Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death

Abstract

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Pseudomonas aeruginosacan diversify after host cell invasion to establish multiple intracellular niches

“…Aligning with our previously published data (9–11, 16), T3SS-expressing intracellular bacteria localized to the cytosol in both epithelial cell types, wherein they replicated rapidly and disseminated throughout the cytoplasm (Fig. 1).…”

“…Differing from other intracellular bacteria that use host cytoskeletal components for intracellular motility (24), P. aeruginosa then utilizes its own pili/twitching motility to disseminate throughout the host cell (25). Meanwhile, ExoS further supports intracellular survival by delaying lytic host cell death effectively preserving the intracellular niche (16), and it drives formation of plasma membrane blebs to which some bacteria traffic and replicate within (9, 19). These blebs can subsequently disconnect from cells, becoming vesicles with intact membranes that can carry enclosed live/swimming bacteria to distant sites (9).…”

Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics

“…It has been reported early that ExoS participates in host cell apoptosis via its GAP region or ADP-ribosyltransferase (ADPr) activity. 114 , 115 Furthermore, the ExoS possesses ADPRT activity, which induces P. aeruginosa -afflicted host cell apoptosis by targeting a variety of Ras proteins. 116 ExoU is the longest P. aeruginosa effector containing 687 amino acids (73.9 kDa).…”

The Lectin LecB Induces Patches with Basolateral Characteristics at the Apical Membrane to Promote Pseudomonas aeruginosa Host Cell Invasion

“…The involvement of Rac1 for P. aeruginosa internalization through the LecB-triggered cascade we describe here will need further clarification. Specifically, the P. aeruginosa exotoxin S and exotoxin T are known to contain N-terminal RhoGTPase activating protein (RhoGAP) domains, which can hydrolyze GTP to GDP in Rho, Rac, and Cdc42, leading to cytoskeletal depolymerization and countering host cell invasion ( 46 , 47 ). It will be interesting to investigate whether varying expression levels of LecB, exotoxin S, and exotoxin T cause more or less invasive behavior of P. aeruginosa .…”