Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin

The beta-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.     

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.     

Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan

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 beta 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: alpha-5N beta OHC(4)7NFmPse-(2-->4)beta-Xyl-(1-->3)-beta-FucNAc-(1-->3)-beta-Ser.     

Glycosylation of Pilin and Nonpilin Protein Constructs by Pseudomonas aeruginosa 1244

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the β carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus with Escherichia coli alkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO. E. coli alkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.Colonization and dissemination of the opportunistic pathogen Pseudomonas aeruginosa rely to a large extent on the ability of this organism to produce functional type IV pili (26). These protein fibers, which radiate from the cell pole, are adhesion factors (51), mediate a form of surface translocation referred to as twitching motility (10, 37), and are important in biofilm formation (39). The pili of this organism are primarily composed of a monomeric subunit called pilin (PilA). Type IV pili can be differentiated into two classes (a or b) on the basis of the PilA sequence and structure (23). Although they display considerable sequence variation, the majority of the type IVa pilins of P. aeruginosa can be placed into one of three groups on the basis of primary structure and antigenicity, as well as by the presence of auxiliary pilin genes found immediately downstream from pilA (8, 33). We previously determined that pilin from P. aeruginosa 1244, which belongs to group I (8), contained an O-antigen repeating unit covalently attached to the β-hydroxyl group of a serine residing at the C terminus of this protein (7). While the specific physiological role of the pilin glycan in this organism is not clear, the presence of this saccharide influences pilus hydrophobicity and has a pronounced effect on virulence, as determined in a mouse respiratory model (47). The metabolic origin of the pilin saccharide is the O-antigen biosynthetic pathway (14), and its attachment is catalyzed by an oligosaccharyl transferase (OTase) called PilO (6). Specific regions of this cytoplasmic membrane protein necessary for glycosylation activity have been identified (42). Topological studies of PilO have shown that these regions face the periplasm, suggesting that pilin glycosylation takes place in this chamber (42). Here the glycan substrate is the O-antigen repeating unit covalently linked to the undecaprenol carrier lipid.PilO has a very relaxed glycan substrate specificity, as indicated by the evidence that it is able to utilize a number of structurally dissimilar O-antigen repeating units as substrate (14), and requires only features of the reducing end sugar to carry out pilin glycosylation (28). WaaL, the enzyme that transfers polymerized O antigen to core lipid A, from Escherichia coli also has a similar broad glycan specificity (19). Recent studies (18) provided evidence that PglL, an OTase of Neisseria meningitidis, recognized only the carrier lipid and was able to attach a variety of saccharides to the pilin of this organism. Although the glycan specificity of PilO is relaxed, this enzyme will not attach other carrier lipid-bound saccharides, such as the peptidoglycan subunit or polymerized O-antigen repeating unit, to pilin. This is indicated by the absence of pilins with increased mass in O-antigen-negative mutants or the production of multiple pilin sizes in the wild-type strain (6).In vivo analysis of mutagenized P. aeruginosa 1244 pilin showed that the C-terminal serine of this protein was a major pilin glycosylation recognition feature of PilO (27). In addition, modification (substitution of the C-terminal amino acid with a 3-residue sequence terminating in serine) of a group II pilin allowed PilO-dependent attachment of the O-antigen repeating unit (27). While these results suggested that the preponderance of pilin structural information was not required for glycosylation, it was not clear whether regions common among the P. aeruginosa pilins were needed. In the present study three types of experiments were carried out in order to answer this question. First, the glycosylation site was extended away from the pilin surface with the addition of a 15-residue peptide which terminates with serine. Second, an engineered periplasmic protein containing the glycosylation residue at its C terminus was fused with pilin and tested for PilO activity. Finally, this periplasmic protein containing no pilin common region was constructed and tested. Evidence presented in this paper suggests that PilO requires only the glycosylation target residue.The work presented also indicated that, in addition to pilins, nonpilin protein free in the periplasm or anchored to the cytoplasmic membrane could be engineered so as to serve as a PilO substrate. These results suggest that a wide range of pilins and nonpilin proteins can be engineered to serve as substrate for glycosylation, a finding that would potentially have practical value, particularly in the area of vaccine construction. In addition to elucidating the protein specificity of the PilO system, the present work determined that the peptide extension used can supply functional epitope information to the modified protein, in addition to providing a site for glycosylation. Altogether, the results presented suggest that engineering of pilins and nonpilin proteins for the biological generation of protein-peptide-saccharide constructs is a potentially important strategy in vaccine design.     

Pseudomonas aeruginosa pilin activates the inflammasome

IL-1β is produced from inactive pro-IL-1β by activation of caspase-1 brought about by a multi-subunit protein platform called the inflammasome. Many bacteria can trigger inflammasome activity through flagellin activation of the host protein NLRC4. However, strains of the common human pathogen Pseudomonas aeruginosa lacking flagellin can still activate the inflammasome. We set out to identify what non-flagellin components could produce this activation. Using mass spectroscopy, we identified an inflammasome-activating factor from P. aeruginosa as pilin, the major component of the type IV bacterial pilus. Purified pilin introduced into mouse macrophages by liposomal delivery activated caspase-1 and led to secretion of mature IL-1β, as did recombinant pilin purified from Escherichia coli. This was dependent on caspase-1 but not on the host inflammasome proteins NLRC4, NLRP3 or ASC. Mutants of P. aeruginosa strain PA103 lacking pilin did not activate the inflammasome following infection of macrophages with live bacteria. Type III secretion remained intact in the absence of pili, showing this was not due to a lack of effector delivery. Our observations show pilin is a novel activator of the inflammasome in addition to flagellin and the recently described PrgJ protein family, the basal body rod component of the type III apparatus.     

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 beta-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.     

X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement.Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme.     

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.     

Two unusual pilin sequences from different isolates of Pseudomonas aeruginosa.

The pilin genes of two Pseudomonas aeruginosa strains isolated from two different patients with cystic fibrosis were cloned and sequenced. The predicted protein sequences of these two pilins had several unusual features compared with other published P. aeruginosa pilin sequences.     

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.     

N-Terminal amino acid sequence of pilin isolated from Pseudomonas aeruginosa.

The amino-terminal amino acid sequence of the pili protein from Pseudomonas aeruginosa K pili is presented. The sequence is compared with those reported by others for pilin obtained from Neisseria gonorrhoeae and Moraxella nonlique-faciens. All three sequences are highly homologous, contain only two hydrophilic residues in the first 22 positions, and contain an unusual amino acid, N-monomethylphenylalanine, at the amino terminus.     

Glycosylation of b-Type flagellin of Pseudomonas aeruginosa: structural and genetic basis

The flagellin of Pseudomonas aeruginosa can be classified into two major types-a-type or b-type-which can be distinguished on the basis of molecular weight and reactivity with type-specific antisera. Flagellin from the a-type strain PAK was shown to be glycosylated with a heterogeneous O-linked glycan attached to Thr189 and Ser260. Here we show that b-type flagellin from strain PAO1 is also posttranslationally modified with an excess mass of up to 700 Da, which cannot be explained through phosphorylation. Two serine residues at positions 191 and 195 were found to be modified. Each site had a deoxyhexose to which is linked a unique modification of 209 Da containing a phosphate moiety. In comparison to strain PAK, which has an extensive flagellar glycosylation island of 14 genes in its genome, the equivalent locus in PAO1 comprises of only four genes. PCR analysis and sequence information suggested that there are few or no polymorphisms among the islands of the b-type strains. Mutations were made in each of the genes, PA1088 to PA1091, and the flagellin from these isogenic mutants was examined by mass spectrometry to determine whether they were involved in posttranslational modification of the type-b flagellin. While mutation of PA1088, PA1089, and PA1090 genes altered the composition of the flagellin glycan, only unmodified flagellin was produced by the PA1091 mutant strain. There were no changes in motility or lipopolysaccharide banding in the mutants, implying a role that is limited to glycosylation.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).     

The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli

Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4- or 8-amino-acid deletion at the N-terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C-terminal truncations having between 36 and 56 amino acids of the N-terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.     

Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa

The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P. aeruginosa genome contains three genes encoding acyl carrier proteins. We have cloned all three genes, expressed the acyl carrier proteins, and characterized each as a substrate for RhlI. A continuous, spectrophotometric assay was developed to facilitate kinetic and mechanistic studies of RhlI. Acp1, which has not been characterized previously, was a good substrate for RhlI, with a K(m) of 7 microM; the reaction proceeded with a k(cat) value of 0.35 s(-1). AcpP, which supports fatty acid biosynthesis, was also a good substrate in the RhlI reaction, where k(cat) was 0.46 s(-1), and the K(m) for AcpP was 6 microM. The third acyl carrier protein, Acp3, was a poor substrate for RhlI, with a K(m) of 280 microM; k(cat) was 0.03 s(-1). Taken together with microarray data from the literature which show that expression of the gene encoding Acp1 is under the control of the quorum-sensing system, our data suggest that Acp1 is likely to be the substrate for RhlI in vivo. Isotope labeling studies were conducted to investigate the chemical mechanism of the RhlI-catalyzed lactonization reaction. Solvent deuterons were not incorporated into product, which implicates a direct attack mechanism in which the carboxylate oxygen of the presumptive N-butyryl-SAM intermediate attacks the methylene carbon adjacent to the sulfonium ion. Alternative mechanisms, in which N-butyrylvinylglycine is formed via elimination of methylthioadenosine, were ruled out on the basis of the observation that RhlI failed to convert authentic N-butyrylvinylglycine to N-butyryl-L-homoserine lactone.     

Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin

Pilin assembly into type IV pili is required for virulence by bacterial pathogens that cause diseases such as cholera, pneumonia, gonorrhea, and meningitis. Crystal structures of soluble, N-terminally truncated pilin from Vibrio cholera toxin-coregulated pilus (TCP) and full-length PAK pilin from Pseudomonas aeruginosa reveal a novel TCP fold, yet a shared architecture for the type IV pilins. In each pilin subunit a conserved, extended, N-terminal alpha helix wrapped by beta strands anchors the structurally variable globular head. Inside the assembled pilus, characterized by cryo-electron microscopy and crystallography, the extended hydrophobic alpha helices make multisubunit contacts to provide mechanical strength and flexibility. Outside, distinct interactions of adaptable heads contribute surface variation for specificity of pilus function in antigenicity, motility, adhesion, and colony formation.