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Volume 157, Issue 7

Repression of -glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Free

Abstract

is the most common airborne fungal pathogen, causing fatal invasive aspergillosis in immunocompromised patients. The crude mortality is 60–90 % and remains around 29–42 % even with treatment. The main reason for patient death is the low efficiency of the drug therapies. As protein -glycosylation is involved in cell wall biogenesis in , a deeper understanding of its role in cell wall biogenesis will help to develop new drug targets. The Af gene encodes the essential catalytic subunit of oligosaccharyltransferase, an enzyme complex responsible for the transfer of the -glycan to nascent polypeptides. To evaluate the role of -glycosylation in cell wall biosynthesis, we constructed the conditional mutant strain CPR-stt3 by replacing the endogenous promoter of Af with the nitrogen-dependent promoter. Repression of the Af gene in the CPR-stt3 strain led to a severe retardation of growth and a slight defect in cell wall integrity (CWI). One of the most interesting findings was that upregulation of the cell wall-related genes was not accompanied by an activation of the MpkA kinase, which has been shown to be a central element in the CWI signalling pathway in both and . Considering that the unfolded protein response (UPR) was found to be activated, which might upregulate the expression of cell wall protein and chitin, our data suggest that the UPR, instead of the MpkA-dependent CWI signalling pathway, is the major compensatory mechanism induced by repression but not abolition of -glycosylation in . Our finding is a key to understanding the complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Funding
This study was supported by the:
  • National Natural Science Foundation of China (Award 31011130030 and 31030025)
© 2011 SGM
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2011-07-01
2024-04-25
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References

  1. Amaar Y. G., Moore M. M. ( 1998). Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus . Curr Genet 33:206–215 [View Article][PubMed]
     [Google Scholar]
  2. Beauvais A., Bruneau J. M., Mol P. C., Buitrago M. J., Legrand R., Latgé J. P. ( 2000). Glucan synthase complex of Aspergillus fumigatus . J Bacteriol 183:2273–2279[PubMed] [CrossRef]
     [Google Scholar]
  3. Beauvais A., Maubon D., Park S., Morelle W., Tanguy M., Huerre M., Perlin D. S., Latgé J. P. ( 2005). Two α(1-3) glucan synthases with different functions in Aspergillus fumigatus . Appl Environ Microbiol 71:1531–1538 [View Article][PubMed]
     [Google Scholar]
  4. Bowman J. C., Hicks P. S., Kurtz M. B., Rosen H., Schmatz D. M., Liberator P. A., Douglas C. M. ( 2002). The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 46:3001–3012 [View Article][PubMed]
     [Google Scholar]
  5. Bradford M. M. ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
     [Google Scholar]
  6. Branza-Nichita N., Negroiu G., Petrescu A. J., Garman E. F., Platt F. M., Wormald M. R., Dwek R. A., Petrescu S. M. ( 2000). Mutations at critical N-glycosylation sites reduce tyrosinase activity by altering folding and quality control. J Biol Chem 275:8169–8175 [View Article][PubMed]
     [Google Scholar]
  7. Bulawa C. E. ( 1992). CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis . Mol Cell Biol 12:1764–1776[PubMed]
     [Google Scholar]
  8. Carotti C., Ferrario L., Roncero C., Valdivieso M. H., Duran A., Popolo L. ( 2002). Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization. Yeast 19:1113–1124 [View Article][PubMed]
     [Google Scholar]
  9. Cove D. J. ( 1966). The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. . Biochim Biophys Acta 113:51–56 [CrossRef]
     [Google Scholar]
  10. Denning D. W. ( 2003). Echinocandin antifungal drugs. Lancet 362:1142–1151 [View Article][PubMed]
     [Google Scholar]
  11. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. ( 1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356 [View Article]
     [Google Scholar]
  12. Elorza M. V., Murgui A., Sentandreu R. ( 1985). Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls. J Gen Microbiol 131:2209–2216[PubMed]
     [Google Scholar]
  13. Fujioka T., Mizutani O., Furukawa K., Sato N., Yoshimi A., Yamagata Y., Nakajima T., Abe K. ( 2007). MpkA-dependent and -independent cell wall integrity signaling in Aspergillus nidulans . Eukaryot Cell 6:1497–1510 [View Article][PubMed]
     [Google Scholar]
  14. Hearn V. M., Sietsma J. H. ( 1994). Chemical and immunological analysis of the Aspergillus fumigatus cell wall. Microbiology 140:789–795 [View Article][PubMed]
     [Google Scholar]
  15. Hebert D. N., Garman S. C., Molinari M. ( 2005). The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags. Trends Cell Biol 15:364–370 [View Article][PubMed]
     [Google Scholar]
  16. Helenius A., Aebi M. ( 2001). Intracellular functions of N-linked glycans. Science 291:2364–2369 [View Article][PubMed]
     [Google Scholar]
  17. Helenius A., Aebi M. ( 2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049 [View Article][PubMed]
     [Google Scholar]
  18. Hu W., Sillaots S., Lemieux S., Davison J., Kauffman S., Breton A., Linteau A., Xin C., Bowman J. et al. ( 2007). Essential gene identification and drug target prioritization in Aspergillus fumigatus . PLoS Pathog 3:e24 [View Article][PubMed]
     [Google Scholar]
  19. Hutzler F., Gerstl R., Lommel M., Strahl S. ( 2008). Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2p. Mol Microbiol 68:1438–1449 [View Article][PubMed]
     [Google Scholar]
  20. Kelleher D. J., Gilmore R. ( 2006). An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology 16:47R–62R [View Article][PubMed]
     [Google Scholar]
  21. Kelleher D. J., Karaoglu D., Mandon E. C., Gilmore R. ( 2003). Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties. Mol Cell 12:101–111 [View Article][PubMed]
     [Google Scholar]
  22. Kim R., Emi M., Tanabe K., Murakami S. ( 2006). Role of the unfolded protein response in cell death. Apoptosis 11:5–13 [View Article][PubMed]
     [Google Scholar]
  23. Knauer R., Lehle L. ( 1999). The oligosaccharyltransferase complex from yeast. Biochim Biophys Acta 1426:259–273[PubMed] [CrossRef]
     [Google Scholar]
  24. Lagorce A., Le Berre-Anton V., Aguilar-Uscanga B., Martin-Yken H., Dagkessamanskaia A., François J. ( 2002). Involvement of GFA1, which encodes glutamine-fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae . Eur J Biochem 269:1697–1707 [View Article][PubMed]
     [Google Scholar]
  25. Latgé J. P. ( 1999). Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12:310–350[PubMed]
     [Google Scholar]
  26. Latgé J. P. ( 2001). The pathobiology of Aspergillus fumigatus . Trends Microbiol 9:382–389 [View Article][PubMed]
     [Google Scholar]
  27. Lee J. I., Yu Y. M., Rho Y. M., Park B. C., Choi J. H., Park H. M., Maeng P. J. ( 2005). Differential expression of the chsE gene encoding a chitin synthase of Aspergillus nidulans in response to developmental status and growth conditions. FEMS Microbiol Lett 249:121–129 [View Article][PubMed]
     [Google Scholar]
  28. Levin D. E. ( 2005). Cell wall integrity signaling in Saccharomyces cerevisiae . Microbiol Mol Biol Rev 69:262–291 [View Article][PubMed]
     [Google Scholar]
  29. Livak K. J., Schmittgen T. D. ( 2001). Analysis of relative gene expression data using real-time quantitative PCR and method. Methods 25:402–408 [View Article][PubMed]
     [Google Scholar]
  30. Malhotra J. D., Kaufman R. J. ( 2007). The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 18:716–731 [View Article][PubMed]
     [Google Scholar]
  31. Maubon D., Park S., Tanguy M., Huerre M., Schmitt C., Prévost M. C., Perlin D. S., Latgé J. P., Beauvais A. ( 2006). AGS3, an α(1-3)glucan synthase gene family member of Aspergillus fumigatus, modulates mycelium growth in the lung of experimentally infected mice. Fungal Genet Biol 43:366–375 [View Article][PubMed]
     [Google Scholar]
  32. Mellado E., Dubreucq G., Mol P., Sarfati J., Paris S., Diaquin M., Holden D. W., Rodriguez-Tudela J. L., Latgé J. P. ( 2003). Cell wall biogenesis in a double chitin synthase mutant (chsG−/chsE−) of Aspergillus fumigatus . Fungal Genet Biol 38:98–109 [View Article][PubMed]
     [Google Scholar]
  33. Mesaeli N., Nakamura K., Zvaritch E., Dickie P., Dziak E., Krause K. H., Opas M., MacLennan D. H., Michalak M. ( 1999). Calreticulin is essential for cardiac development. J Cell Biol 144:857–868 [View Article][PubMed]
     [Google Scholar]
  34. Meusser B., Hirsch C., Jarosch E., Sommer T. ( 2005). ERAD: the long road to destruction. Nat Cell Biol 7:766–772 [View Article][PubMed]
     [Google Scholar]
  35. Mouyna I., Monod M., Fontaine T., Henrissat B., Léchenne B., Latgé J. P. ( 2000). Identification of the catalytic residues of the first family of β(1-3)glucanosyltransferases identified in fungi. Biochem J 347:741–747 [View Article][PubMed]
     [Google Scholar]
  36. Mouyna I., Morelle W., Vai M., Monod M., Léchenne B., Fontaine T., Beauvais A., Sarfati J., Prévost M. C. et al. ( 2005). Deletion of GEL2 encoding for a β(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus . Mol Microbiol 56:1675–1688 [View Article][PubMed]
     [Google Scholar]
  37. Muro-Pastor M. I., Gonzalez R., Strauss J., Narendja F., Scazzocchio C. ( 1999). The GATA factor AreA is essential for chromatin remodelling in a eukaryotic bidirectional promoter. EMBO J 18:1584–1597 [View Article][PubMed]
     [Google Scholar]
  38. Nilsson I., Kelleher D. J., Miao Y., Shao Y., Kreibich G., Gilmore R., von Heijne G., Johnson A. E. ( 2003). Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J Cell Biol 161:715–725 [View Article][PubMed]
     [Google Scholar]
  39. Parodi A. J. ( 1999). Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells. Biochim Biophys Acta 1426:287–295[PubMed] [CrossRef]
     [Google Scholar]
  40. Popolo L., Gilardelli D., Bonfante P., Vai M. ( 1997). Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae . J Bacteriol 179:463–469[PubMed]
     [Google Scholar]
  41. Ram A. F. J., Klis F. M. ( 2006). Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc 1:2253–2256 [View Article][PubMed]
     [Google Scholar]
  42. Ram A. F. J., Wolters A., Ten Hoopen R., Klis F. M. ( 1994). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10:1019–1030 [View Article][PubMed]
     [Google Scholar]
  43. Ram A. F. J., Arentshorst M., Damveld R. A., vanKuyk P. A., Klis F. M., van den Hondel C. A. ( 2004). The cell wall stress response in Aspergillus niger involves increased expression of the glutamine : fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall. Microbiology 150:3315–3326 [View Article][PubMed]
     [Google Scholar]
  44. Richie D. L., Miley M. D., Bhabhra R., Robson G. D., Rhodes J. C., Askew D. S. ( 2007). The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress. Mol Microbiol 63:591–604 [View Article][PubMed]
     [Google Scholar]
  45. Richie D. L., Hartl L., Aimanianda V., Winters M. S., Fuller K. K., Miley M. D., White S., McCarthy J. W., Latgé J. P. et al. ( 2009). A role for the unfolded protein response (UPR) in virulence and antifungal susceptibility in Aspergillus fumigatus . PLoS Pathog 5:e1000258 [View Article][PubMed]
     [Google Scholar]
  46. Romero B., Turner G., Olivas I., Laborda F., De Lucas J. R. ( 2003). The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen. Fungal Genet Biol 40:103–114 [View Article][PubMed]
     [Google Scholar]
  47. Römisch K. ( 2005). Endoplasmic reticulum-associated degradation. Annu Rev Cell Dev Biol 21:435–456 [View Article][PubMed]
     [Google Scholar]
  48. Ron D., Walter P. ( 2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8:519–529 [View Article][PubMed]
     [Google Scholar]
  49. Roncero C. ( 2002). The genetic complexity of chitin synthesis in fungi. Curr Genet 41:367–378 [View Article][PubMed]
     [Google Scholar]
  50. Ruddock L. W., Molinari M. ( 2006). N-Glycan processing in ER quality control. J Cell Sci 119:4373–4380 [View Article][PubMed]
     [Google Scholar]
  51. Schoffelmeer E. A., Klis F. M., Sietsma J. H., Cornelissen B. J. ( 1999). The cell wall of Fusarium oxysporum . Fungal Genet Biol 27:275–282 [View Article][PubMed]
     [Google Scholar]
  52. Silberstein S., Gilmore R. ( 1996). Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J 10:849–858[PubMed]
     [Google Scholar]
  53. Terashima H., Yabuki N., Arisawa M., Hamada K., Kitada K. ( 2000). Up-regulation of genes encoding glycosylphosphatidylinositol (GPI)-attached proteins in response to cell wall damage caused by disruption of FKS1 in Saccharomyces cerevisiae . Mol Gen Genet 264:64–74 [View Article][PubMed]
     [Google Scholar]
  54. Travers K. J., Patil C. K., Wodicka L., Lockhart D. J., Weissman J. S., Walter P. ( 2000). Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258 [View Article][PubMed]
     [Google Scholar]
  55. Valdivia R. H., Schekman R. ( 2003). The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci U S A 100:10287–10292 [View Article][PubMed]
     [Google Scholar]
  56. Valdivieso M. H., Mol P. C., Shaw J. A., Cabib E., Durán A. ( 1991). CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae . J Cell Biol 114:101–109 [View Article][PubMed]
     [Google Scholar]
  57. Valiante V., Heinekamp T., Jain R., Härtl A., Brakhage A. A. ( 2008). The mitogen-activated protein kinase MpkA of Aspergillus fumigatus regulates cell wall signaling and oxidative stress response. Fungal Genet Biol 45:618–627 [View Article][PubMed]
     [Google Scholar]
  58. Valiante V., Jain R., Heinekamp T., Brakhage A. A. ( 2009). The MpkA MAP kinase module regulates cell wall integrity signaling and pyomelanin formation in Aspergillus fumigatus . Fungal Genet Biol 46:909–918 [View Article][PubMed]
     [Google Scholar]
  59. Weerapana E., Imperiali B. ( 2006). Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16:91R–101R [View Article][PubMed]
     [Google Scholar]
  60. Weidner G., d'Enfert C., Koch A., Mol P. C., Brakhage A. A. ( 1998). Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′-monophosphate decarboxylase. Curr Genet 33:378–385 [View Article][PubMed]
     [Google Scholar]
  61. Xia G., Jin C., Zhou J., Yang S., Zhang S., Jin C. ( 2001). A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. Eur J Biochem 268:4079–4085 [View Article][PubMed]
     [Google Scholar]
  62. Yan Q., Lennarz W. J. ( 2002). Studies on the function of oligosaccharyl transferase subunits. Stt3p is directly involved in the glycosylation process. J Biol Chem 277:47692–47700 [View Article][PubMed]
     [Google Scholar]
  63. Yan A., Lennarz W. J. ( 2005). Unraveling the mechanism of protein N-glycosylation. J Biol Chem 280:3121–3124 [View Article][PubMed]
     [Google Scholar]
  64. Yoshida S., Ohya Y., Nakano A., Anraku Y. ( 1995). STT3, a novel essential gene related to the PKC1/STT1 protein kinase pathway, is involved in protein glycosylation in yeast. Gene 164:167–172 [View Article][PubMed]
     [Google Scholar]
  65. Zhang L., Zhou H., Ouyang H., Li Y., Jin C. ( 2008). Afcwh41 is required for cell wall synthesis, conidiation, and polarity in Aspergillus fumigatus . FEMS Microbiol Lett 289:155–165 [View Article][PubMed]
     [Google Scholar]
  66. Zhang L., Feng D., Fang W., Ouyang H., Luo Y., Du T., Jin C. ( 2009). Comparative proteomic analysis of an Aspergillus fumigatus mutant deficient in glucosidase I (AfCwh41). Microbiology 155:2157–2167 [View Article][PubMed]
     [Google Scholar]
  67. Zhuo H., Hu H., Zhang L., Li R., Ouyang H., Ming J., Jin C. ( 2007). O-Mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) is crucial for cell wall integrity and conidium morphology, especially at an elevated temperature. Eukaryot Cell 6:2260–2268 [View Article][PubMed]
     [Google Scholar]
  68. Zmeili O. S., Soubani A. O. ( 2007). Pulmonary aspergillosis: a clinical update. QJM 100:317–334 [View Article][PubMed]
     [Google Scholar]
  69. Zufferey R., Knauer R., Burda P., Stagljar I., te Heesen S., Lehle L., Aebi M. ( 1995). STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo. EMBO J 14:4949–4960[PubMed]
     [Google Scholar]
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Repression of N-glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Aspergillus fumigatus
Microbiology 157, 1968 (2011); https://doi.org/10.1099/mic.0.047712-0
/content/journal/micro/10.1099/mic.0.047712-0
/content/journal/micro/10.1099/mic.0.047712-0
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