Truncation of type IV pilin induces mucoidy in Pseudomonas aeruginosa strain PAO579

Abstract

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ22). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosa strain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA108), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA108 confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA108, algW, algU, and rpoN encoding the alternative sigma factor σ54. We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway.

Truncated type IV pilin PilA108 activates the intramembrane protease AlgW to cleave MucA and PilA108 itself in vitro

“…In this study, we demonstrated that the truncated pilin PilA 108 with the FTF motif at its carboxyl terminus (Withers et al 2013), and this is the first trial to activate AlgW by another protein in vitro. This demonstrated intuitionally the possibly real reaction in the cell.…”

“…In vivo genetic analyses suggested that PilA 108 might cause the mucoid conversion through AlgW activation (Withers et al 2013), but we had no idea whether PilA 108 acted directly on AlgW. Moreover, the use of a purified protein to activate AlgW to cleave MucA directly in vitro has never been reported.…”

“…The stable mucoid strain PAO579 was isolated using carbenicillin selection from mucoid variants of PAO381 (Govan and Fyfe 1978), a non-mucoid derivative of strain PAO1. Whole genome resequencing revealed that three tandem single point mutations in the pilA gene generates a truncated PilA (PilA 108 ), which is the cause of mucoidy through an AlgW/AlgU-dependent pathway (Withers et al 2013). Based on genetic analyses, it was proposed that PilA 108 with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF) is able to activate AlgW to cleave MucA (Withers et al 2013).…”

Draft Genome Sequence of a Mucoid Isolate of Pseudomonas aeruginosa Strain C7447m from a Patient with Cystic Fibrosis

“…Despite the mucoid phenotype, the strain C7447m has wild-type algU and mucA . Furthermore, among previously reported alginate production-related genes (3–6), the amino acid sequences of MucC, MucD, AlgL, and KinB each have 1 amino acid change; AlgX has 2 amino acid changes; and AlgI and AlgP have 5 amino acid changes. We also found some SNPs located in the intergenic region before algB , algC , algD , amrZ , clpP2 , mucE , mucR , and pilA .…”

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

“…with fibrosis patients). [13][14][15] This mucoid strain is characterized by the overproduction of alginate polysaccharides, which are copolymers of 1-4 linked D-mannuronic and L-guluronic acid residues. 16 The adhesion of mucoid alginate to the lung of patient is a major virulence factor that causes a series of problems including hosting dormant bacteria, reducing the effectiveness of antibiotics, and difficulty in breathing.…”