In vitro assays to monitor the activity of Pseudomonas aeruginosa Type III secreted proteins.
In vitro assays to monitor the activity of Pseudomonas aeruginosa Type III secreted proteins.
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DOI：
10.1007/978-1-4939-0473-0_14
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Frank DW
中科院分区：
文献类型：
--
作者：
Rolsma SL;Frank DW
铜绿假单胞菌分泌许多毒素和破坏性酶，在发病机制中发挥不同的作用。假单胞菌的III型分泌系统（T3 SS）是将毒素的子集直接递送到真核细胞的细胞质中的系统。分泌的效应物包括ExoS、ExoT、ExoU和ExoY。在这一章中，我们描述的方法来诱导T3 S的表达和测量在体外试验中的每个效应的酶活性。ExoU是一种磷脂酶，其活性可以在基于荧光的测定中测量，该测定监测荧光底物PED 6的裂解。ExoS和ExoT都具有ADP-核糖基转移酶（ADPRT）和GTP酶激活蛋白（GAP）活性。ADPRT活性可以通过使用放射性标记的烟酰胺腺嘌呤二核苷酸（NAD+）并测量ADP-核糖共价掺入靶蛋白来评估。通过从[γ-32 P] GTP结合的靶蛋白释放放射性标记的磷酸盐来测量GAP活性。根据最近的趋势，减少在实验室中使用放射性，替代检测使用荧光或生物素标记的试剂进行了说明。ExoY是核苷酸环化酶;由ExoY刺激的cAMP产生可使用反相HPLC或用市售免疫测定法监测。
Pseudomonas aeruginosa secretes numerous toxins and destructive enzymes that play distinct roles in pathogenesis. The Type III secretion system (T3SS) of Pseudomonas is a system that delivers a subset of toxins directly into the cytoplasm of eukaryotic cells. The secreted effectors include ExoS, ExoT, ExoU, and ExoY. In this chapter, we describe methods to induce T3S expression and measure the enzymatic activities of each effector in in vitro assays. ExoU is a phospholipase and its activity can be measured in a fluorescence-based assay monitoring the cleavage of the fluorogenic substrate, PED6. ExoS and ExoT both possess ADP-ribosyltransferase (ADPRT) and GTPase-activating protein (GAP) activity. ADPRT activity can be assessed by using radiolabeled nicotinamide adenine dinucleotide (NAD+) and measuring the covalent incorporation of ADP-ribose into a target protein. GAP activity is measured by the release of radiolabeled phosphate from [γ-32P]GTP-bound target proteins. In accordance with recent trends towards reducing the use of radioactivity in the laboratory, alternative assays using fluorescent or biotin-labeled reagents are described. ExoY is a nucleotidyl cyclase; cAMP production stimulated by ExoY can be monitored using reverse-phase HPLC or with commercially available immunological assays.