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 Pseudomonas aeruginosa 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 algT (algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 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, algO, also known as prc or PA3257, and mucP Interestingly, our algO mutations were complemented by both mucP and algO, leading us to delete, clone, and overexpress mucP, algO, mucE, and mucD 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 Pseudomonas aeruginosaIMPORTANCE Infection by the opportunistic pathogen Pseudomonas aeruginosa 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.

Keywords: AlgT/U; Prc/AlgO/Tsp; RseP/MucP/YaeL; anti-sigma factor; cystic fibrosis; mucoid conversion; sigma factors; sigma-22; sigma-E; σ22; σE.

FIG 1

Regulated intramembrane proteolysis pathway of P. aeruginosa. The C-terminal WVF motif of MucE indirectly activates the AlgW protease to cleave MucA. This cleavage requires the removal of the MucB protein from the MucA C terminus. Subsequent cleavage of MucA is performed by the AlgO and MucP proteases, which releases the AlgT/U sigma factor. Further processing by SspA, ClpX, and ClpP removes the remaining MucA fragment, allowing AlgT/U to interact with RNA polymerase (RNAP) and begin transcription of the alginate pathway genes. This process is negatively regulated in the periplasmic space by MucD. In the mucoid mucA22 mutant, the truncated C terminus of the protein is not bound by MucB, allowing for cleavage by the AlgO protease (see the text for details). The cleaved MucA22 protein is then processed by SspA, ClpX, and ClpP. Both wild-type and MucA22 pathways also undergo regulation by the LptD outer membrane protein (46). AlgO, MucD, AlgW, and MucP all contain PDZ domains (yellow boxes) involved in protein-protein interactions. Scissors indicate proteolytic cleavage of MucA or MucA22 by AlgW (green), AlgO (burgundy), and MucP (orange).

FIG 2

Complementation of sap17 and sap20 mutants with cosmid pMO013722 on LBTc plates. (A) The P. aeruginosa genes found in cosmid pMO013722 (corresponding to nucleotides 4068269 to 409343 of the PAO1 genome) are shown. The red symbol indicates the position of the EZ::Tn transposon insertion. (B) Phenotype of wild-type, mutant, and complemented strains inoculated on LB plates. The parental PDO300 strain displays a mucoid phenotype which is lost in the sap mutants but restored upon transformation of pMO013722. The prototypic PAO1 strain, transformed with the empty vector plasmid pLAFR3, is included as a nonmucoid control. Cells were streaked on LB plates containing tetracycline and were incubated at 37°C for 24 h.

FIG 3

Alginate levels in P. aeruginosa strains. Assays were performed in triplicate, and statistics were calculated using the two-tailed Student t test; asterisks indicate P values of <0.05 (A) Alginate levels of wild-type (PAO1), mucoid (PDO300), sap17, and sap20 overnight LB cultures were measured using the uronic acid assay standardized with purified sodium alginate, expressed in micrograms of alginate per milliliter of culture supernatant. Strains lacked plasmid (No Vector), contained plasmid pLAFR3 (Vector), contained pMO013722 (pCosmid), or contained pJG293 (pAlgT/U) as indicated. (B) sap mutants containing pLAFR3 (Vector), pMO013722 (pCosmid), pMO013722 containing the transposon insertion (pCosmid::Tn), pMF54 (P_trc Vector), and pLVF54 containing mucP (P_trc-mucP) were grown overnight in LB medium containing carbenicillin (except the no vector control) and induced at the indicated IPTG concentrations. (C) Wild-type PAO1 and mucoid strains PDO300 and PA2192 were transformed with the mucP-containing pLVF52 plasmid and grown in LB broth containing carbenicillin with increasing concentrations of IPTG. The inset image indicates the mucoid phenotype of pLVF52-transformed PA2192 in the presence and absence of 1 mM IPTG.

FIG 4

Linear representations of the AlgO (A) and MucP (B) proteins and their mutant variants. Signal sequence (SS), zinc-binding (Zn-BS), protein-protein association (PDZ), transmembrane (TM), catalytic and tail-specific protease (TSP), and RIP motif domains are indicated. Changes in the sequence due to frameshifting or single-amino-acid changes are indicated in grey (AlgO) and pink (MucP), and the specific deletion in AlgO336 is indicated as a gap in the sequence. The positioning of MucP in the inner membrane is indicated at the bottom of panel B. The zinc-binding and RIP metalloprotease motifs are indicated as HEFGH and LDGG, respectively. The C1N and MRE β-loop domains (62) are situated between TM1 and TM2, respectively. The conserved motif of the MRE β-loop (63) is indicated as PLGG.