Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guerry, P. Articles by Logan, S. M. Search for Related Content PubMed PubMed Citation Articles by Guerry, P. Articles by Logan, S. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

 Previous Article  |  Next Article 

Journal of Bacteriology, September 2007, p. 6731-6733, Vol. 189, No. 18
0021-9193/07/$08.00+0     doi:10.1128/JB.00642-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Protein Glycosylation in Campylobacter jejuni: Partial Suppression of pglF by Mutation of pseC

Patricia Guerry,^1* Cheryl P. Ewing,¹ Ian C. Schoenhofen,² and Susan M. Logan²

Enteric Diseases Department, Naval Medical Research Center, Silver Spring, Maryland 20910,¹ Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada²

Received 24 April 2007/ Accepted 6 July 2007

Top ABSTRACT TEXT REFERENCES

 
Campylobacter jejuni has systems for N- and O-linked protein glycosylation. Although biochemical evidence demonstrated that a pseC mutant in the O-linked pathway accumulated the product of pglF in the N-linked pathway, analyses of transformation frequencies and glycosylation statuses of N-glycosylated proteins indicated a partial suppression of pglF by pseC.

Top ABSTRACT TEXT REFERENCES

 
Campylobacter jejuni has two protein glycosylation systems (18). Campylobacter flagellins, like those of many other polar flagellates, are decorated with O-linked glycans (12). One such sugar is pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero--L-manno-nonulosonic acid; Pse5Ac7Ac) (13). Genetic analyses have identified the genes responsible for the production of Pse5Ac7Ac (4, 5, 20), and recently, the biosynthetic pathway of Pse5Ac7Ac within Helicobacter pylori and C. jejuni was elucidated (16). Glycosylation of flagellin subunits is required for filament biogenesis in C. jejuni (4), and this O-linked system appears to specifically glycosylate flagellin (12). The N-linked glycosylation system modifies numerous periplasmic proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy--D-glucopyranose (2,4-diacetamido-Bac) at the reducing end of the glycan (23). The N-linked system includes an oligosaccharide transferase that resembles that of eukaryotes, and it has been characterized biochemically (2, 3, 7, 11, 15, 21). The phenotype of C. jejuni mutants defective in the N-linked system is pleiotropic, likely reflecting the variety of proteins glycosylated by this system. Although fully motile (19), mutants defective in N-linked glycosylation have a reduced ability to invade intestinal epithelial cells in vitro (19), reduced levels of colonization in animals (6, 19), and a significant reduction in natural transformability (9).

Synthesis of Pse5Ac7Ac and 2,4-diacetamido-Bac begins with the modification of UDP-GlcNAc by distinct pairs of dehydratase/aminotransferase enzymes (17). These are Cj1293 (PseB) and Cj1294 (PseC) for the Pse5Ac7Ac pathway and Cj1120c (PglF) and Cj1121c (PglE) for the 2,4-diacetamido-Bac pathway (17). PseB has C4,6 dehydratase/C5 epimerase activity that results in the production of UDP-2-acetamido-2,6-dideoxy-ß-L-arabino-hexos-4-ulose, which is the substrate for the second of the enzyme pair, PseC, an aminotransferase which produces UDP-4-amino-4,6-dideoxy-ß-L-AltNAc. Upon accumulation of the UDP-arabino-ketone product, it was demonstrated that the PseB enzyme can also perform a second epimerization, which results in the production of UDP-2-acetamido-2,6-dideoxy--D-xylo-hexos-4-ulose (17) (Fig. 1). The latter sugar is also the product of PglF, a UDP--D-GlcNAc C6 dehydratase (17; Fig. 1). A recent metabolomic study confirmed the accumulation of UDP-2,4-diacetamido-Bac in a pseC mutant in vivo (14). The accumulation of this intermediate led us to determine whether a pseC mutation could suppress pglF by supplying the missing intermediate in the pgl pathway (see Fig. 1).

View larger version (13K): [in this window] [in a new window]

FIG. 1. CMP-pseudaminic acid and UDP-2,4-diacetamido-Bac pathways in C. jejuni. The enzymes and biosynthetic intermediates of the initial steps in each pathway, as determined by Schoenhofen et al. (16, 17), are indicated.

A double mutant of 81-176 was constructed by transformation of the pglF::aph3 (19) gene into 81-176 pseC::cat (5). The construction was confirmed by PCR analysis using primers that bracketed the insertion point of aph3 into pglF (data not shown). The original pglF::aph3 mutant was fully motile (19), but the double mutant, like the pseC::cat parent (4), was nonmotile (data not shown). Figure 2 shows that loss of glycosylation in the pglF mutant resulted in reduced reactivity with soybean agglutinin (SBA), which binds to terminal GalNAc residues (7), compared to those for wild-type 81-176 and the pseC mutant. Lectin reactivity appeared to be restored to the wild-type pattern in the pglF pseC double mutant (lane 3), consistent with a restoration of N-linked glycosylation (7, 19).

View larger version (56K): [in this window] [in a new window]

FIG. 2. SBA lectin blot of whole cells of C. jejuni. Whole-cell proteins were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with SBA lectin as described by Kelly et al. (7). Lane 1, 81-176; lane 2, pglF mutant; lane 3, pglF pseC mutant; lane 4, pseC mutant. The positions of molecular mass markers (in kilodaltons) are shown on the left. wt, wild type.

In order to study this apparent suppression in more detail, we examined glycosylation of two unrelated proteins directly. VirB10, a periplasmic component of a plasmid-encoded type IV secretion system in 81-176, has been shown experimentally to contain two sites of N-linked glycosylation (9). In wild-type 81-176 and the pseC mutant, VirB10 migrates as a doublet that represents glycosylation at one or two sites (9) (Fig. 3). In pglF, VirB10 runs at the predicted mass of the unglycosylated protein; in a pglF pseC double mutant, VirB10 migrates at a position consistent with either no glycosylation or glycosylation at a single site (Fig. 3). The consensus site for N-linked glycosylation has recently been defined (8), and CmeC (Cj0365c), an outer membrane component of an efflux pump (10), is predicted to have two sites of N-linked glycosylation. As shown in Fig. 3, in wild-type 81-176 and the pseC mutant, a single band is visible in immunoblots with anti-CmeC antiserum. However, in the pglF mutant, the CmeC band migrates more rapidly, consistent with the loss of glycosylation. In the double mutant, there are three bands. One corresponds to the band seen in pglF (unglycosylated), one is comparable to that of the wild type (fully glycosylated), and one is intermediate in size (one glycosylation site). Thus, in the wild type, CmeC appears to be glycosylated at two sites; in the double mutant, there appears to be a mixture, with one, two, or no sites glycosylated.

View larger version (51K): [in this window] [in a new window]

FIG. 3. Immunoblot of glycosylated proteins. Glycine-extracted antigens of C. jejuni strains were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels and immunoblotted with a polyclonal rabbit antiserum against recombinant VirB10 (9) or CmeC (D. Rockabrand and P. Guerry, unpublished data). Equal loading of samples was confirmed by staining with Gel Code Blue (Pierce, Rockford, IL) prior to blotting. WT, wild type.

Mutants in the pgl glycosylation system are defective in natural transformation (9). Natural transformation of C. jejuni is dependent on a type II secretion system (22), and multiple components of this secretion system have potential sites for N-linked glycans (8). Table 1 compares the natural transformation frequencies of wild-type 81-176 with those of the mutants. There was a significant difference between results for the wild type and for the pglF mutant (P < 0.05), as previously reported for other pgl mutants (9), and there was no difference between results for the wild type and the pseC mutant. The transformation frequency of the pglF pseC double mutant was higher than that of the pglF mutant but did not reach the level seen with the wild type.

View this table: [in this window] [in a new window]

TABLE 1. Natural transformation frequencies

PglF and PseB belong to a family of dehydratases that can be divided into two subfamilies. The first subfamily, which includes PglF and WbpM, consists of large proteins associated with the inner membrane of the cell (1). Thus, it is likely that the biosynthesis of the 2,4-diacetamido-Bac is closely associated with the cytoplasmic face of the inner membrane. Additionally, 2,4-diacetamido-Bac is transferred onto a membrane-associated lipid carrier by PglC (2). In contrast, PseB belongs to the second subfamily of dehydratase enzymes, whose members are smaller than and lack the membrane-anchoring domain associated with the first subfamily. This difference in cellular localization may contribute in part to the inability of PseB to supply sufficient precursor to the pgl system to fully glycosylate N-linked proteins in C. jejuni.

 
This work was supported by National Institute of Allergy and Infectious Disease grant RO1 AI43559 (to P.G.) and Navy Work Unit no. 6000.RAD1.DA3.A0308 from the Military Infectious Diseases Program.

We thank David Rockabrand for the CmeC antiserum.

 
* Corresponding author. Mailing address: Enteric Diseases Department, Naval Medical Research Center, 503 Robert Grant Ave., Silver Spring, MD 20910. Phone: (301) 319-7662. Fax: (301) 319-7679. E-mail: guerryp{at}nmrc.navy.mil

Published ahead of print on 13 July 2007.

Top ABSTRACT TEXT REFERENCES

 

1
1. Creuzenet, C., and J. S. Lam. 2001. Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa. Mol. Microbiol. 41:1295-1310.[CrossRef][Medline]
2
2. Glover, K. J., E. Weerapana, M. M. Chen, and B. Imperiali. 2006. Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphase transferase in the Campylobacter jejuni N-linked glycosylation pathway. Biochemistry 45:5343-5350.[CrossRef][Medline]
3
3. Glover, K. J., E. Weerapana, S. Numao, and B. Imperiali. 2005. Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni. Chem. Biol. 12:1311-1315.[CrossRef][Medline]
4
4. Goon, S., J. F. Kelly, S. M. Logan, C. P. Ewing, and P. Guerry. 2003. Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene. Mol. Microbiol. 50:659-671.[CrossRef][Medline]
5
5. Guerry, P., C. P. Ewing, M. Schirm, M. Lorenzo, J. Kelly, D. Pattarini, G. Majam, P. Thibault, and S. M. Logan. 2006. Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence. Mol. Microbiol. 60:299-311.[CrossRef][Medline]
6
6. Hendrixson, D. R., and V. J. DiRita. 2004. Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract. Mol. Microbiol. 52:471-484.[CrossRef][Medline]
7
7. Kelly, J., H. Jarrell, L. Millar, L. Tessier, L. M. Fiori, P. C. Lau, B. Allan, and C. M. Szymanski. 2006. Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer. J. Bacteriol. 188:2427-2434.[Abstract/Free Full Text]
8
8. Kowarik, M., N. M. Yound, S. Numao, B. L. Schulz, I. Hug, N. Callewaert, D. C. Mills, D. C. Watson, M. Hernandez, J. F. Kelly, M. Wacker, and M. Aebi. 2006. Definition of the bacterial N-glycosylation site consensus sequence. EMBO J. 25:1957-1966.[CrossRef][Medline]
9
9. Larsen, J. C., C. M. Szymanski, and P. Guerry. 2004. N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176. J. Bacteriol. 186:6508-6514.[Abstract/Free Full Text]
10
10. Lin, J., L. O. Michel, and Q. Zhang. 2002. CmeABC functions as a multidrug efflux system in Campylobacter jejuni. Antimicrob. Agents Chemother. 46:2124-2134.[Abstract/Free Full Text]
11
11. Linton, D., N. Dorrell, P. G. Hitchen, S. Amber, A. V. Karlyshev, H. R. Morris, A. Dell, M. A. Valvano, M. Aebi, and B. W. Wren. 2005. Functional analysis of the Campylobacter jejuni N-linked protein glycosylation system. Mol. Microbiol. 55:1695-1703.[CrossRef][Medline]
12
12. Logan, S. M. 2006. Flagellar glycosylation—a new component of the motility repertoire. Microbiology 152:1249-1262.[Abstract/Free Full Text]
13
13. Logan, S. M., J. F. Kelly, P. Thibault, C. P. Ewing, and P. Guerry. 2002. Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins. Mol. Microbiol. 46:587-597.[CrossRef][Medline]
14
14. McNally, D. J., J. P. M. Hui, A. J. Aubrey, K. K. K. Mui, P. Guerry, J.-R. Brisson, S. M. Logan, and E. C. Soo. 2006. Functional characterization of the flagellar glycosylation locus in Campylobacter jejuni 81-176 using a focused metabolomics approach. J. Biol. Chem. 281:18489-18498.[Abstract/Free Full Text]
15
15. Olivier, N. B., M. M. Chen, J. R. Behr, and B. Imperiali. 2006. In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system. Biochemistry 45:13659-13669.[CrossRef][Medline]
16
16. Schoenhofen, I. C., D. J. McNally, J.-R. Brisson, and S. M. Logan. 2006. Elucidation of the CMP-pseudaminic acid pathway in Helicobacter pylori: synthesis from UDP-N-acetyl-glucosamine by a single enzymatic reaction. Glycobiology 16:8C-14C.[Abstract/Free Full Text]
17
17. Schoenhofen, I. C., D. J. McNally, E. Vinogradov, D. Whitfield, N. M. Young, S. Dick, W. W. Wararchuk, J.-R. Brisson, and S. M. Logan. 2006. Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter. J. Biol. Chem. 281:723-732.[Abstract/Free Full Text]
18
18. Szymanski, C. M., S. M. Logan, D. Linton, and B. W. Wren. 2003. Campylobacter—a tale of two protein glycosylation systems. Trends Microbiol. 11:233-238.[Medline]
19
19. Szymanski, C. M., R. Yao, C. P. Ewing, T. J. Trust, and P. Guerry. 1999. Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol. Microbiol. 32:1022-1030.[CrossRef][Medline]
20
20. Thibault, P., S. M. Logan, J. F. Kelly, J.-R. Brisson, C. P. Ewing, T. J. Trust, and P. Guerry. 2001. Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin. J. Biol. Chem. 276:34862-34870.[Abstract/Free Full Text]
21
21. Wacker, M., D. Linton, P. G. Hitchen, M. Nita-Lazar, S. M. Haslam, S. J. North, M. Panico, H. R. Morris, A. Dell, B. W. Wren, and M. Aebi. 2002. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298:1790-1793.[Abstract/Free Full Text]
22
22. Wiesner, R. S., D. R. Hendrixson, and V. J. DiRita. 2003. Natural transformation of Campylobacter jejuni requires components of a type II secretion system. J. Bacteriol. 185:5408-5418.[Abstract/Free Full Text]
23
23. Young, N. M., J. R. Brisson, J. Kelly, D. C. Watson, L. Tessier, P. H. Lanthier, H. C. Jarrell, N. Cadotte, F. St. Michael, E. Aberg, and C. M. Szymanski. 2002. Structure of the N-linked glycan on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277:42530-42539.[Abstract/Free Full Text]

---

Journal of Bacteriology, September 2007, p. 6731-6733, Vol. 189, No. 18
0021-9193/07/$08.00+0     doi:10.1128/JB.00642-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Nothaft, H., Liu, X., McNally, D. J., Li, J., Szymanski, C. M. (2009). Study of free oligosaccharides derived from the bacterial N-glycosylation pathway. Proc. Natl. Acad. Sci. USA 106: 15019-15024 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guerry, P. Articles by Logan, S. M. Search for Related Content PubMed PubMed Citation Articles by Guerry, P. Articles by Logan, S. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH