相似。与CyaA和EF不同，rExoY的活性不受钙调蛋白的刺激或激活。添加中国仓鼠卵巢（CHO）细胞的胞质提取物后，检测到活性有500倍的刺激。这些结果表明，一种不同于钙调蛋白的真核因子增强了rExoY的催化作用。对ExoY假定活性位点内的残基进行定点诱变消除了腺苷酸环化酶活性。用产生ExoY的铜绿假单胞菌菌株感染CHO细胞导致细胞内cAMP积累。CHO细胞内的cAMP积累依赖于完整的III型转运装置，表明ExoY直接转运到真核细胞质中。
