1887

Abstract

, an opportunistic fungal pathogen, infects the human host via inhalation of airborne conidia. Adhesion of fungal conidia, to host cells and extracellular matrix (ECM) components associated with host tissue surfaces, is thought to be the primary step in the pathogenesis and dissemination of infection. To identify novel adhesion proteins (adhesins) of , we screened its proteome using (software program for prediction of adhesins and adhesin-like proteins using neural networks). One of the predicted adhesin-encoding genes with a (probability of being adhesin) value >0.9, the gene encoding extracellular thaumatin domain protein (AfCalA), was cloned and expressed in . Recombinant AfCalAp showed significant binding with laminin and murine lung cells. Anti-AfCalAp antibodies inhibited the binding of AfCalAp to laminin in a dose-dependent manner. Significant binding of anti-AfCalAp antibodies to 2 h swollen conidia suggests the presence of AfCalAp on the conidial surface. transcript was not detectable in resting conidia but was detected in conidia incubated with RPMI 1640 medium in the presence and absence of lung epithelial cell line (A539)-derived ECM. Elevated levels of IgE antibodies specific to AfCalAp were observed in the sera of two out of seven patients with allergic bronchopulmonary aspergillosis. The study confirms the relevance of the bioinformatic approach for predicting fungal adhesins and establishes AfCalAp as a novel laminin-binding protein of .

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2009-06-01
2019-10-13
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References

  1. Annaix, V., Bouchara, J. P., Larcher, G., Chabasse, D. & Tronchin, G. ( 1992; ). Specific binding of human fibrinogen fragment D to Aspergillus fumigatus conidia. Infect Immun 60, 1747–1755.
    [Google Scholar]
  2. Belaish, R., Sharon, H., Levdansky, E., Greenstein, S., Shadkchan, Y. & Osherov, N. ( 2008; ). The Aspergillus nidulans cetA and calA genes are involved in conidial germination and cell wall morphogenesis. Fungal Genet Biol 45, 232–242.[CrossRef]
    [Google Scholar]
  3. Bodey, G. P. & Vartivarian, S. ( 1989; ). Aspergillosis. Eur J Clin Microbiol Infect Dis 8, 413–437.[CrossRef]
    [Google Scholar]
  4. Bouchara, J. P., Sanchez, M., Chevailler, A., Marot-Leblond, A., Lissitzky, J. C., Tronchin, G. & Chabasse, D. ( 1997; ). Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus fumigatus conidia. Infect Immun 65, 2717–2724.
    [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.[CrossRef]
    [Google Scholar]
  6. Brandhorst, T. & Klein, B. ( 2000; ). Cell wall biogenesis of Blastomyces dermatitidis. Evidence for a novel mechanism of cell surface localization of a virulence-associated adhesin via extracellular release and reassociation with cell wall chitin. J Biol Chem 275, 7925–7934.[CrossRef]
    [Google Scholar]
  7. Bromley, I. M. & Donaldson, K. ( 1996; ). Binding of Aspergillus fumigatus spores to lung epithelial cells and basement membrane proteins, relevance to the asthmatic lung. Thorax 51, 1203–1209.[CrossRef]
    [Google Scholar]
  8. Chaturvedi, A. K., Kavishwar, A., Shiva Keshava, G. B. & Shukla, P. K. ( 2005; ). Monoclonal immunoglobulin G1 directed against Aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis. Clin Diagn Lab Immunol 12, 1063–1068.
    [Google Scholar]
  9. Cheng, G., Wozniak, K., Walling, M. A., Fidel, P. L., Jr, Trupin, S. R. & Hoyer, L. L. ( 2005; ). Comparison between Candida albicans agglutinin-like sequence gene expression patterns in human clinical specimens and models of vaginal candidiasis. Infect Immun 73, 1656–1663.[CrossRef]
    [Google Scholar]
  10. Coulot, P., Bouchara, J. P., Renier, G., Annaix, V., Planchenault, C., Tronchin, G. & Chabasse, D. ( 1994; ). Specific interaction of Aspergillus fumigatus with fibrinogen and its role in cell adhesion. Infect Immun 62, 2169–2177.
    [Google Scholar]
  11. Gaur, N. K. & Klotz, S. A. ( 1997; ). Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins. Infect Immun 65, 5289–5294.
    [Google Scholar]
  12. Gil, M. L., Penalver, M. C., Lopez-Ribot, J. L., O'Connor, J. E. & Martinez, J. P. ( 1996; ). Binding of extracellular matrix proteins to Aspergillus fumigatus conidia. Infect Immun 64, 5239–5247.
    [Google Scholar]
  13. Gonzalez, A., Gomez, B. L., Restrepo, A., Hamilton, A. J. & Cano, L. E. ( 2005; ). Recognition of extracellular matrix proteins by Paracoccidioides brasiliensis yeast cells. Med Mycol 43, 637–645.[CrossRef]
    [Google Scholar]
  14. Green, C. B., Cheng, G., Chandra, J., Mukherjee, P., Ghannoum, M. A. & Hoyer, L. L. ( 2004; ). RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms. Microbiology 150, 267–275.[CrossRef]
    [Google Scholar]
  15. Greenstein, S., Shadkchan, Y., Jadoun, J., Sharon, C., Markovich, S. & Osherov, N. ( 2006; ). Analysis of the Aspergillus nidulans thaumatin-like cetA gene and evidence for transcriptional repression of pyr4 expression in the cetA-disrupted strain. Fungal Genet Biol 43, 42–53.[CrossRef]
    [Google Scholar]
  16. Holmes, K. & Fowlkes, B. J. ( 1996; ). Immunofluorescence and cell sorting. In Current Protocols in Immunology, 2nd edn. Edited by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach & W. Strober. New York: Wiley.
  17. Klein, B. S. ( 2000; ). Molecular basis of pathogenicity in Blastomyces dermatitidis: the importance of adhesion. Curr Opin Microbiol 3, 339–343.[CrossRef]
    [Google Scholar]
  18. Koiwa, H., Kato, H., Nakatsu, T., Oda, J., Yamada, Y. & Sato, F. ( 1999; ). Crystal structure of tobacco PR-5d protein at 1.8 Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins. J Mol Biol 286, 1137–1145.[CrossRef]
    [Google Scholar]
  19. Krebitz, M., Wagner, B., Ferreira, F., Peterbauer, C., Campillo, N., Witty, M., Kolarich, D., Steinkellner, H., Scheiner, O. & Breiteneder, H. ( 2003; ). Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein. J Mol Biol 329, 721–730.[CrossRef]
    [Google Scholar]
  20. Levdansky, E., Romano, J., Shadkchan, Y., Sharon, H., Verstrepen, K. J., Fink, G. R. & Osherov, N. ( 2007; ). Coding tandem repeats generate diversity in Aspergillus fumigatus genes. Eukaryot Cell 6, 1380–1391.[CrossRef]
    [Google Scholar]
  21. Lopez-Ribot, J. L. & Chaffin, W. L. ( 1994; ). Binding of the extracellular matrix component entactin to Candida albicans. Infect Immun 62, 4564–4571.
    [Google Scholar]
  22. Masuoka, J., Wu, G., Glee, P. M. & Hazen, K. C. ( 1999; ). Inhibition of Candida albicans attachment to extracellular matrix by antibodies which recognize hydrophobic cell wall proteins. FEMS Immunol Med Microbiol 24, 421–429.[CrossRef]
    [Google Scholar]
  23. Mendes-Giannini, M. J., Andreotti, P. F., Vincenzi, L. R., da Silva, J. L., Lenzi, H. L., Benard, G., Zancope-Oliveira, R., de Matos Guedes, H. L. & Soares, C. P. ( 2006; ). Binding of extracellular matrix proteins to Paracoccidioides brasiliensis. Microbes Infect 8, 1550–1559.[CrossRef]
    [Google Scholar]
  24. Osmond, R. I., Hrmova, M., Fontaine, F., Imberty, A. & Fincher, G. B. ( 2001; ). Binding interactions between barley thaumatin-like proteins and (1,3)-β-d-glucans. Kinetics, specificity, structural analysis and biological implications. Eur J Biochem 268, 4190–4199.[CrossRef]
    [Google Scholar]
  25. Penalver, M. C., O'Connor, J. E., Martinez, J. P. & Gil, M. L. ( 1996; ). Binding of human fibronectin to Aspergillus fumigatus conidia. Infect Immun 64, 1146–1153.
    [Google Scholar]
  26. Rosenberg, M., Patterson, R., Mintzer, R., Cooper, B. J., Roberts, M. & Harris, K. E. ( 1977; ). Clinical and immunologic criteria for the diagnosis of allergic bronchopulmonary aspergillosis. Ann Intern Med 86, 405–414.[CrossRef]
    [Google Scholar]
  27. Sachdeva, G., Kumar, K., Jain, P. & Ramachandran, S. ( 2005; ). SPAAN: a software program for prediction of adhesins and adhesin-like proteins using neural networks. Bioinformatics 21, 483–491.[CrossRef]
    [Google Scholar]
  28. Saxena, S., Madan, T., Muralidhar, K. & Sarma, P. U. ( 2003; ). cDNA cloning, expression and characterization of an allergenic L3 ribosomal protein of Aspergillus fumigatus. Clin Exp Immunol 134, 86–91.[CrossRef]
    [Google Scholar]
  29. Sturtevant, J. E. & Latge, J. P. ( 1992; ). Interactions between conidia of Aspergillus fumigatus and human complement component C3. Infect Immun 60, 1913–1918.
    [Google Scholar]
  30. Thau, N., Monod, M., Crestani, B., Rolland, C., Tronchin, G., Latge, J. P. & Paris, S. ( 1994; ). rodletless mutants of Aspergillus fumigatus. Infect Immun 62, 4380–4388.
    [Google Scholar]
  31. Tronchin, G., Esnault, K., Renier, G., Filmon, R., Chabasse, D. & Bouchara, J. P. ( 1997; ). Expression and identification of a laminin-binding protein in Aspergillus fumigatus conidia. Infect Immun 65, 9–15.
    [Google Scholar]
  32. Trudel, J., Grenier, J., Potvin, C. & Asselin, A. ( 1998; ). Several thaumatin-like proteins bind to β-1,3-glucans. Plant Physiol 118, 1431–1438.[CrossRef]
    [Google Scholar]
  33. Wasylnka, J. A. & Moore, M. M. ( 2000; ). Adhesion of Aspergillus species to extracellular matrix proteins, evidence for involvement of negatively charged carbohydrates on the conidial surface. Infect Immun 68, 3377–3384.[CrossRef]
    [Google Scholar]
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AfCalAp aligned with previously characterized thaumatin domain proteins. (a) Alignment with PR-5d, antifungal protein from , and (b) alignment with thaumatin A, sweet tasting protein from . Residues crucial for antifungal activity have been highlighted in green, whereas those described as being important for sweet taste are highlighted in pink. Yellow highlighting indicates the position of the signal peptide in AfCalAp. Stretches of amino acids shown in orange indicate the position of the thaumatin consensus sequence in AfCalAp as determined using InterProScan. [ PDF file] (33 KB)

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Expression of and is not modulated in swollen conidia, by the presence of A549-derived extracellular matrix. Lane 1, 100 bp DNA ladder (New England Biolabs); lanes 2 and 3, RT-PCR of using RNA isolated from 1 h swollen conidia in the presence and absence of A549-derived ECM, respectively; lanes 4 and 5, RT-PCR of using RNA isolated from 1 h swollen conidia in the presence and absence of A549-derived ECM, respectively. [ PDF file] (37 KB)

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