In vitro assays to monitor the activity of Pseudomonas aeruginosa Type III secreted proteins. Methods Mol Biol 2014;1149:171-84
Date
05/14/2014Pubmed ID
24818904Pubmed Central ID
PMC5860653DOI
10.1007/978-1-4939-0473-0_14Scopus ID
2-s2.0-84927145450 (requires institutional sign-in at Scopus site) 7 CitationsAbstract
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-(32)P]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.
Author List
Rolsma SL, Frank DWAuthor
Dara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
ADP Ribose TransferasesBacterial Proteins
Bacterial Secretion Systems
Biological Assay
Electrophoresis, Polyacrylamide Gel
Enzyme Assays
Phospholipases
Pseudomonas aeruginosa