Pseudomonas aeruginosa Regulated Intramembrane Proteolysis: Protease MucP Can Overcome Mutations in the AlgO Periplasmic Protease To Restore Alginate Production in Nonmucoid Revertants

Abstract

The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen from a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the () gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes,, also known as or, and Interestingly, our mutations were complemented by both and, leading us to delete, clone, and overexpress ,, , and in both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Infection by the opportunistic pathogen is the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

Overproduction of the AlgT sigma factor is lethal to mucoidPseudomonas aeruginosa

“…It has been observed that mucoid strains frequently revert to nonmucoid by acquiring mutations in algT (19, 25, 38). To determine what other mutations we could identify in algT that result in nonmucoid reversion, we grew mucoid PDO300 under low aeration, a stressful condition that selects for reversion (19). We isolated 18 PDO300 nonmucoid revertants and then sequenced algT to determine what proportion had acquired mutations in this gene.…”

“…We found that 3 out of the 6 PDO300 and PDO300 Δ algD suppressors contained mutations in the MucA protease, mucP. MucP is a zinc metalloprotease that participates in the proteolytic cascade that degrades MucA (18, 19, 42). Delgado et al analyzed the MucP sequence and found four possible transmembrane domains, one beta-loop domain, a metalloprotease zinc-binding motif, two PDZ binding domains, and a RIP motif (19).…”

“…MucP is a zinc metalloprotease that participates in the proteolytic cascade that degrades MucA (18, 19, 42). Delgado et al analyzed the MucP sequence and found four possible transmembrane domains, one beta-loop domain, a metalloprotease zinc-binding motif, two PDZ binding domains, and a RIP motif (19). Many previous studies have predicted the secondary structure of the membrane bound region of MucP but failed to provide a three-dimensional atomistic model.…”

“…AlgT is made available and alginate biosynthesis is triggered by a regulated intermembrane proteolysis (RIP) cascade that is activated by proteases (17). Briefly, activated AlgW cleaves the periplasmic C-terminus of MucA making the truncated inner membrane portion of MucA available for cleavage by MucP (18, 19). This releases the cytosolic N-terminus AlgT-bound fragment of MucA where it is finally completely degraded by the ClpPX proteasome.…”

Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

“…Similarly, the abundance of PA14_01490 (PA0122/RahU), a Rhl activated lipid binding protein, was decreased with both lactonase treatments but to a greater extent with SsoPox W263I (fold change of −959 and −107 vs. control and GcL) (Miklavič et al, 2015). Conversely, GcL induced a stronger decrease of Tsp (or AlgO), a protein part of alginate synthesis regulation, than SsoPox W263I (fold change of −115 and −35 vs. control and SsoPox W263I) (Qiu et al, 2007;Hay et al, 2014;Delgado et al, 2018). However, AlgU, the alginate regulator, and OprF, a pleiotropic porin also involved in biofilm formation, were only significantly reduced with SsoPox W263I (Hay et al, 2014;Chevalier et al, 2017).…”