RESUMO
Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a large percentage of airway infections that cause morbidity and mortality in immunocompromised patients, especially those with cystic fibrosis (CF). One important P. aeruginosa virulence factor is a type III secretion system (T3SS) that translocates effectors into host cells. ExoS is a T3SS effector with ADP ribosyltransferase (ADPRT) activity. The ADPRT activity of ExoS promotes P. aeruginosa virulence by inhibiting phagocytosis and limiting the oxidative burst in neutrophils. The P. aeruginosa T3SS also translocates flagellin, which can activate the NLRC4 inflammasome, resulting in: 1) gasdermin-D (GSDMD) pores, release of IL-1ß and pyroptosis; and 2) histone 3 citrullination (CitH3) and decondensation and expansion of nuclear DNA into the cytosol. However, recent studies with the P. aeruginosa laboratory strain PAO1 indicate that ExoS ADPRT activity inhibits activation of the NLRC4 inflammasome in neutrophils. Here, an ExoS+ CF clinical isolate of P. aeruginosa with a hyperactive T3SS was identified. Variants of the hyperactive T3SS mutant or PAO1 were used to infect neutrophils from C57BL/6 mice or mice engineered to have a CF genotype or a defect in inflammasome assembly. Responses to NLRC4 inflammasome assembly or ExoS ADPRT activity were assayed, results of which were found to be similar for C57BL/6 or CF neutrophils. The hyperactive T3SS mutant had enhanced resistance to neutrophil killing, like previously identified hypervirulent P. aeruginosa isolates. ExoS ADPRT activity in the hyperactive T3SS mutant regulated inflammasome and nuclear DNA decondensation responses like PAO1 but promoted enhanced CitH3 and plasma membrane rupture (PMR). Glycine supplementation inhibited PMR caused 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 a hypervirulent P. aeruginosa isolate.
RESUMO
The Burkholderia cepacia complex contains opportunistic pathogens that cause chronic infections and inflammation in lungs of people with cystic fibrosis. Two closely related species within this complex are Burkholderia cenocepacia and the recently classified Burkholderia orbicola. B. cenocepacia and B. orbicola encode a type VI secretion system and the effector TecA, which is detected by the pyrin/caspase-1 inflammasome, and triggers macrophage inflammatory death. In our earlier study the pyrin inflammasome was dispensable for lung inflammation in mice infected with B. orbicola AU1054, indicating this species activates an alternative pathway of macrophage inflammatory death. Notably, B. cenocepacia J2315 and K56-2 can damage macrophage phagosomes and K56-2 triggers activation of the caspase-11 inflammasome, which detects cytosolic LPS. Here we investigated inflammatory cell death in pyrin-deficient ( Mefv -/- ) mouse macrophages infected with B. cenocepacia J2315 or K56-2 or B. orbicola AU1054 or PC184. Macrophage inflammatory death was measured by cleavage of gasdermin D protein, release of cytokines IL-1α and IL-1ß and plasma membrane rupture. Findings suggest that J2315 and K56-2 are detected by the caspase-11 inflammasome in Mefv -/- macrophages, resulting in IL-1ß release. In contrast, inflammasome activation is not detected in Mefv -/- macrophages infected with AU1054 or PC184. Instead, AU1054 triggers an alternative macrophage inflammatory death pathway that requires TecA and results in plasma membrane rupture and IL-1α release. Amino acid variation between TecA isoforms in B. cenocepacia and B. orbicola may explain how the latter species triggers a non-inflammasome macrophage death pathway.