Pseudomonas aeruginosa PilY1 Binds Integrin in an RGD- and Calcium-Dependent Manner

Abstract

PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.

Calcium Binding Properties of the Kingella kingae PilC1 and PilC2 Proteins Have Differential Effects on Type IV Pilus-Mediated Adherence and Twitching Motility

“…The PilC1 and PilC2 proteins in the pathogenic Neisseria species have been identified as the adhesive component of the pilus fiber and are located at the tip of the fiber in N. gonorrhoeae (29)(30)(31). Recent observations suggest that P. aeruginosa PilY1 (the single PilC-like protein in P. aeruginosa) is also an adhesin (25,32). In earlier work, we observed a reduction in adherence to human cells by K. kingae strain 269-492 derivatives that expressed only PilC1 or PilC2, supporting a po-tential role for these proteins in adherence (14).…”

Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

“…The fimU promoter is upstream of six open reading frames (ORFs) (fimU-pilVWXY1E), which encode prepilins and a calcium-dependent retraction protein required for the normal assembly and function of the type IVa pilus apparatus (36,(53)(54)(55)(56). Since the expression of the fimU operon directly affects type IVa pilus function, the twitching motility phenotype in the context of the algR and algZ mutants was tested.…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…Those data led to a focus on development of anti-P. aeruginosa vaccines containing peptides corresponding to the D region (71,72,75,162,216). Newer information showing that the minor pilins are present in sheared pilus fractions (154,416) and that the orthologous PilC1 or PilC2 (Neisseria and Kingella) and PilY1 (Pseudomonas) proteins are potentially pilus associated and required for adherence to-and manipulation of-the host (174,204,217,288,289,384) suggests that additional studies are needed to unequivocally identify all T4aP adhesins. Given the wide range of surfaces to which T4P bind, there may be multiple players that contribute to adherence under specific circumstances.…”

“…N. meningitidis isolates use PilC1 and PilC2 to adhere to the uropods of neutrophils in order to escape phagocytosis (362). P. aeruginosa expresses an integrinbinding PilC homolog, called PilY1, that requires the T4P system for surface localization, although its direct association with pili has not yet been demonstrated convincingly (13,53,174,204). Mutation of PilY1 homologs in plant pathogens has also been associated with defects in motility and biofilm formation (252,310).…”