Peter Castric

pilo, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin

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(r) 50,862. This ORF, called pilO, was flanked by a tRNAthr gene, which was followed by a transcriptional termination sequence. The tRNAthr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P. aeruginosa strains. A 2200 base mRNA strand, which contained both the pilO 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 pilus-specific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparent M(r) and a more neutral pl compared to that produced by a strain containing a functional pilO gene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functional pilO gene.

Influence of Pilin Glycosylation on Pseudomonas aeruginosa 1244 Pilus Function

ABSTRACT 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.

Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity.

The 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 O‐antigen 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.

Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site

ABSTRACT Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.

Influence of oxygen on thePseudomonas aeruginosa hydrogen cyanide synthase

Partially purified HCN Synthase (HCS) required exogenous electron acceptors for activity. Phenazine methosulfate (PMS) provided the greatest activity, whereas oxygen allowed only a limited response. TheP. aeruginosa secondary metabolite pyocyanin supported HCS-mediated cyanide production. HCN production by whole cells operated maximally at low oxygen levels, whereas moderate oxygen levels limited HCS activity. Respiration and cyanogenesis by whole cells were equally sensitive to azide; HCS was completely resistant.

Pseudomonas aeruginosa 1244 Pilin Glycosylation: Glycan Substrate Recognition

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (beta-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (beta-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO(1244) in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.

Glycosylation Substrate Specificity of Pseudomonas aeruginosa 1244 Pilin

The β-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, whereas that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C terminus allowed glycosylation to take place. Extending the “tail” region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature and that this residue cannot be substituted at its carboxyl group. Although no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur.

Immunization with a Pseudomonas aeruginosa 1244 Pilin Provides O-Antigen-Specific Protection

ABSTRACT The O antigen is both a major structural outer membrane component and the dominant epitope of most gram-negative bacteria. Pseudomonas aeruginosa 1244 produces a type IV pilus and covalently links an O-antigen repeating unit to each pilin monomer. Here we show that immunization of mice with pure pilin from strain 1244 by use of either the mouse respiratory model or the thermal injury model resulted in protection from challenge with a pilus-null O-antigen-producing 1244 mutant. These results provide evidence that the pilin glycan stimulates a protective response that targets the O antigen, suggesting that this system could be used as the basis for the development of a variety of bioconjugate vaccines protective against gram-negative bacteria.

PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment

PilO of Pseudomonas aeruginosa 1244 catalyses the attachment of an O‐antigen repeating unit to the β‐carbon of the pilin C‐terminal residue, a serine. The present study was conducted to locate the regions of this enzyme important in catalysis and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable in extracts of P. aeruginosa or Escherichia coli, even under conditions of overexpression, it was found that an intact MalE–PilO fusion protein was produced in significant amounts. This fusion complemented a P. aeruginosa 1244 mutant containing a pilO deletion and targeted to the cytoplasmic membrane of E. coli. Wzy and WaaL, enzymes that also utilize the O‐antigen repeating unit as substrate, were found to share a sequence pattern with PilO even though these proteins have little overall sequence similarity. PilO constructs in which portions of this common sequence were deleted or altered by site‐directed mutagenesis lacked pilin glycosylating activity. Deletions of segments downstream from the common region also prevented enzyme activity. Topology studies showed that the two PilO regions associated with enzyme activity were located in the periplasm. These results establish regions of this enzyme important for catalysis and present evidence that pilin glycosylation occurs in the periplasmic space of this organism.

The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10−6 M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.