Genetic characterization of mycobacterial ,-transpeptidases Free

Abstract

,-Transpeptidases (Ldts) catalyse the formation of 3–3 cross-links in peptidoglycans (PGs); however, the role of these enzymes in cell envelope physiology is not well understood. Mycobacterial PG contains a higher percentage of 3–3 cross-links (~30–80 %) than the PG in most other bacteria, suggesting that they are particularly important to mycobacterial cell wall biology. The genomes of and encode multiple Ldt genes, but it is not clear if they are redundant. We compared the sequences of the Ldt proteins from 18 mycobacterial genomes and found that they can be grouped into six classes. We then constructed strains lacking single or multiple Ldt genes to determine the physiological consequence of the loss of these enzymes. We report that of the single mutants, only one, Δ (, class 5), displayed an increased susceptibility to imipenem – a carbapenem antibiotic that inhibits the Ldt enzymes. The invariant cysteine in the active site of LdtC was required for function, consistent with its role as an Ldt. A triple mutant missing and both of the class 2 genes displayed hypersusceptibility to antibiotics, lysozyme and -methionine, and had an altered cellular morphology. These data demonstrated that the distinct classes of mycobacterial Ldts may reflect different, non-redundant functions and that the class 5 Ldt was peculiar in that its loss, alone and with the class 2 proteins, had the most profound effect on phenotype.

Funding
This study was supported by the:
  • National Institutes of Health (Award T32 AI007362 and AI073772)
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2014-08-01
2024-03-28
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References

  1. Asubel F., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. ( 1987). Current Protocols in Molecular Biology New York: Greene/Wiley Interscience;
    [Google Scholar]
  2. Azuma I., Thomas D. W., Adam A., Ghuysen J. M., Bonaly R., Petit J. F., Lederer E. ( 1970). Occurrence of N-glycolylmuramic acid in bacterial cell walls. A preliminary survey. Biochim Biophys Acta 208:444–451 [View Article][PubMed]
    [Google Scholar]
  3. Biarrotte-Sorin S., Hugonnet J. E., Delfosse V., Mainardi J. L., Gutmann L., Arthur M., Mayer C. ( 2006). Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase. J Mol Biol 359:533–538 [View Article][PubMed]
    [Google Scholar]
  4. Böth D., Steiner E. M., Stadler D., Lindqvist Y., Schnell R., Schneider G. ( 2013). Structure of LdtMt2, an l,d-transpeptidase from Mycobacterium tuberculosis . Acta Crystallogr D Biol Crystallogr 69:432–441 [View Article][PubMed]
    [Google Scholar]
  5. Brennan P. J. ( 1989). Structure of mycobacteria: recent developments in defining cell wall carbohydrates and proteins. Rev Infect Dis 11:Suppl 2S420–S430 [View Article][PubMed]
    [Google Scholar]
  6. Cava F., de Pedro M. A., Lam H., Davis B. M., Waldor M. K. ( 2011). Distinct pathways for modification of the bacterial cell wall by non-canonical d-amino acids. EMBO J 30:3442–3453 [View Article][PubMed]
    [Google Scholar]
  7. Cordillot M., Dubée V., Triboulet S., Dubost L., Marie A., Hugonnet J. E., Arthur M., Mainardi J. L. ( 2013). In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by l,d-transpeptidases and inactivation of these enzymes by carbapenems. Antimicrob Agents Chemother 57:5940–5945 [View Article][PubMed]
    [Google Scholar]
  8. Correale S., Ruggiero A., Capparelli R., Pedone E., Berisio R. ( 2013). Structures of free and inhibited forms of the l,d-transpeptidase LdtMt1 from Mycobacterium tuberculosis . Acta Crystallogr D Biol Crystallogr 69:1697–1706 [View Article][PubMed]
    [Google Scholar]
  9. Dramsi S., Magnet S., Davison S., Arthur M. ( 2008). Covalent attachment of proteins to peptidoglycan. FEMS Microbiol Rev 32:307–320 [View Article][PubMed]
    [Google Scholar]
  10. Erdemli S. B., Gupta R., Bishai W. R., Lamichhane G., Amzel L. M., Bianchet M. A. ( 2012). Targeting the cell wall of Mycobacterium tuberculosis: structure and mechanism of l,d-transpeptidase 2. Structure 20:2103–2115 [View Article][PubMed]
    [Google Scholar]
  11. Gupta R., Lavollay M., Mainardi J. L., Arthur M., Bishai W. R., Lamichhane G. ( 2010). The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin. Nat Med 16:466–469 [View Article][PubMed]
    [Google Scholar]
  12. Hett E. C., Rubin E. J. ( 2008). Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev 72:126–156 [View Article][PubMed]
    [Google Scholar]
  13. Hirschfield G. R., McNeil M., Brennan P. J. ( 1990). Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis . J Bacteriol 172:1005–1013[PubMed]
    [Google Scholar]
  14. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. ( 1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59 [View Article][PubMed]
    [Google Scholar]
  15. Horcajo P., de Pedro M. A., Cava F. ( 2012). Peptidoglycan plasticity in bacteria: stress-induced peptidoglycan editing by noncanonical d-amino acids. Microb Drug Resist 18:306–313 [View Article][PubMed]
    [Google Scholar]
  16. Kay B. K., Williamson M. P., Sudol M. ( 2000). The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J 14:231–241[PubMed]
    [Google Scholar]
  17. Kim H. S., Kim J., Im H. N., Yoon J. Y., An D. R., Yoon H. J., Kim J. Y., Min H. K., Kim S. J. & other authors ( 2013). Structural basis for the inhibition of Mycobacterium tuberculosis l,d-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains. Acta Crystallogr D Biol Crystallogr 69:420–431 [View Article][PubMed]
    [Google Scholar]
  18. Kumar P., Arora K., Lloyd J. R., Lee I. Y., Nair V., Fischer E., Boshoff H. I., Barry C. E. III ( 2012). Meropenem inhibits d,d-carboxypeptidase activity in Mycobacterium tuberculosis . Mol Microbiol 86:367–381 [View Article][PubMed]
    [Google Scholar]
  19. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A. & other authors ( 2007). Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 [View Article][PubMed]
    [Google Scholar]
  20. Lavollay M., Arthur M., Fourgeaud M., Dubost L., Marie A., Veziris N., Blanot D., Gutmann L., Mainardi J. L. ( 2008). The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by l,d-transpeptidation. J Bacteriol 190:4360–4366 [View Article][PubMed]
    [Google Scholar]
  21. Lavollay M., Fourgeaud M., Herrmann J. L., Dubost L., Marie A., Gutmann L., Arthur M., Mainardi J. L. ( 2011). The peptidoglycan of Mycobacterium abscessus is predominantly cross-linked by l,d-transpeptidases. J Bacteriol 193:778–782 [View Article][PubMed]
    [Google Scholar]
  22. Lederer E. ( 1971). The mycobacterial cell wall. Pure Appl Chem 25:135–165 [View Article][PubMed]
    [Google Scholar]
  23. Li W. J., Li D. F., Hu Y. L., Zhang X. E., Bi L. J., Wang D. C. ( 2013). Crystal structure of l,d-transpeptidase LdtMt2 in complex with meropenem reveals the mechanism of carbapenem against Mycobacterium tuberculosis . Cell Res 23:728–731 [View Article][PubMed]
    [Google Scholar]
  24. Lu J. Z., Fujiwara T., Komatsuzawa H., Sugai M., Sakon J. ( 2006). Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges. J Biol Chem 281:549–558 [View Article][PubMed]
    [Google Scholar]
  25. Magnet S., Bellais S., Dubost L., Fourgeaud M., Mainardi J. L., Petit-Frère S., Marie A., Mengin-Lecreulx D., Arthur M., Gutmann L. ( 2007a). Identification of the l,d-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan. J Bacteriol 189:3927–3931 [View Article][PubMed]
    [Google Scholar]
  26. Magnet S., Arbeloa A., Mainardi J. L., Hugonnet J. E., Fourgeaud M., Dubost L., Marie A., Delfosse V., Mayer C. & other authors ( 2007b). Specificity of l,d-transpeptidases from Gram-positive bacteria producing different peptidoglycan chemotypes. J Biol Chem 282:13151–13159 [View Article][PubMed]
    [Google Scholar]
  27. Mainardi J. L., Morel V., Fourgeaud M., Cremniter J., Blanot D., Legrand R., Frehel C., Arthur M., Van Heijenoort J., Gutmann L. ( 2002). Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium . J Biol Chem 277:35801–35807 [View Article][PubMed]
    [Google Scholar]
  28. Mainardi J. L., Hugonnet J. E., Rusconi F., Fourgeaud M., Dubost L., Moumi A. N., Delfosse V., Mayer C., Gutmann L. & other authors ( 2007). Unexpected inhibition of peptidoglycan ld-transpeptidase from Enterococcus faecium by the beta-lactam imipenem. J Biol Chem 282:30414–30422 [View Article][PubMed]
    [Google Scholar]
  29. McNeil M., Daffe M., Brennan P. J. ( 1990). Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls. J Biol Chem 265:18200–18206[PubMed]
    [Google Scholar]
  30. Patru M. M., Pavelka M. S. Jr ( 2010). A role for the class A penicillin-binding protein PonA2 in the survival of Mycobacterium smegmatis under conditions of nonreplication. J Bacteriol 192:3043–3054 [View Article][PubMed]
    [Google Scholar]
  31. Purdy G. E., Niederweis M., Russell D. G. ( 2009). Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides. Mol Microbiol 73:844–857 [View Article][PubMed]
    [Google Scholar]
  32. Quintela J. C., Caparrós M., de Pedro M. A. ( 1995). Variability of peptidoglycan structural parameters in Gram-negative bacteria. FEMS Microbiol Lett 125:95–100 [View Article][PubMed]
    [Google Scholar]
  33. Raymond J. B., Mahapatra S., Crick D. C., Pavelka M. S. Jr ( 2005). Identification of the namH gene, encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. J Biol Chem 280:326–333[PubMed] [CrossRef]
    [Google Scholar]
  34. Sanders A. N., Pavelka M. S. Jr ( 2013). Phenotypic analysis of Escherichia coli mutants lacking l,d-transpeptidases. Microbiology 159:1842–1852 [View Article][PubMed]
    [Google Scholar]
  35. Schleifer K. H., Kandler O. ( 1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477[PubMed]
    [Google Scholar]
  36. Schoonmaker M. K., Bishai W. R., Lamichhane G. ( 2014). Nonclassical transpeptidases of Mycobacterium tuberculosis alter cell size, morphology, the cytosolic matrix, protein localization, virulence, and resistance to β-lactams. J Bacteriol 196:1394–1402 [View Article][PubMed]
    [Google Scholar]
  37. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr ( 1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis . Mol Microbiol 4:1911–1919 [View Article][PubMed]
    [Google Scholar]
  38. Song H., Sandie R., Wang Y., Andrade-Navarro M. A., Niederweis M. ( 2008). Identification of outer membrane proteins of Mycobacterium tuberculosis . Tuberculosis (Edinb) 88:526–544 [View Article][PubMed]
    [Google Scholar]
  39. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H. & other authors ( 1991). New use of BCG for recombinant vaccines. Nature 351:456–460 [View Article][PubMed]
    [Google Scholar]
  40. van Kessel J. C., Hatfull G. F. ( 2008). Mycobacterial recombineering. Methods Mol Biol 435:203–215 [View Article][PubMed]
    [Google Scholar]
  41. Whisstock J. C., Lesk A. M. ( 1999). SH3 domains in prokaryotes. Trends Biochem Sci 24:132–133 [View Article][PubMed]
    [Google Scholar]
  42. Wietzerbin J., Das B. C., Petit J. F., Lederer E., Leyh-Bouille M., Ghuysen J. M. ( 1974). Occurrence of d-alanyl-(d)-meso-diaminopimelic acid and meso-diaminopimelyl-meso-diaminopimelic acid interpeptide linkages in the peptidoglycan of Mycobacteria. Biochemistry 13:3471–3476 [View Article][PubMed]
    [Google Scholar]
  43. Williamson M. P. ( 1994). The structure and function of proline-rich regions in proteins. Biochem J 297:249–260[PubMed]
    [Google Scholar]
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