In vitro Assays to Monitor the Activity of Pseudomonas aeruginosa Type III Secreted Proteins

Abstract

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.

An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections

“…It is possible that fumarate may reduce the level of cytotoxicity of P. aeruginosa to the airway cells by tuning down virulence factors and influencing the level of toxins secreted by P. aeruginosa. It is known, for example, that at least certain strains of P. aeruginosa utilize the type III secretion system (T3SS) to deliver effector proteins that are cytotoxic to mammalian cells during infection to avoid innate immune clearance (42,43). As a further indication of the potential importance of the observed cytoprotective effect of fumarate, azithromycin is often given concurrently with TOB primarily for its anti-inflammatory effect (and possible inhibition of bacterial communication through quorum sensing) (34), even though it appears clinically to antagonize/ reduce the anti-infective effect of TOB (36).…”

Rational Design of a Chimeric Derivative of PcrV as a Subunit Vaccine Against Pseudomonas aeruginosa

“…Then the content of flagellin was determine by Western Blot and used as a control. Meanwhile, ExoU in the supernatant was collected and concentrated as described previously (25). The quantity of ExoU was analyzed by Western blot using anti-ExoU antibodies(self-developed) as primary antibodies.…”

Exoproteomics for Better Understanding Pseudomonas aeruginosa Virulence

“…In P. aeruginosa, T3SS is induced in vitro by calcium depletion. This depletion mimics the cell medium in which calcium is quenched and this signal is sufficient to induce T3SS [12,38]. T6SS is activated by close contact with target cells for interbacterial competition or eukaryotic host pathogenesis [39].…”