IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.01576-06v1
75/4/2084    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kraus, D.
Right arrow Articles by Peschel, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kraus, D.
Right arrow Articles by Peschel, A.
Infection and Immunity, April 2007, p. 2084-2087, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01576-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Muropeptide Modification-Amidation of Peptidoglycan D-Glutamate Does Not Affect the Proinflammatory Activity of Staphylococcus aureus{triangledown}

Dirk Kraus,1 Hubert Kalbacher,2 Julia Buschmann,3 Brigitte Berger-Bächi,4 Friedrich Götz,3 and Andreas Peschel1*

Cellular and Molecular Microbiology Division, Medical Microbiology and Hygiene Institute, University of Tübingen, Tübingen, Germany,1 Medical and Natural Sciences Research Center, University of Tübingen, Tübingen, Germany,2 Microbial Genetics, University of Tübingen, Tübingen, Germany,3 Medical Microbiology Institute, University of Zürich, Zürich, Switzerland4

Received 29 September 2006/ Returned for modification 30 November 2006/ Accepted 18 January 2007


   ABSTRACT
Top
 Abstract
Text
 References
 
Peptidoglycan muropeptides, potent proinflammatory components, are amidated in Staphylococcus aureus for unknown reasons. To study whether this modification may modulate proinflammatory capacity, cytokine induction by isogenic S. aureus strains with different amidation levels and by synthetic amidated/nonamidated muramyldipeptides was evaluated. However, amidation did not significantly affect cytokine induction. This finding contributes to defining peptidoglycan receptor specificities and indicates that further rationales for muropeptide amidation have to be considered.


   TEXT
Top
 Abstract
 Text
References
 
Staphylococcus aureus is a frequent constituent of human nasal microflora and a major cause of severe endogenous infections (11). The staphylococcal cell wall determines several key aspects of the infection process, such as adherence (11, 28), immune recognition (13), and resistance to host defenses (21, 22). While many adhesive surface proteins have been investigated (12), the structure, function, and variability of nonproteinaceous polymers, such as peptidoglycan (PG) or teichoic acids, have remained parts of a neglected field of research. S. aureus is known to modify the canonical PG structure by O acetylation of the glycan strand and by amidation of the {alpha}-carboxyl group of the D-glutamate (D-Glu) residue in muropeptides, resulting in the formation of D-isoglutamine (16, 29). While O acetylation confers lysozyme resistance (3), the primary role of D-Glu amidation has remained unclear. Nevertheless, the latter modification is known to affect the level of methicillin resistance (4, 17) and to contribute to vancomycin susceptibility in S. aureus (8). The amino group for D-Glu amidation in S. aureus muropeptides is most probably derived from free glutamine (17, 23). However, the responsible transamidase enzyme has not yet been identified. Gustafson et al. described a femC mutant with 48% decreased muropeptide amidation (17, 26). The femC mutant has a disrupted glnR gene encoding the regulator of the glutamine synthetase (Fig. 1A), which results in reduced glutamine synthetase activity. As a consequence of lower amounts of the amino group donor, muropeptide amidation is reduced in this mutant (17).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 1. The S. aureus glutamine synthetase operon, with the insertion site of Tn551 in the femC mutant and the deleted region in the glnRA mutant (A), and structures of amidated MDP(iQ) and nonamidated MDP(E) (B).

 
PG has potent proinflammatory properties (5). It is sensed by the human innate immune system via NOD1 or NOD2 proteins (15, 18). An additional role of the TLR2 receptor in PG sensing has been described previously (10) but remains a matter of debate (27). The minimal structure required for NOD1 recognition is a dipeptide consisting of D-Glu and meso-diaminopimelate (mesoDAP), which is mainly produced by gram-negative bacteria (14, 15). D-Glu amidation (leading to D-isoglutamine) strongly impairs PG recognition by NOD1 (7), and the amidation of mesoDAP completely abrogates the ability of NOD1 to detect PG (15). NOD2 has a different substrate specificity as it requires a muramyldipeptide (MDP) composed of N-acetylmuramic acid, L-alanine, and D-Glu (Fig. 1B) (19, 27). S. aureus PG is recognized by NOD2 but not by NOD1 since it contains L-lysine instead of meso-DAP (7, 15). However, the relevance of D-Glu amidation for the efficiency of NOD2 recognition has remained unclear. MDP amidation has a profound impact on the physicochemical properties of the molecule since it removes the negative charge from the D-Glu {alpha}-carboxyl group (Fig. 1B). Accordingly, the net charge of MDP changes from –2 to –1 at physiological pH as a result of D-Glu amidation. In an attempt to study whether this structural difference has an impact on proinflammatory activity, we evaluated the tumor necrosis factor alpha (TNF-{alpha})-stimulating capacities of S. aureus strains with different levels of PG amidation and of synthetic MDPs with and without amidation.

Proinflammatory capacities of S. aureus wild type and femC mutant. S. aureus NCTC8325 wild type and the isogenic femC mutant (17) were grown overnight in glutamine-deficient, lipopolysaccharide-free RPMI 1640 medium (Sigma), harvested, and washed twice in phosphate-buffered saline (PBS). The mutant grew slower than the wild type did, but both strains reached similar bacterial densities. After heat inactivation at 90°C for 15 min, bacteria were washed again with PBS and adjusted to 109 bacteria/ml by using a Neubauer chamber. Endotoxin contamination of these preparations was below the detection limit of 10 pg/ml of the Limulus amoebocyte lysate test (Cambrex). A total of 360 µl whole blood from healthy volunteers was incubated with 40 µl of PBS containing increasing numbers of bacteria at 37°C for 8 h. Subsequently, samples were centrifuged and TNF-{alpha} levels in the supernatant were determined by enzyme-linked immunosorbent assay (R&D Systems). Both bacterial strains stimulated TNF-{alpha} production in a dose-dependent fashion (Fig. 2A). However, there was no significant difference in TNF-{alpha} levels induced by wild-type or femC mutant bacteria, suggesting that the modulation of muropeptide structure by D-Glu amidation may not affect the inflammatory activity of S. aureus cells.


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 2. TNF-{alpha} induction by S. aureus strains (A) and synthetic MDPs (B). (A) Increasing numbers of S. aureus wild-type (gray bars) or femC mutant bacteria (white bars) were incubated with whole human blood, and TNF-{alpha} levels were determined. (B) MDP(iQ) (gray bars) or MDP(E) (white bars) was incubated with whole human blood, and TNF-{alpha} levels were determined. The means and standard errors of the means (error bars) of four independent experiments, run in duplicates, are shown. The small differences between the strains in panel A are not significant as calculated by Student's t test.

 
In an attempt to obtain a mutant strain that completely lacks D-Glu amidation and might cause a stronger phenotypic difference, we deleted the entire operon composed of glutamine synthetase (glnA) and a corresponding regulator gene (glnR) (Fig. 1A). However, the resulting mutant grew only upon supplementation with significant amounts of glutamine and did not have any advantage relative to the femC mutant (data not shown).

Proinflammatory capacities of synthetic MDPs with or without D-Glu amidation. In order to study whether S. aureus MDPs with or without D-Glu amidation differ in their levels of proinflammatory activity when administered in the absence of other bacterial molecules, we chemically synthesized a nonamidated MDP with a D-Glu residue at position 2 of the peptide strand MDP(E), which is not commercially available. Synthesis of the acetylmuramyl-L-alanyl-D-glutamyl peptide was accomplished using Fmoc/tBu solid-phase peptide synthesis methodology (1) on acid-sensitive chlorotrityl chloride resin (2). Briefly, to 0.5 g 2-chlorotrityl chloride resin (0.6 mmol chloride/g resin) in 5 ml dichloromethane, 1 mmol of Fmoc-D-Glu(OBut)-OH and 2.5 mmol diisopropylethylamine (DIPEA) in 4 ml dichloromethane were added. After vigorous shaking for 60 min, the resin was filtered off, washed twice with dimethylformamide (DMF), and deactivated with a mixture of dichloromethane-methanol-DIPEA (80:15:5) for 15 min. After washing with DMF, the Fmoc group was deprotected with 5 ml of 20% piperidine in DMF for 5 min. After intensive washing with DMF, isopropanol, and diethyl ether, 1 mmol Fmoc-Ala-OH, 1 mmol diisoproyplcarbodiimide, and 1 mmol 1-hydroxybenzotriazole in 5 ml DMF were added and incubated for 60 min. Deprotection with piperidine and further coupling with N-acetylmuramic acid yielded the protected peptide. Removal of the side chain-protecting group and detachment from the resin were accomplished by treatment with trifluoroacetic acid-water-phenol (90:5:5). The peptide was precipitated by the addition of diethyl ether and lyophilized. The crude peptide was purified by preparative, reversed-phase, high-performance liquid chromatography on a Reprosil C8 column (150 x 10 mm) using a flat gradient from 100% A to 40% B in 40 min (A, 0.055% trifluoroacetic acid; B, 80% acetonitrile-0.05% trifluoroacetic acid) while monitoring absorbance at 214 nm. Appropriate fractions were lyophilized, and the identity of the product was confirmed by electrospray ionization mass spectrometry (ESI-MS). Our synthetic MDP(E) and the amidated MDP(iQ) purchased from Bachem were found to have the expected mass difference of 1 (Fig. 3).


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 3. Averaged negative ion mode ESI-MS of MDP(iQ) and MDP(E). Peaks with the expected masses for MDP(iQ) (491.3) or MDP(E) (492.3) are indicated.

 
MDP(E) and MDP(iQ) were dissolved in PBS. Again, the endotoxin contamination of the preparations was below the detection limit. Forty microliters of PBS containing increasing concentrations of these substances was incubated with 360 µl whole human blood at 37°C for 8 h to stimulate TNF-{alpha} production. TNF-{alpha} was quantified as described above. Both compounds stimulated TNF-{alpha} production in a dose-dependent manner. However, amidated and nonamidated MDP did not differ in their potency levels (Fig. 2B), which is in accordance with the equal proinflammatory activities of S. aureus wild type and the femC mutant.

Concluding comments. The inflammation-eliciting capacity of S. aureus plays a crucial role in the host response to infections and contributes to life-threatening complications in septicemia (5, 13). However, the diversity, relative importance, and variability of proinflammatory staphylococcal molecules, such as peptidoglycan, lipopeptides, lipoteichoic acid, formylated peptides, and others have remained uncertain. While the relative importance of lipoproteins and formylated peptides has recently been confirmed using defined mutants lacking the corresponding classes of molecules (6, 9, 25), such a strategy is impossible for PG, which is essential for bacterial viability. However, bacterial mutants with altered PG structures might help to investigate the relative role of PG, along with the modulation of PG structure. This study demonstrates that the amidation of muropeptides does not influence the capacity of S. aureus or its MDP to stimulate the production of TNF-{alpha}, which contributes to our understanding of the specificity of PG-sensing host receptors. Why S. aureus amidates its muropeptides remains an open question. A possible reason may be the concomitant alteration of the net charge in the S. aureus cell envelope, which may play a role in the maintenance and turnover of the bacterial cell wall. Accordingly, muropeptide amidation has been implicated in PG cross-linking efficiency (20, 24). Identifying the muropeptide amidase gene would enable the construction of an S. aureus mutant lacking any muropeptide amidation without affecting the intracellular glutamine pool. Such an approach represents an important challenge for future studies.


   ACKNOWLEDGMENTS
 
We thank Gabriele Hornig and Katja Henseler for excellent technical assistance and Daniel Bächle and Alexander Beck (Panatecs, Tübingen) for ESI-MS analysis.

Our research is supported by grants from the German Research Foundation to A.P. and F.G. (FOR449, GRK685, and TR34) and to A.P. and H.K. (SFB685); the European Union (LSHM-CT-2004-512093) to A.P.; the German Ministry of Education and Research (NGFN2) to A.P. and F.G.; and the IZKF program of the Medical Faculty, University of Tübingen, to A.P.


   FOOTNOTES
 
* Corresponding author. Mailing address: Medical Microbiology and Hygiene Institute, University of Tübingen, Elfriede-Aulhorn-Strasse 6, 72076 Tübingen, Germany. Phone: (49)-7071-2981515. Fax: (49)-7071-293435. E-mail: andreas.peschel{at}uni-tuebingen.de. Back

{triangledown} Published ahead of print on 29 January 2007. Back

Editor: J. N. Weiser


   REFERENCES
Top
 Abstract
 Text
 References
 
1. Baechle, D., A. Cansier, R. Fischer, J. Brandenburg, T. Burster, C. Driessen, and H. Kalbacher. 2005. Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity. J. Pept. Sci. 11:166-174.[CrossRef][Medline]
2. Barlos, K., O. Chatzi, D. Gatos, and G. Stavropoulos. 1991. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Pept. Protein Res. 37:513-520.[Medline]
3. Bera, A., S. Herbert, A. Jakob, W. Vollmer, and F. Götz. 2005. Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol. Microbiol. 55:778-787.[CrossRef][Medline]
4. Berger-Bächi, B., A. Strassle, J. E. Gustafson, and F. H. Kayser. 1992. Mapping and characterization of multiple chromosomal factors involved in methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 36:1367-1373.[Abstract/Free Full Text]
5. Boneca, I. G. 2005. The role of peptidoglycan in pathogenesis. Curr. Opin. Microbiol. 8:46-53.[CrossRef][Medline]
6. Bubeck Wardenburg, J., W. A. Williams, and D. Missiakas. 2006. Host defenses against Staphylococcus aureus infection require recognition of bacterial lipoproteins. Proc. Natl. Acad. Sci. USA 103:13831-13836.[Abstract/Free Full Text]
7. Chamaillard, M., M. Hashimoto, Y. Horie, J. Masumoto, S. Qiu, L. Saab, Y. Ogura, A. Kawasaki, K. Fukase, S. Kusumoto, M. A. Valvano, S. J. Foster, T. W. Mak, G. Nunez, and N. Inohara. 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4:702-707.[CrossRef][Medline]
8. Cui, L., H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu. 2000. Contribution of a thickened cell wall and its glutamine nonamidated component to the vancomycin resistance expressed by Staphylococcus aureus Mu50. Antimicrob. Agents Chemother. 44:2276-2285.[Abstract/Free Full Text]
9. Dürr, M. C., S. A. Kristian, M. Otto, G. Matteoli, P. S. Margolis, J. Trias, K. P. Van Kessel, J. A. Van Strijp, E. Bohn, R. Landmann, and A. Peschel. 2006. Neutrophil chemotaxis by pathogen-associated molecular patterns—formylated peptides are crucial but not the sole neutrophil attractants produced by Staphylococcus aureus. Cell Microbiol. 8:207-217.[CrossRef][Medline]
10. Dziarski, R., and D. Gupta. 2005. Staphylococcus aureus peptidoglycan is a Toll-like receptor 2 activator: a reevaluation. Infect. Immun. 73:5212-5216.[Abstract/Free Full Text]
11. Foster, T. J. 2004. The Staphylococcus aureus "superbug." J. Clin. Investig. 114:1693-1696.[CrossRef][Medline]
12. Foster, T. J., and M. Hook. 1998. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 6:484-488.[CrossRef][Medline]
13. Fournier, B., and D. J. Philpott. 2005. Recognition of Staphylococcus aureus by the innate immune system. Clin. Microbiol. Rev. 18:521-540.[Abstract/Free Full Text]
14. Girardin, S. E., I. G. Boneca, L. A. Carneiro, A. Antignac, M. Jehanno, J. Viala, K. Tedin, M. K. Taha, A. Labigne, U. Zahringer, A. J. Coyle, P. S. DiStefano, J. Bertin, P. J. Sansonetti, and D. J. Philpott. 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300:1584-1587.[Abstract/Free Full Text]
15. Girardin, S. E., L. H. Travassos, M. Herve, D. Blanot, I. G. Boneca, D. J. Philpott, P. J. Sansonetti, and D. Mengin-Lecreulx. 2003. Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J. Biol. Chem. 278:41702-41708.[Abstract/Free Full Text]
16. Götz, F., T. Bannerman, and K.-H. Schleifer. 2006. The genera Staphylococcus and Macrococcus, p. 45-75. In M. Dworkin (ed.), The prokaryotes: an evolving electronic resource for the microbiological community. Springer Verlag, New York, NY.
17. Gustafson, J., A. Strassle, H. Hachler, F. H. Kayser, and B. Berger-Bachi. 1994. The femC locus of Staphylococcus aureus required for methicillin resistance includes the glutamine synthetase operon. J. Bacteriol. 176:1460-1467.[Abstract/Free Full Text]
18. Inohara, N., M. Chamaillard, C. McDonald, and G. Nunez. 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu. Rev. Biochem. 74:355-383.[CrossRef][Medline]
19. Inohara, N., Y. Ogura, A. Fontalba, O. Gutierrez, F. Pons, J. Crespo, K. Fukase, S. Inamura, S. Kusumoto, M. Hashimoto, S. J. Foster, A. P. Moran, J. L. Fernandez-Luna, and G. Nunez. 2003. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J. Biol. Chem. 278:5509-5512.[Abstract/Free Full Text]
20. Komatsuzawa, H., K. Ohta, H. Labischinski, M. Sugai, and H. Suginaka. 1999. Characterization of fmtA, a gene that modulates the expression of methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 43:2121-2125.[Abstract/Free Full Text]
21. Kraus, D., and A. Peschel. 2006. Molecular mechanisms of bacterial resistance to antimicrobial peptides. Curr. Top. Microbiol. Immunol. 306:231-250.[Medline]
22. Peschel, A., and H. G. Sahl. 2006. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nat. Rev. Microbiol. 4:529-536.[CrossRef][Medline]
23. Siewert, G., and J. L. Strominger. 1968. Biosynthesis of the peptidoglycan of bacterial cell walls. XI. Formation of the isoglutamine amide group in the cell walls of Staphylococcus aureus. J. Biol. Chem. 243:783-790.[Abstract/Free Full Text]
24. Sinha, R. K., and F. C. Neuhaus. 1991. Biosynthesis of peptidoglycan in Gaffkya homari: on the target(s) of benzylpenicillin. Antimicrob. Agents Chemother. 35:1753-1759.[Abstract/Free Full Text]
25. Stoll, H., J. Dengjel, C. Nerz, and F. Götz. 2005. Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation. Infect. Immun. 73:2411-2423.[Abstract/Free Full Text]
26. Strandén, A. M., M. Roos, and B. Berger-Bachi. 1996. Glutamine synthetase and heteroresistance in methicillin-resistant Staphylococcus aureus. Microb. Drug Resist. 2:201-207.[Medline]
27. Travassos, L. H., S. E. Girardin, D. J. Philpott, D. Blanot, M. A. Nahori, C. Werts, and I. G. Boneca. 2004. Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition. EMBO Rep. 5:1000-1006.[CrossRef][Medline]
28. Weidenmaier, C., J. F. Kokai-Kun, S. A. Kristian, T. Chanturyia, H. Kalbacher, M. Gross, G. Nicholson, B. Neumeister, J. J. Mond, and A. Peschel. 2004. Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat. Med. 10:243-245.[CrossRef][Medline]
29. Wilkinson, B. J. 1997. Biology, p. 1-38. In K. B. Crossley and G. L. Archer (ed.), The staphylococci in human disease. Churchill Livingstone, New York, NY.

Infection and Immunity, April 2007, p. 2084-2087, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01576-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.01576-06v1
75/4/2084    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kraus, D.
Right arrow Articles by Peschel, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kraus, D.
Right arrow Articles by Peschel, A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals