An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P. aeruginosa bearing glycosylated pili. One-dimensional NMR analysis of the glycan indicated the sugars to be 5N␤OHC 4 7NfmPse, Xyl, and FucNAc. The complete proton assignments of these sugars as well as the serine residue were determined by COSY and TOCSY. Electrospray ionization mass spectrometry (MS) determined the mass of this molecule to be 771.5. The ROESY NMR spectrum, tandem MS/MS analysis, and methylation analysis provided information on linkage and the sequence of oligosaccharide components. These data indicated that the molecule had the following structure: ␣-5N␤OHC 4 7NFmPse-(234)-␤-Xyl-(133)-␤-FucNAc-(133)-␤-Ser.Pseudomonas aeruginosa is an opportunistic pathogen capable of causing severe infections in individuals with compromised defense mechanisms (1). The somatic pili, protein filaments that extend as bundles from one or both of the cell poles, are considered to be a major virulence factor, promoting adherence and invasiveness (2, 3). These fibers are composed of a monomeric subunit, pilin, which has a strain-dependent molecular weight of ϳ16,000. The mature form of this protein is produced by the removal of a six-residue leader sequence, a process that is accompanied by methylation of the nascent amino-terminal phenylalanine (4). Although this had initially been considered the only post-translational modification of this protein, evidence has been generated indicating that P. aeruginosa strain 1244 pilin is also glycosylated (5). This process is dependent on pilO, a gene present as part of an operon that also contains the pilin structural gene, pilA.Although archeal and eubacterial S-layers commonly contain covalently bound glycan (6 -8), other examples of glycosylated prokaryotic surface proteins are rare. Cell wall-associated glycoproteins have been demonstrated in Streptococcus sanguis (9) and Mycobacterium tuberculosis (10). Among Gram-negative bacteria, Campylobacter species have been shown to contain a general protein glycosylation system that modifies a number of surface proteins including flagellin (11-13). Evidence has been presented that a glycan is associated with P. aeruginosa flagellin (14). As with P. aeruginosa (5), the pili of Neisseria meningitidis and Neisseria gonorrhoeae have been shown to be glycosylated. X-ray diffraction studies indicate that the N. gonorrhoeae pilin glycan is a disaccharide (15). In addition to glycerol phosphate (16), N. meningitidis pilin contains covalently bound trisaccharide (17).Although the detection of glycosylated bacterial surface proteins is uncommon, the comprehensive determination of their carbohydrate structure has been even more rare. Results present...

Seventy-four of 110 strains of Pseudomonas aeruginosa tested produced detectable amounts of HCN from growth in 2% peptone or nutrient agar. Of the 25 species of12 bacterial and fungal genera tested, other than P. aeruginosa, only P. fluorescens and P. polycolor gave positive HCN tests. Cyanide is produced after cessation of active growth. Iron was stimulatory to cyanogenesis in concentration above 1 muM, while copper, zinc, cobalt, and manganese at concentrations of 20 muM had no effect. Cyanogenesis id dependent on the temperature of incubation within ranges which allow complete growth. Inorganic phosphate in concentrations between 90 and 300 mM allows growth but inhibits HCN production. Growth of cells anaerobically, using nitrate as the electron acceptor, results in low cyanide yields, which can be partially reversed by subsequent aerobic incubation. These results indicate that HCN is a secondary metabolite of P. aeruginosa.

Nucleotide sequencing of a region downstream from the Pseudomonas aeruginosa 1244 pilin structural gene, pilA, revealed an ORF potentially able to code for a protein of M, 50862. This ORF, called pilo, was flanked by a tRNA* gene, which was followed by a transcriptional termination sequence. The tRNA*' gene and the termination sequem were nearly identical to sequences found immediately adjacent to the pilA gene of several P. aewginosa strains.A 2200 base mRNA strand, which contained both the pil0 and pilA transcripts, was produced from this region, while a 650 base transcript containing only pilA was present in a 100-fold excess over the longer transcript. Hyperexpression of the pilA gene in a PilO-strain resulted in normal pilusspecific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparent M, and a more neutral pl compared to that produced by a strain containing a functional pil0 gene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functional pil0 gene.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.

SummaryThe structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the Oantigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.