The Pseudomonas aeruginosa pathogenicity island PAPI-1 is transferred via a novel type IV pilus

Abstract

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments, including humans, is in part due to its large and diverse genomic repertoire. The genomes of most strains contain a significant number of large and small genomic islands, including those carrying virulence determinants (pathogenicity islands). The pathogenicity island PAPI-1 of strain PA14 is a cluster of 115 genes, and some have been shown to be responsible for virulence phenotypes in a number of infection models. We have previously demonstrated that PAPI-1 can be transferred to other P. aeruginosa strains following excision from the chromosome of the donor. Here we show that PAPI-1 is transferred into recipient P. aeruginosa by a conjugative mechanism, via a type IV pilus, encoded in PAPI-1 by a 10-gene cluster which is closely related to the genes in the enterobacterial plasmid R64. We also demonstrate that the precursor of the major pilus subunit, PilS2, is processed by the chromosomally encoded prepillin peptidase PilD but not its paralog FppA. Our results suggest that the pathogenicity island PAPI-1 may have evolved by acquisition of a conjugation system but that because of its dependence on an essential chromosomal determinant, its transfer is restricted to P. aeruginosa or other species capable of providing a functional prepilin peptidase.

FIG. 1.

Conservation of the PAPI-1 pil gene clusters in different microorganisms. The putative function of each PAPI-1 pilus protein is presented in Table 2. Homologous proteins in various bacteria are indicated by the same color. The numbers indicate the sequence similarity of each protein with its PAPI-1 homolog. The black arrows represent those genes lacking homologs in the PAPI-1 island. The gaps in PilQ2 and PilR2 of the P. aeruginosa (Pa) PAGI-5 represent the disruptions of ORFs by insertions. The pil cluster sequences were retrieved from GenBank: PAPI-1, accession no. AY273869; pKLC102, AY257538; PAGI-5, EF611301; R64 from Salmonella enterica serovar Typhimurium (St), NC_005014; and YAPI from Yersinia pseudotuberculosis (Yp), AJ627388. The pil cluster sequence of P. aeruginosa strain PA7, P. syringae (Ps) strain B782a, and P. fluorescens (Pf) strain Pf-5 were obtained from the Pseudomonas Genome Database (38).

FIG. 2.

(A) Chromosomal locations of three P. aeruginosa pilin genes, pilA, flp, and pilS2, in strain PA14. The numbers indicate the chromosome positions. (B) Phylogenetic analyses of the three P. aeruginosa pilin proteins and some pilin proteins from other organisms. The alignment was performed by the Jotun Hein method using DNASTAR Lasergene software. The pilin protein sequences were retrieved from GenBank. The type IVb pilin proteins are represented by PAPI-1 PilS2 (accession o. YP_792926), pKLC102 PilS (AAP22599), PAGI-5 PilS (translated protein from EF611301, protein ID ABR13424.1), PilS of P. fluorescens Pf-5 (Pf) (YP_261763), PilS of P. syringae B728a (Ps) (AAY36562), R64 PilS (NP_863479), YAPI-1 PilS (YP_001402683), BfpA of Escherichia coli (Ec) (YP_001965379), TcpA of Vibrio cholerae (Vc) (AAA88688), P. aeruginosa Flp (YP_792651), and Flp of Caulobacter crescentus (Cc) (AAF40189). Type IVa pilin proteins used in the analysis are PA14 PilA (YP_792871), PilA of Burkholderia dolosa (Bd) (ZP_04944829), and PilE from Neisseria gonorrhoeae (Ng) (CAA47349).

FIG. 3.

(A) Leader peptides of representative type IV prepilin proteins. The arrow indicates the site of processing by the prepilin peptidase. (B) Processing of the PilS2 leader peptide by chromosomally encoded PilD. Western immunoblot analysis of cleavage of His₆-tagged PilS2 in strain PA14 ΔpilS2ppilS2-His expressing wild-type levels of PilD (lane 1), in a PilD-deficient mutant (PA14 ΔpilDΔpilS2ppilS2-His (lane 2), and in the complemented strain PA14 ΔpilDΔpilS2att::pilD ppilS2-His (lane 3) is shown.

FIG. 4.

Expression of the PAPI-1 pil genes. β-Galactosidase levels in P. aeruginosa strains PAO1 (open bars) and PA14 (filled bars) carrying promoter-lacZ fusions are shown. The putative promoters of the PAPI-1 pil operon (pilL2P) and the pilS2 gene (pilS2P) were fused to the reporter lacZ gene, and the expression of β-galactosidase was monitored in both PAO1, a strain lacking of the PAPI-1 island, and strain PA14. Error bars indicate standard deviations.

FIG. 5.

Examination of PAPI-1-encoded pili by transmission electron microscopy (TEM). Long, thin, bundled pili of the control cells (PA14TnC2 ΔpilS2ΔpilA ppilS2) (A) and gold-labeled PAPI-1 pili (arrow with open head) of Flag-tagged cells (PA14TnC2 ΔpilS2ΔpilA ppilS2-flag) beside the flagella (filled arrow) (B) are seen. The immunogold labeling appears to be specific to the Flag tag, since there is little labeling of the native pili (no Flag-tagged PilS2) (A).