Glycosylation of b-Type Flagellin of Pseudomonas aeruginosa ...

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Oct 26, 2005 - J. Thomas-Oates. 1998. Mass spectrometric ... Thibault, P., S. M. Logan, J. F. Kelly, J. R. Brisson, C. P. Ewing, T. J. Trust, and P. Guerry. 2001.
JOURNAL OF BACTERIOLOGY, June 2006, p. 4395–4403 0021-9193/06/$08.00⫹0 doi:10.1128/JB.01642-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 188, No. 12

Glycosylation of b-Type Flagellin of Pseudomonas aeruginosa: Structural and Genetic Basis Amrisha Verma,1† Michael Schirm,2† Shiwani K. Arora,1 P. Thibault,3 Susan M. Logan,4 and Reuben Ramphal1* Department of Medicine/Infectious Diseases, University of Florida, Gainesville, Florida 326101; Caprion Pharmaceuticals, Montreal, Quebec, Canada2; Institute for Research in Immunology and Cancer, University of Montreal, Quebec, Canada3; and NRC Institute for Biological Sciences, Ottawa, Ontario, Canada4 Received 26 October 2005/Accepted 24 March 2006

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.

However, the genetic and biochemical basis of the glycosylation process is still poorly understood. Some genetic systems responsible for glycosylation of selected bacterial proteins have been described. Both N-linked and O-linked glycosylation pathways have been described in C. jejuni (35). Similarly, in N. meningitidis, a polymorphic locus designated as the pgl locus required for the glycosylation of class II pili was identified (13). A genomic island consisting of 14 open reading frames (ORFs) involved in the glycosylation of flagellin in P. aeruginosa was also identified (2). In most cases, the genes responsible for glycosylation are located in close vicinity of the gene encoding the target protein. The biological role(s) associated with bacterial protein glycosylation is still not precisely understood. However, glycosylation defective mutants of several bacteria have recently been shown to be attenuated in virulence attributes, such as adhesion and invasion (34), colonization (14), and burn wound infection (4), suggesting a role for protein-associated glycans in bacterial pathogenesis. P. aeruginosa is an opportunistic pathogen which causes serious infections in immunocompromised human hosts. In addition to possessing a number of known virulence factors such as exotoxins, proteases, lipases, lipopolysaccharide (LPS), and pili, its flagellin protein, like others, has been shown to be a potent stimulator of the inflammatory response via Toll-like receptor 5 (40, 44) and has been suggested to be an inflammatory virulence factor (9). P. aeruginosa flagellins can be classified into two groups (a and b types) based on their molecular weights and reactivity with specific sera (1, 18). The a-type flagellins have more variable molecular masses (45 to 52

Protein glycosylation has been long recognized as an important posttranslational modification in eukaryotic cells. However, a number of recent studies have shown glycosylation of cell surface-located and secreted proteins in prokaryotes. Pathogenic bacteria such as Neisseria gonorrhoeae (33), Neisseria meningitidis (25), and one strain of Pseudomonas aeruginosa (6) are now known to glycosylate pili. Similarly, Chlamydia (17) and Escherichia coli (21) glycosylate at least one of their adhesins, and two species of Ehrlichia (23) have been reported to glycosylate a surface-exposed immunodominant protein. Moreover, flagellin, the major subunit of the flagellum, is glycosylated in many bacteria, including P. aeruginosa (2, 5), Campylobacter coli (22), Campylobacter jejuni (8), Treponema pallidum (43), Borrelia burgdorferi (10), Helicobacter felis (12), Caulobacter crescentus (19), Agrobacterium tumefaciens (7), and Listeria monocytogenes (31). In spite of this explosion of information concerning the extent of bacterial protein glycosylation, very limited information is available regarding the actual glycan structures found on these glycoproteins. Recently, the glycan structures on the flagellins of C. jejuni (38) and H. pylori (29) and the a-type flagellin of P. aeruginosa have been elucidated (30); the glycans on neisserial pili (25, 33) and a specific Pseudomonas pilus (6) have also been characterized.

* Corresponding author. Mailing address: Department of Medicine/ Infectious Diseases, P.O. Box 100277, JHMHC, University of Florida, Gainesville, FL 32610. Phone: (352) 392-2932. Fax: (352) 392-6481. E-mail: [email protected]. † A.V. and M.S. contributed equally to this study. 4395

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J. BACTERIOL. TABLE 1. Bacterial strains and plasmids used in this study

Strains, plasmids, and primers

Strains E. coliDH5␣ P. aeruginosa PAO1 PAOrfbC PA1088 PA1089 PA1090 Plasmids pBluescript pEX18Gm pBS206207 pBS206207G pEX⌬PA1089 pEX⌬PA1090 Primers RER206 RER207 RER243 RER244 RER258 RER259 RER274 RER275 RER276 RER277 RER278 RER279 RER280 RER281 a

Description, origin, and/or sequencea

Source or reference

F⫺ ␾80lacZ⌬M15 ⌬(lacZYA-argF)U169 endA1 recA1 hsdR17 (rK⫺ mK⫹) supE44 thi-1 gyrA96 relA1 phoA; RP4-2-Tc::Mu-Km::Tn7; Tpr Smr pro res⫺ mod⫹

Invitrogen

Wild-type laboratory strain Gentamicin insertion mutant in the rfbCgene of P. aeruginosa PAO1 Gentamicin insertion mutant in PA1088 In-frame deletion mutant of PA1089 In-frame deletion mutant of PA1090

M. Vasil

Cloning vector Apr; oriT⫹ sacB⫹, gene replacement vector with MCS from pUC18 pBluescript containing 2.2-kb PAO1 DNA with PA1088 and flanking regions pBS206207 with gentamicin cassette inserted at the StuI site in PA1088 pEX18Gm containing ca. 2-kb PAO1 DNA flanking PA1089 from which aa 2 to 180 were deleted pEX18Gm containing ca. 2-kb PAO1 DNA flanking PA1089 from which aa 14 to 208 were deleted

Invitrogen/Life Technologies 15 This study This study This study

5⬘-CCCAAACTCGAGAAGTACCTGTTCTCCGGCAGCCAGGG-3⬘ 5⬘-CCCAAAGAGCTCTTGCCGGTCACGACGAAGCATTGCCG-3⬘ 5⬘-CGGAGTCGACAGCAGGCTACGGAATG 5⬘-CCCGGCGATTTTCTTTAGCCCTCCCG 5⬘-GTCTTCCACCAGAAGCACCGCCGAAG 5⬘-CATTATGCGGCCTGCTTCGCCCATCG 5⬘-CCCAAAAAGCTTGACGACGTGACCCTGGTGAATACCGC-3⬘ 5⬘-CCCAAATCTAGACACAGAGTCATCCCCGTCGCTGCAG-3⬘ 5⬘-CCCAAATCTAGAGGCCTGAGCGATTTCGTGGTCCATGAAG-3⬘ 5⬘-CCCAAAGAGCTCGATACCGAGAGAGCCTCCTCCAGCAG--3⬘ 5⬘-CCCAAAAAGCTTGAGCGGCTGCTTTTCCAGCGGATGTC-3⬘ 5⬘-CCCAAATCTAGAGCCCGCCGCGCTGATCACAGCATGTTC-3⬘ 5⬘-CCCAAATCTAGACGGCGCGTGCACCTGAATAACTGGAAC-3⬘ 5⬘-CCCAAAGAGCTCCCTTGCGGATCAATGCCACCACACGC-3⬘

This This This This This This This This This This This This This This

This study This study This study

This study

study study study study study study study study study study study study study study

Tpr, trimethoprim resistance; Smr, streptomycin resistance; Apr, ampicillin resistance. Restriction sites are indicated in boldface. aa, amino acids.

kDa) and are further classified into A1 and A2 subtypes based on the differences in their amino acid sequences (3). The b-type flagellins have a more conserved sequence and show an invariant molecular mass of about 53 kDa (5). The discrepancy between the predicted molecular mass of the a-type flagellin (39 kDa) and the observed molecular mass was attributed to a posttranslational modification (39), which was later shown to be an addition of glycan chains (5, 30). Precisely, a serine residue at position 189 and a threonine residue at position 260 were shown to be modified with a heterogeneous glycan comprising of up to 11 monosaccharide units that were O linked through a rhamnose residue to the flagellin backbone. In addition, two genes, orfA and orfN, belonging to the glycosylation island (GI) gene cluster considered to be involved in flagellin glycosylation, were shown to be required for attachment of the heterogeneous glycan and the proximal rhamnose residue, respectively (30). The b-type flagellin was thought to be nonglycosylated since any possible modification did not significantly alter the molecular weight of the flagellin and therefore could not be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, these flagellins were reported to possess phosphotyrosine (15). In addition, the glycosylation machinery of the a-type flagellin did not appear to be able to

modify b-type flagellin (2). Nevertheless, the identification of a possible small GI near the flagellin gene on the PAO1 genome suggested that the PAO1 flagellin might also be glycosylated. The aim of the present study therefore was to determine whether the b-type flagellins are glycosylated and to investigate the genetic basis for this modification. We provide here evidence that the b-type flagellin produced by P. aeruginosa strain PAO1 and other similar strains is glycosylated and that four ORFs constituting the GI are needed for this posttranslational modification. Compared to the a-type flagellin, which is glycosylated at threonine189 and serine 260, the b-type flagellin is glycosylated at two serine residues that are located close to each other (amino acids 191 and 195). Furthermore, the glycan added to the b-type flagellin is only 709 Da. in molecular mass and is less heterogeneous compared to that of a-type flagellin of strain PAK.

MATERIALS AND METHODS Bacterial strains and antibiotics used. The bacterial strains, plasmid constructs, and primers used in the present study are listed in Table 1. For Escherichia coli DH5␣ 100 ␮g of ampicillin and 10 ␮g of gentamicin/ml was used, and for P. aeruginosa 50 ␮g of gentamicin and 300 ␮g of carbenicillin/ml was used. Construction of mutants. Strain PAO1 is a commonly used strain whose genome has been sequenced. It is known to carry b-type flagellin. The PA1091

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FIG. 1. Schematic diagram showing the structure of GIs of a- and b-type P. aeruginosa strains. The diagram shows the region of the P. aeruginosa chromosome where the flagellar genes and the GIs are located. The a-type P. aeruginosa strain PAK has the GI located on an ⬃16-kb fragment of DNA containing 14 ORFs, while the b-type strain PAO1 consists of 4 ORFs located on an ⬃8-kb piece of DNA.

mutant has been described before (4). A PA1088 mutant was constructed by inserting a gentamicin resistance cassette into the PA1088 ORF at the unique StuI site. The primer pair RER206 and RER207 (RER206/207) (Table 1) was used to amplify a 2.2-kb DNA fragment from P. aeruginosa PAO1. This fragment contained the complete PA1088 ORF and some flanking DNA. This fragment was cloned into the vector pBluescript at HindIII and XhoI sites to yield pBS206207. A gentamicin resistance cassette was then cloned into the unique StuI site in PA1088 as a blunt-ended DNA fragment, leading to the construction of pBS206207G. This construct was introduced into P. aeruginosa PAO1 by electroporation to allow homologous recombination. Double crossovers were selected that were resistant to 50 ␮g of gentamicin/ml and sensitive to 300 ␮g of carbenicillin/ml. The insertion of the gentamicin cassette in the PA1088 ORF was confirmed by PCR (data not shown). The two in-frame deletion mutants in ORFs PA1089 and PA1090 were constructed by deleting amino acid residues 2 to 180 from PA1089 and amino acid residues 14 to 208 from PA1090. Primer pairs RER274/275 and RER276/277 (Table 1) were used to amplify approximately 1 kb of DNA flanking the region to be deleted from PA1089. Similarly, the primer pairs RER278/279 and RER280/281 were used to amplify approximately 1 kb of DNA flanking the region to be deleted from PA1090. The two 1-kb DNA pieces were spliced together as HindIII/XbaI and XbaI/SstI fragments and were cloned as approximately 2-kb HindIII/SstI inserts into the vector pEX18Gm (11), leading to the construction of two plasmids, pEX⌬PA1089 and pEX⌬1090. These two plasmids were introduced into P. aeruginosa PAO1 by electroporation, and single crossovers were selected on plates containing 50 ␮g of gentamicin/ml. These clones were tested for sucrose sensitivity, and sucrose-sensitive clones were resolved to sucrose-resistant clones as a result of a second recombination event and excision of the pEX18Gm plasmid backbone. These clones were confirmed by PCR, which gave smaller PCR products from the PA1089 and PA1090 mutant strains compared to their wild-type parent strain PAO1 (data not shown). Flagellum purification. Flagella were purified from Pseudomonas strains grown overnight in L broth. Flagella were sheared from the surface of the bacteria and collected by ultracentrifugation as previously described (39). In brief, bacteria were grown overnight in L broth, harvested by centrifugation, and resuspended in cold phosphate-buffered saline containing 10 mM MgCl2. Flagella were removed from the cells by shearing in a cold Waring blender for 20 s. The cells were separated from the flagella by centrifugation at 12,000 ⫻ g for 30 min. The supernatant was then filtered through a 0.45-␮m-pore-size Millipore filter and recentrifuged at 12,000 ⫻ g for 30 min. The supernatant thus obtained was then ultracentrifuged at 100, 000 ⫻ g for 1 h. Flagella, obtained as a pellet, were then suspended in minimum amount of phosphate-buffered saline containing 10 mM MgCl2. Purity was assessed by SDS-PAGE analysis. Contaminating bands approximating the size of pilin (ca. 15.5 kDa) were occasionally seen and were generally less than 5% of the flagellar preparation. No pilin fragments were found on peptide analysis and when mass measurements were made on proteins in the 49- to 50-kDa range. Mass spectrometry (MS). For intact mass analysis, purified flagellins were dialyzed in aqueous 0.2% (vol/vol) formic acid by using a Centricon YM-10 membrane filter (Millipore, Mississauga, Ontario, Canada). The solution was infused into the mass spectrometer at a flow rate of 0.5 ␮l/min. Sample digestion

were performed overnight in 50 mM NH4HCO3 (5% acetonitrile) using sequence-grade trypsin (Promega, Madison, WI) at a enzyme/substrate ratio of 1:50. When required, a part of this tryptic digest was digested with chymotrypsin (Promega) for 4 h in 100 mM ammonium bicarbonate. All digests were analyzed with a Waters CapLC liquid chromatograph coupled to nanoelectrospray on a Q-TOF Ultima instrument (Waters, Milford, Mass). Peptides were separated and analyzed as described previously (30). The glycosylation site was identified by using ␤-elimination with ammonium hydroxide, which leaves a modified Ser or Thr residue that could be located by using tandem MS (MS/MS) (26). For the analysis of intact flagellin, dialyzed proteins were infused into a Waters Q-TOF Ultima mass spectrometer at a flow rate of 0.5 ␮l/min similar to that described previously (30). MS/MS experiments were performed using argon as the collision gas with collision energies ranging from 10 to 25 V. Second-generation fragment ion spectra were obtained by increasing the RF lens1 voltage from 50 to 125 V, thereby forming fragment ions in the high-pressure region of the skimmer/cone region of the mass spectrometer that were subsequently selected as precursor ions for MS/MS analyses. MS/MS spectra were searched against a nonredundant NCBI database using Mascot (Matrix Science, London, United Kingdom), selecting all known bacteria. Parent ion and fragment ion mass tolerances were both set at ⫾0.4 Da. External calibration was performed by infusing 150 fmol/␮l of solution of Glu-fibrinopeptide B (50% aqueous methanol, 0.2% formic acid), providing a mass accuracy of ⫾50 ppm. Accurate mass assignments in MS/MS mode were performed using fragment ions of known composition to bracket the ion of interest and provided a mass accuracy within 10 ppm of the predicted values.

RESULTS Comparison of the GI of P. aeruginosa strains PAO1 (b type) and PAK (a type). The GI of P. aeruginosa strain PAK, which possesses a-type flagellin, has been described previously (2) and consists of 14 ORFs. This island is located between the flgL and fliC genes of strain PAK. When the corresponding chromosomal region of the b-type flagellin strain PAO1 was analyzed, a much shorter putative GI consisting of only four ORFs was found. A diagrammatic comparison of the GIs in P. aeruginosa strains PAK and PAO1 is depicted in Fig. 1. The homologies of the four ORFs comprising the PAO1 GI with other proteins in the microbial genome databases are shown in Table 2. Although we were not able to detect glycosylation of PAO1 flagellin earlier by Western blot analyses, the new information about the PAO1 GI and the homologies of these ORFs to a glycosyl transferase (PA1091) a phosphoserine phosphatase (PA1089), and a nucleotidyltransferase (PA1090) suggested that the PAO1 flagellin may also be glycosylated.

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TABLE 2. Similarities of P. aeruginosa PAO1 ORFs to proteins in the microbial genome databasea COG/PFAM prediction PA ORF (identity)

Protein or product

Localization Function

Homologue

PA1088 (NP_249779) Hypothetical protein Cytoplasmic (class 4)

Methylase involved in ubiquinone/ 53% identical to SAM-dependent menaquinone biosynthesis methyltransferases (Magnetospirillum (coenzyme Q metabolism) magnetotacticum) PA1089 (NP_249780) Hypothetical protein Cytoplasmic Hydrolase, haloacid dehalogenase-like 56% similar to Phosphoserine (class 4) hydrolase phosphatase (serB) (Methanococcus jannaschii); amino acid transport and metabolism PA1090 (NP_249781) Hypothetical protein Cytoplasmic Predicted sugar 49% similar to UDP-N-acetylglucosamine (class 4) nucleotidyltransferases (cell pyrophosphorylase (Neisseria envelope biogenesis, outer gonorrhoeae) membrane) PA1091 (NP_249782) Hypothetical protein Cytoplasmic Putative glycosyl/ glycerophosphate 45% similar to O- antigen biosynthesis (class 4) membrane transferases involved in teichoic protein RfbC (Myxococcus xanthus); acid biosynthesis (cell envelope 53% similar to C-terminal fragment of biogenesis outer membrane) RfbC (Riftia pachyptila endosymbiont); 42% similar to putative glycosyltransferase in a locus involved in synthesis of a carbohydrate antigen (Enterococcus faecalis)

a Class 4 genes encode hypothetical proteins. These genes are defined in the Pseudomonas genome database version 2 as homologs of previously reported genes of unknown function or with no similarity to any previously reported sequences.

It was reported earlier that the GIs in the a-type P. aeruginosa strains are polymorphic, being either long or short (3). In order to explore whether similar polymorphisms exist in the b-type strains, 12 b-type strains were chosen from diverse locations—environmental, blood or urine, and cystic fibrosis (CF)—and were analyzed by PCR. Two sets of primers, RER243/258 and RER244/259, were used to amplify two overlapping pieces of DNA of 3.685 and 3.575 kb, respectively, from the wild-type PAO1. All of the 12 strains analyzed yielded PCR products with sizes identical to those for strain PAO1 (data not shown). The complete GI of one clinical strain, JG3, was sequenced and was found to be identical to the wild PAO1 sequence, suggesting that the putative GI in the b-type P. aeruginosa strains is highly conserved (data not shown). Furthermore, the nucleotide sequence of the GI of another b-type strain, PA14, whose genome has been recently sequenced (www.tigr.org), was also found to be 99% identical to that of PAO1, further confirming that the b-type strains have a highly conserved GI. Intact mass analysis of P. aeruginosa PAO flagellin protein. To ascertain whether the predicted mass of this flagellin was equal to the measured mass, nanoelectrospray MS analysis of purified flagellin from strain PAO was performed. This revealed three well-defined peaks at 49,820 Da and two minor peaks at 49,402 Da and 49,611 Da, corresponding to excess masses of 707, 289, and 498 Da, respectively, from the predicted protein mass (Mtheo ⫽ 49,111 Da). The peak at 49,402 Da is consistent with the addition of two deoxyhexose residues to the flagellin protein. This is similar to what was observed previously for a-type flagellin protein from P. aeruginosa strain JJ692 (30). The other two peaks (49,611 and 49,802 Da) correspond to flagellin modified with two deoxyhexose residues to which is added either one (49,611 Da) or two (49,820 Da) residues of mass 209 Da (Fig. 2A). Liquid chromatography-nanoelectrospray MS/MS analysis of chymotryptic or tryptic digest of PAO flagellin protein. To

more precisely determine the type and location of glycosylation, the flagellin protein was digested with trypsin and analyzed by capillary liquid chromatography-nanoelectrospray MS. A database search of the acquired MS/MS spectra was performed and confirmed the identification of the flagellin protein with 85% sequence coverage (Fig. 3). Only a few small tryptic peptides of less than 800 Da and a large peptide in the central region of the protein were not identified (T136-207, theoretical mass of 6,809.3 Da). In general, peptides of this size are difficult to separate by reversed-phase C18 chromatography and additional enzymatic degradation using chymotrypsin was required to obtain a proteolytic fragment of adequate size. Analysis of the chymotryptic or tryptic digest identified a peptide (sequence segment 186-207) modified with either a deoxyhexose as found in strain JJ692 or with an abundant oxonium ion at m/z 356 as shown in Fig. 4a. The second-generation fragment ion spectrum of the oxonium ion at m/z 356 is shown in Fig. 4B and provides information on the composition of the glycan moiety. Most notably, a second-generation fragment ion of m/z 130 is obvious in the spectrum (Fig. 4b). The loss of 226 Da from the m/z 356 ion, which results in the appearance of this m/z 130 fragment ion, likely corresponds to loss of a deoxyhexose plus a phosphate group from the glycan moiety. It is noteworthy that no loss of H3PO4 is observed, suggesting that the phosphate group could be linked to the carbohydrate and the unknown 129 Da residue via a phosphoester bond. An exact mass measurement by top-down analysis (30) of the oxonium ion at m/z 356 was determined to be 356.112 ⫾ 10 ppm. The second-generation fragmentation pattern suggests that the O-linked glycan comprises a deoxyhexose to which is attached a phosphate group and an unknown residue of 129.085 Da. The finding of a phosphate group confirms a previous report that b-type flagellin was phosphorylated. However, in that study the phosphate was suggested to be attached to a tyrosine residue (15). The occurrence of an O-linked glycan containing an amino acid is a potential assignment since glutamine and

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FIG. 2. Intact mass analysis of P. aeruginosa flagellin. Reconstructed molecular mass profiles of flagellin from PAO1 (A), PAO1 rfbC (B), PA1088 (C), and PA1089 (D).

glycine substituents were previously reported on pseudaminic acid of the flagellar glycan of Campylobacter sp. (32) and on the core oligosaccharide of H. influenzae LPS (20). In addition, the N-linked flagellar glycan of the archaeon Methanococcus voltae was recently shown to contain a threonine addition to the glycan trisaccharide (41). However, structural elucidation by NMR analysis of the purified glycan will be required for the unequivocal assignment of this novel moiety. Determination of glycan attachment site(s). To precisely determine the site of attachment of this novel glycan, the flagellin peptide 173-207 (for amino acids 173 to 207) was subjected to ␤-elimination using NH4OH. This analysis iden-

tified serine residues 191 and 195 as the sites of modification. The MS/MS spectra of the ␤-eliminated chymotryptic or tryptic peptide 172-207 is shown in Fig. 5. The y fragments extending beyond y9 all showed an increase of ⫹1 Da resulting from the deamidation of asparagine to aspartic acid. The y14 and y17 ions showed, respectively, a mass shift of ⫺1 Da from the predicted mass, indicating that serines 191 and 195 are modified with O-linked glycans. Functional characterization of PA1088, PA1089, PA1090, and PA1091 from the GI of PAO1. In contrast to strain PAK, which has an extended GI containing 14 genes, the equivalent region in PAO displays four ORFs: PA1088, PA1089, PA1090,

FIG. 3. Assignment map of PAO flagellin. The sites of O-linked glycosylation are enclosed in boxes, and assigned peptides are in boldface, whereas peptides that have not been assigned are in normal typeface.

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FIG. 4. Characterization of PAO glycopeptide 186-207 from chymotryptic digestion. (a) MS/MS spectrum of m/z 1,328.5 corresponding to the doubly protonated peptide ion GTATASGIASGTVNLVG GQVK modified with two neutral 355-Da residues. In the low mass region an oxonium ion at m/z 356 from the O-linked glycan is observed. (b) MS/MS spectrum of oxonium ion m/z 356 formed from collisional activation in the ion source.

and PA1091. Purified flagellin from PA1088, PA1089, PA1090, and PA1091 mutant strains were analyzed by infusion MS. Intact mass analysis of flagellin from PA1091 revealed a single peak (49,109 Da) corresponding to flagellin that is nonglycosylated (Mr predicted 49,111 Da; Fig. 2B). As we had shown with PAK flagellin (30), inactivation of this ORF in PAO also results in an inability to attach the glycan/deoxyhexose moiety at the site of glycosylation, and the flagellin protein remains unmodified. Flagellin from PA1088 displayed a mass of 49,400 Da corresponding to flagellin protein modified with only two deoxyhexose residues, indicating a role for the PA1088 gene product either in the biosynthetic pathway or in the subsequent addition of the novel 209 Da moiety to each O-linked deoxyhexose monosaccharide (Fig. 2C). In contrast, analysis of flagellin from PA1089 revealed a more heterogeneous pattern with O-linked glycans bearing either a single deoxyhexose residue (49,400 Da) or the 356 Da residue at the two glycosylation sites (49,819 Da). Compared to native PAO flagellin, where a single major glycosylated form of mass 49,820 Da was obvious, the flagellin from PA1089 displayed a much greater heterogeneity in size distribution (Fig. 2D), which is indicative of an inability to efficiently glycosylate the flagellin protein. We were unsuccessful in obtaining an intact mass spectrum for flagellin purified from PA1090 (data not shown). Although we are currently unable to explain this result, the failure of this flagellin

J. BACTERIOL.

protein to efficiently ionize under the conditions used indicates a potential change in glycan composition. The four mutants were examined for phenotypic changes such as motility and LPS banding pattern, which are known to occur when mutations are made in the glycosylation genes of other organisms. None of the mutants were affected in these phenotypes, similar to what was seen previously with mutations in the GI genes of strain PAK. Complementation of PA1091 mutant of PAO1 with the homologous PAK orfN gene. Structural analysis of flagellin from PAO1 had revealed the presence of a deoxyhexose monosaccharide as a component of the 335-Da glycan moiety. The orfN gene from PAK is 41% identical to PA1091 and has been shown to be responsible for the addition of rhamnose to the PAK flagellin protein. To determine whether the product of PA1091 is indeed a functional homolog, a plasmid containing the orfN gene from PAK was inserted into the PA1091 mutant and flagellin from the resulting transconjugant was examined by top-down MS. Intact mass analysis revealed a single species of flagellin of the predicted mass lacking glycosylation and no evidence for restoration of the wild-type glycosylation profile (data not shown). In contrast, complementation of PAK orfN mutant restored a wild-type glycosylation profile. These preliminary data are suggestive of a unique specificity for the enzymatic products of the PA1091 gene and orfN from the PAK glycosylation locus. As a consequence of this observation, in addition to the structural analysis of flagellin from a PA1091 mutant which revealed that glycosylation had been abolished, we now propose that the gene be renamed to fgtA (for flagellar glycosyl transferase). Flagellar glycan structures of P. aeruginosa. It appears that the flagellar glycan produced by PAO is unique in structure and quite distinct from the glycan described on PAK flagellin (Fig. 6). As with the flagellar glycan of PAK, the linking sugar appears to be a deoxyhexose that appears to be transferred to the peptide backbone by the glycosyltransferase encoded by PA1091 (fgtA). In contrast to PAK, the remaining glycan structure appears to contain a phosphate group to which is attached in phosphoester linkage a terminal modification of 192 Da. Mutational analysis of the first gene in the PAO GI PA1088, which shows homology to a phosphoethanolamine N-methyl transferase, is responsible for the addition of this 209-Da modification to the deoxyhexose monosaccharide. The remaining two genes in the GI PA1089 and PA1090 clearly have an effect on the ability to produce the wild-type structure, although we have been unable to determine the precise role each plays in the biosynthetic pathway. DISCUSSION This study characterizes the unique posttranslational modifications found on the b-type PAO1 flagellin. Prior to this work, it had been suggested based on migration in SDS-PAGE and glycan staining that type b strains did not produce glycosylated flagellin (2). However, the presence of four ORFs in the corresponding region, two of which had homology to genes involved in the synthesis of glycan chains, suggested that these may be the functional equivalents of a GI. Intact mass analysis of the PAO1 b-type flagellin revealed a significant increase in mass of the protein monomer of ca. 1.4% from the predicted.

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FIG. 5. Determination of glycosylation sites on peptide 173-207. MS/MS spectrum of m/z 1,048.3 after ␤-elimination. The MS/MS spectrum of the peptide QVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVK after ␤-elimination is shown. Fragment ions showing a downward shift of either 1 or 2 m/z units (indicated by an asterisk) enabled the identification of the linkage site of the glycan. Note that the asparagine is deamidated to the corresponding isoaspartate residue (y9 ⫹ 1).

FIG. 6. Comparison of P. aeruginosa flagellar glycan structure. (A) PAK flagellin; (B) PAO flagellin. Rha, rhamnose; Pen, pentose; Hex, hexose; dHex, deoxyhexose; HexA, hexuronic acid; dHexN, deoxyhexosamine, PO4, phosphate.

The reconstructed mass profile of flagellin from PAO1 indicated that the majority of flagellin was glycosylated with an additional mass of 709 Da, while minor amounts of alternate glycoforms of additional masses of 291 and 500 Da were also apparent. In contrast to PAK a-type flagellin, PAO1 b-type flagellin produces a shorter, less heterogeneous glycan moiety. Consistent with this finding, while the a-type strains show significant heterogeneity of their GIs (3), the b-type strains show marked conservation of their genetic content. While the glycan characterized on PAK flagellin was a heterogeneous oligosaccharide comprising up to 11 monosaccharide residues, the glycan present on PAO1 flagellin is of mass 356.112 Da and appears to be composed of a single deoxyhexose monosaccharide linked to a unique modification of mass 209 Da which contains a phosphate moiety. Although the structural configuration of this novel substituent is currently unknown, accurate mass analysis and fragmentation pattern have provided preliminary information on the composition. As with the a-type flagellin, it appears that the glycan of b-type flagellin is linked to the protein backbone through a deoxyhexose moiety. In contrast to the remaining structure, this deoxyhexose linkage appears to be a common feature of flagellar glycosylation in Pseudomonas. The identification of nonglycosylated flagellin from the PA1091 mutant was a result identical to that observed in previous studies of the orfN mutant of PAK.

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However, the two genes do not appear to be functional homologs, and this indicates either specificity for a unique deoxyhexose monosaccharide or, alternatively, a unique substrate specificity in terms of the respective flagellin monomers. Mapping of the glycosylation sites revealed a different pattern from that found on PAK flagellin, where the two sites on the primary sequence were a considerable distance apart (T189 and S260). In contrast, the two sites on PAO flagellin are situated in close proximity to each other on the primary sequence (S191 and S195). Unlike N-linked glycosylation of proteins, where a defined consensus sequence has been identified in both eukaryotes (16) and prokaryotes (24, 42), the basis of site specificity for O linkage of glycans to proteins is much more poorly defined, especially with respect to prokaryotic glycoproteins. The site of glycosylation may be related to local hydrophobicity, whereby particular Ser/Thr residues within this local hydrophobic environment project outward and so are accessible to glycosyltransferases. It seems feasible that the location of these sites in the respective PAK and PAO1 folded flagellin protein or chaperone/flagellin complex may indeed be in a similar hydrophobic pocket even though they do not align on the primary sequences. Nonetheless, the localization of glycosylation sites in both PAK and PAO to a central region of the flagellin monomer most likely results in an exposed surface location on the assembled filament rather than buried within the central core. The crystal structure of the flagellin protein from Salmonella enterica serovar Typhimurium had indicated that the less well conserved central primary sequence of flagellin comprises the D2 and D3 domains, which are clearly surface exposed in the flagellar filament (28). Other species of pseudomonads have also been shown to possess similar genomic GIs as part of their flagellar regulons. For example, the flagellar regulon of Pseudomonas syringae carries a small island in a similar location and was also shown to possess glycosylated flagellin (36). Glycosylation of flagellin in this plant pathogen appears to be involved in specific host cell recognition of distinct pathovars (37). Currently, the structure of the glycan is unknown but may be related to that found on a-type flagellin in the present study considering the similarity in GI composition. Although the function(s) of the modification on P. aeruginosa flagellin is unclear at present, there is mounting evidence of a biological role in virulence. Glycosylation has been shown to play a role in virulence in the burn mouse model of P. aeruginosa infection (4) and is also involved in triggering an inflammatory response (40). Future studies will be directed toward determining the precise structure and biosynthetic pathway of the novel glycans synthesized by both a-type and b-type flagellin-bearing strains, the immunological response to these glycans, and the characterization of any precise interactions with host cell molecules during infection. ACKNOWLEDGMENTS This study was supported by NIH grant AI 47852 (R.R.) and by the NRC Genomics and Health Initiative of Canada (S.M.L.). We thank T. Devesceri for assistance with figure preparation. REFERENCES 1. Allison, J. S., M. Dawson, D. Drake, and T. C. Montie. 1985. Electrophoretic separation and molecular weight characterization of Pseudomonas aeruginosa H-antigen flagellins. Infect. Immun. 49:770–774.

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