ExoS effector in Pseudomonas aeruginosa Hyperactive Type III secretion system mutant promotes enhanced Plasma Membrane Rupture in Neutrophils.

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen responsible for airway infections in immunocompromised individuals, including those with cystic fibrosis (CF). P. aeruginosa has a type III secretion system (T3SS) that translocates effectors into host cells. ExoS is a T3SS effector with ADP ribosyltransferase (ADPRT) activity. ExoS ADPRT activity promotes P. aeruginosa virulence by inhibiting phagocytosis and limiting oxidative burst in neutrophils. The P. aeruginosa T3SS also translocates flagellin, which can activate the NLRC4 inflammasome, resulting in: 1) gasdermin-D pores, release of IL-1β and pyroptosis; and 2) histone 3 citrullination (CitH3), nuclear DNA decondensation and expansion into the neutrophil cytosol with incomplete NET extrusion. However, studies with P. aeruginosa PAO1 indicate that ExoS ADPRT activity inhibits the NLRC4 inflammasome in neutrophils. Here, we identified an ExoS+ CF clinical isolate of P. aeruginosa with a hyperactive T3SS. Variants of the hyperactive T3SS mutant or PAO1 were used to infect neutrophils from C57BL/6 mice that were wildtype or engineered to have a CF genotype or defects in inflammasome assembly. Responses to NLRC4 inflammasome assembly or ExoS ADPRT activity were assayed and found to be similar for C57BL/6 or CF neutrophils. ExoS ADPRT activity in the hyperactive T3SS mutant regulated inflammasome, nuclear DNA decondensation and incomplete NET extrusion responses, like PAO1, but promoted enhanced CitH3 and plasma membrane rupture (PMR). Glycine supplementation inhibited PMR by the hyperactive T3SS mutant, suggesting ninjurin-1 is required for this process. These results identify enhanced neutrophil PMR as a pathogenic activity of ExoS ADPRT in hypervirulent P. aeruginosa.