William Paranchych

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell‐surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo‐GM1) and, to a lesser extend, ll3N‐acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid‐phase binding assays. Asialo‐GM1, but not GM1, inhibited both PAK and PAK pili binding to immobilized asialo‐GM1 on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo‐GM1, suggesting the presence of a structurally similar receptor‐binding domain in both pilus types. The interaction between asialo‐GM1 and pili occurs at the pilus tip as asialo‐GM1 coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C‐terminal disulphide‐bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal‐tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)ox‐OH and PAO(128–144)ox‐OH, which correspond to the C‐terminal disulphide‐bonded region of Pseudomonas pilin are able to bind to asialo‐GM1 and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo‐GM1 Monoclonal antibody PK3B had no effect on PAK pili binding to asialo‐GM1 Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip‐associated phenomenon Involving a tip‐exposed C‐terminal region of the pilin structural subunit.

The Physiology and Biochemistry of Pili

Publisher Summary This chapter reviews and discusses the structure and function of pili in light of recent advances employing biochemical, immunological, and genetic approaches. Bacterial “fimbriae” or “pili” are thin (2-1 2 nm diameter) non-flagellar protein filaments found on the surfaces of many types of bacteria. The adhesive properties of pili allow them to bind to other bacteria, bacteriophages, mammalian cells, and inert surfaces. The chapter discusses the classification scheme for pili based on morphology, function, and biochemical properties. It describes three major groups of pilus—namely, conjugative, adhesive, and N-methylphenylalanine (NMePhe) pili. The simplest classification of pili on the basis of function is the division into two broad groups: “conjugative” and “adhesive” pili. It is noted that the biochemical properties help to identify the subpopulations of these pili. The conjugative pili show variable preferences for promoting bacterial mating in liquid or on solid media. Flexible pili generally confer the “universal mating type” and promote mating in liquid media and on solid surfaces equally well, whereas rigid pili are usually associated with “surface preferred” or “surface obligatory” mating types.

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo‐GM1 and asialo‐GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo‐GM1 (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo‐GM1 and asialo‐GM2 proposed by Krivan et al., 1988a) using solid‐phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal‐NAc(1–4)βGal‐Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C‐terminal disulphide‐looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C‐terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal‐(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc‐(1–4)βGal‐BSA was inhibited by PAK pili, Ac‐KCTSDQDEOFIPKGCSK‐OH (AcPAK(128–144)ox‐OH) and Ac‐ACKSTQDPMFTPKGCDN‐OH (AcPAO(128–144)ox‐OH) peptides. (In these peptides Ac denotes Nα ‐acetylation of the N‐terminus, ‐OH means a peptide with a free a‐carboxyl group at the C‐terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal‐biotin to the corresponding BSA‐Peptide(128–144)ox‐OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc‐biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C‐termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo‐GM1 receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.

Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene

A 1.2‐kilobase (kb) HindIII restriction fragment containing the pilin gene from Pseudomonas aeruginosa PAK has been cloned and sequenced. The pilin protein is 144 amino acids in length with a positively charged leader sequence of 6 amino acids. There is probably only one copy of the gene per chromosome.

Stages in phage R17 infection: VI. Injection of a protein and RNA into the host cell

Abstract Phage R17 was specifically labeled in the A protein with 3H-histidine, and in the RNA with 32P. Studies were then carried out to determine the fate of these two phage components during R17 infection. It was found that the interaction of phage particles with F piliated bacteria at 37° leads to the cleavage of A protein (MW 39,000) into two smaller components of molecular weights about 24,000 and 15,000, and that these polypeptide fragments are transferred into the cell along with the phage RNA. In addition, it was observed that phage RNA and A protein are injected into the cell in approximately equimolar amounts, and that their kinetics of penetration are similar. It was suggested that the penetration of phage RNA into the host cell may involve the injection of an A protein-RNA complex rather than free RNA.

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C‐terminal region for adherence to human buccal epithelial cells

The adherence of non‐mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15000 Oaltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti‐PAK pilus anti‐serum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface‐exposed. The PAK 128‐144‐OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C‐terminal region of PAK piiin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor‐binding domain of the PAK pilus.

The TraM protein of the conjugative plasmid F binds to the origin of transfer of the F and ColE1 plasmids.

The gene encoding the TraM protein of the conjugative plasmid F was cloned, overexpressed and the gene product was purified. The TraM protein was found in the cytoplasm of cells carrying the F plasmid with a smaller amount in the inner membrane. DNase I footprinting experiments showed that the purified protein protects three regions in the F ori T locus with different affinity for the upper and lower strands of DNA. A 15‐nucleotide motif was identified within the protected regions that represented the DNA‐binding site. The TraM protein was also found to bind to a sequence in the oriT region of the non‐conjugative plasmid ColE1 that resembles the three binding sites in the F oriT region.

Amino acid sequence of pilin isolated from pseudomonas aeruginosa PAK.

Pseudomonas aeruginosa strain PAK have polar pili which are flexible filaments of about 5.4 nm diameter and 2.5 pm average length [l-3]. These pili consist of a single subunit, pilin, which was originally reported to have a A4, of 18 100 on the basis of SDS-polyacrylamide gel electrophoresis and amino acid compositional studies [4]. The sequence of the first 22 amino acids of the Nterminal region of PAK pilin have been reported [5] and shown to be strikingly homologous to the N-terminus of pili [6] isolated from Moraxella nonliquefaciens and Neisseria gonorrhoeae [7]. The Nterminal amino acid is the unusual N-methylphenylalanine [7,8]. [9]. This strain was kindly provided by Dr D.E. Bradley (Memorial University, St. John’s, Newfoundland).

Stages in phage R17 infection: The role of divalent cations

Abstract The cations Mg ++ , Ca ++ , Sr ++ , and Ba ++ are equally effective in promoting R17 infection, whereas no phage growth occurs in the presence of the cations Mn ++ , Zn ++ , Ni ++ , or Co ++ . Maximum yields of phage R17 are obtained when the concentration of divalent ion in the growth medium is 0.7 m M or greater; increasing the concentration of Mg ++ to as high as 50 m M results in no further increase of phage yield over that found at the 0.7 m M concentration. Divalent cations are not required for phage attachment to host bacteria, nor for the stage of phage infection involving disruption of coat protein and release of RNA from the phage particle. Divalent cations are required, however, for the penetration of phage RNA into the host cell. The impairment of phage RNA penetration which occurs as a result of a lack of divalent cations during the initial stages of infection cannot be reversed by the later addition of divalent cations to the medium. On the other hand, if divalent cations are removed from the medium subsequent to the initiation of infection, phage replication proceeds at a normal rate. It is concluded that the only stage of the R17 infective process which is dependent upon the extracellular level of divalent cations is the penetration of phage RNA into the host cell.

Stages in phage R17 infection

Abstract An examination of phage R17 attachment to F-piliated bacteria at 4 ° and at 37 ° has demonstrated that apparently homogeneous phage preparations are heterogeneous with respect to the attachment function. Approximately 50% of the phage population is defective in its ability to adsorb to F pili. Most of these defective particles react only weakly with host bacteria. This weak interaction is sufficient to make the particles sensitive to ribonuclease, but insufficient to allow the release of RNA from the virion. The remaining 50% of the phage population is competent in its attachment function. The attachment process at 4 °C is a reversible one having an association constant of about 1.75. At 37 °, the rapid interaction of competent particles with host bacteria is followed by a very rapid dissociation of the phage-cell complex. The eluted particles do not dissociate into coat protein subunits, but remain intact as empty, or partially empty capsids. Approximate estimates indicate that only 10% of the total phage population succeeds in releasing intact RNA molecules to host bacteria during the course of infection.