Abstract
The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell-cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein-protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8-11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9-14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell.
Highlights
In ⌬R15 and ⌬R8, some PelA was detected in the periplasmic fraction, PelA was enriched in the outer membrane fraction; we propose that these repeats indicate the C- and N-terminal boundaries, respectively, of the tetratricopeptide repeats (TPRs) required for the PelA–PelB interaction (Fig. 3B)
We report the detailed characterization of an interaction between the periplasmic exopolysaccharide-modifying enzyme PelA and the TPR-containing domain of PelB
Using systematic TPR-deletion studies and copurification experiments, we found that R9 –R14 of the PelB TPR-containing domain are required for this interaction
Summary
The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. Our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell. TPR motifs are found in proteins from all domains of life and function as protein–protein interaction modules, acting as scaffolds for large protein complexes involved in diverse cellular processes [15] In bacteria, these include the maintenance of outer membrane integrity [16], chaperone activity in the type III secretion system [17], and assembly of the type IV pilus [18]. Our results suggest that the TPR-containing domain of PelB acts as a scaffold, localizing PelA within the periplasm to the export machinery to increase polysaccharide deacetylation during PEL biosynthesis and biofilm development
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