A truncated mutation of MucA in Pseudomonas aeruginosa from a bronchiectasis patient affects T3SS expression and inflammasome activation

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic airway infection in bronchiectasis patients and is closely associated with poor prognosis. Strains isolated from chronically infected patients typically have a mucoid phenotype due to the overproduction of alginate. In this study, we isolate a P. aeruginosa strain from the sputum of a patient with bronchiectasis and find that a truncated mutation occurred in mucA, which is named mucA117. mucA117 causes the strain to transform into a mucoid phenotype, downregulates the expression of T3SS and inflammasome ligands such as fliC and allows it to avoid inflammasome activation. The truncated mutation of the MucA protein may help P. aeruginosa escape clearance by the immune system, enabling long-term colonization.

Keywords: MucA; Type III secretion systems; bronchiectasis; inflammasome.

Figure 1

P. aeruginosa strain L012 is mucoid colonies and a truncated mutation of mucA occurs in strain L012 (A) P. aeruginosa strains PAO1 and L012 were grown on LB agar and imaged after 24 h to observe mucus formation. (B) Schematic diagram of the MucA structure. Green, AlgU binding domain (AlgU BD); yellow, the transmembrane domain (TM); and blue, the MucB binding domain (MucB BD). The red line indicates the mutation position of P. aeruginosa strain L012, and the green line indicates the mutation position of mucA22.

Figure 2

mucA117 turns the strain into mucoid and reduces the its ability to induce pyroptosis and the activation of inflammasomes in PMs (A) The P. aeruginosa strains PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA, PAO1Δ mucA/L012 mucA, L012/EV and L012/PAO1 mucA were grown on LB agar without arabinose and imaged after 24 h to observe mucus formation. (B) PMs were infected with PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA and PAO1Δ mucA/L012 mucA (MOI=10) for 6 h. Cell death was determined by measuring the LDH released into the supernatants. (C) Caspase-1 cleavage in the supernatants and cell lysates of PMs infected with PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA and PAO1Δ mucA/L012 mucA for 6 h. * P<0.05, ** P<0.01 (unpaired t test).

Figure 3

mucA117 downregulates the expression of T3SS and inflammasome ligand, which may be related to the increased expressions of algU and algR (A) Under T3SS induction conditions, the PcrV levels in the P. aeruginosa strains PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA, PAO1Δ mucA/L012 mucA, L012/EV and L012/PAO1 mucA were detected in culture supernatants and whole-cell extracts. (B) Relative expression of pcrV, fliC, algU and algR in these strains. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (unpaired t test).