ExoS effector in Pseudomonas aeruginosa Hyperactive Type III secretion system mutant promotes enhanced Plasma
Arianna D Reuven1, Sarah Katzenell1, Bethany W Mwaura1
1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth College, Hanover, New Hampshire, United States of America.
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.
Insights
Pseudomonas aeruginosa
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen causing infections in immunocompromised individuals, notably those with cystic fibrosis (CF).
- The bacterium utilizes a type III secretion system (T3SS) to deliver effector proteins, such as ExoS, into host cells.
- ExoS possesses ADP ribosyltransferase (ADPRT) activity, contributing to virulence by interfering with neutrophil functions like phagocytosis and oxidative burst.
Purpose of the Study:
- To investigate the role of ExoS ADPRT activity in a hypervirulent Pseudomonas aeruginosa CF clinical isolate with a hyperactive T3SS.
- To determine how ExoS impacts neutrophil responses, including inflammasome activation, NET extrusion, and cell death mechanisms.
Main Methods:
- Infection of wildtype and CF-genotype mouse neutrophils with P. aeruginosa strains (PAO1 and a hyperactive T3SS CF isolate).
- Assay of NLRC4 inflammasome activation, histone 3 citrullination (CitH3), nuclear DNA decondensation, and plasma membrane rupture (PMR).
- Evaluation of glycine's effect on PMR and assessment of ninjurin-1 involvement.
Main Results:
- ExoS ADPRT activity in the hyperactive T3SS mutant modulated inflammasome responses, DNA decondensation, and NET extrusion, similar to PAO1.
- The hyperactive T3SS mutant exhibited enhanced CitH3 and PMR compared to PAO1.
- Glycine supplementation mitigated PMR, indicating a role for ninjurin-1 in this process.
Conclusions:
- Enhanced neutrophil PMR is a novel pathogenic activity of ExoS ADPRT in hypervirulent P. aeruginosa.
- ExoS ADPRT activity plays a significant role in modulating neutrophil cell death pathways during infection.
- Targeting ExoS or related pathways may offer therapeutic strategies against severe P. aeruginosa infections.
