Blastomyces dermatitidis: Chitinase Homology Model, in Silico Docking, and Inhibition Assay

Amanda J. Searle, Vern Winston, Gene M. Scalarone

Department of Biological Science, Idaho State University, Idaho, USA.

DOI: 10.4236/ojmm.2012.21001                

Abstract

Blastomyces dermatitidis is a thermally dimorphic fungus that causes the disease blastomycosis. Currently there are a limited number of effective treatments, many of which have harsh side effects. Chitin, a component of the fungal cell wall is often broken down and recycled for cell wall remodeling and growth. Chitinase is the digestive enzyme capable of chitin hydrolysis. By inhibiting the chitinase we predicted that cells wouldn’t be able to divide and multiply normally, thereby leading to possible anti-fungal treatments. For this study we modeled the structure of B. dermatitidis chitinase, using homology modeling. By predicting a three-dimensional structure we were able to do additional analyses of the active site of the chitinase and predict the binding of a possible small molecule, acetazolamide, in silico. This binding allowed us to predict that this molecule might be capable of inhibiting the chitinase of B. dermatitidis. This inhibition was tested in vivo. No difference in the growth curves of the test and control organisms was observed, however there was a difference within the cell walls of the yeast cells. The cell walls appeared thicker with additional differences in cell wall orderly growth. These changes are consistent with changes that may occur as B. dermatitidis chitinases are inhibited.

Keywords

Blastomyces dermatitidis; Chitinase; Homology Modeling; Acetazolamide; Inhibition

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	D. J. Baumgardner and D. P. Paretsky, “The in Vitro Isolation of Blastomyces dermatitidis from a Woodpile in North Central Wisconsin,” Medical Mycology, Vol. 37, No. 3, 1999, pp. 163-168. doi:10.1080/j.1365-280X.1999.00214.x
[2]	A. F. DiSalvo, “Topley and Wilson’s Microbiology and Microbial Infections,” 9th Edition, Arnold Publishers, London, 1998.
[3]	B. S. Klein, J. M. Vergeront, R. J. Weeks, U. N. Kumar, G. Mathai, B. Varkey, L. Kaufman, R. W. Bradsher, J. F. Stoebig and J. P. Davis, “Isolation of Blastomyces dermatitidis in Soil Associated with a Large Out-Break of Blastomycosis in Wisconsin,” New England Journal of Medicine, Vol. 314, No. 9, 1986, pp. 529-534. doi:10.1056/NEJM198602273140901
[4]	D. J. Baumgardner and D. P. Paretsky, “Identification of Blastomyces dermatitidis in the Stool of a Dog with Acute Pulmonary Blastomycosis,” Journal of Medical and Veterinary Mycology, Vol. 35, No. 6, 1997, pp. 419-421. doi:10.1080/02681219780001521
[5]	R. W. Bradsher, “Clinical Features of Blastomycosis,” Seminars in Respiratory Infections, Vol. 12, No. 3, 1997, pp. 229-234.
[6]	J. F. Shurley and G. M. Scalarone, “Isoelectric Focusing and ELISA Evaluation of a Blastomyces dermatitidis Human Isolate,” Mycopathologia, Vol. 164, No. 2, 2007, pp. 73-76. doi:10.1007/s11046-007-9033-8
[7]	M. A. Pfaller and D. J. Diekema, “Epidemiology of Invasive Mycoses in North America,” Critical Reviews in Microbiology, Vol. 36, No. 1, 2010, pp. 1-53. doi:10.3109/10408410903241444
[8]	M. C. Dobre, W. R. K. Smoker and P. Kirby, “A Case of Solitary Blastomyces dermatitidis Meningitis,” Clinical Neurology and Neurosurgery, Vol. 113, No. 8, 2011, pp. 665-667. doi:10.1016/j.clineuro.2011.03.012
[9]	B .S. Klein, R. A. Squires, J. K. Lloyd, D. R. Ruge and A. M. Legendre, “Canine Antibody Response to Blastomyces dermatitidis WI-1 Antigen,” American Journal of Veterinary Research, Vol. 61, No. 5, 2000, pp. 554-558. doi:10.2460/ajvr.2000.61.554
[10]	N. M. Rocco, J. C. Carmen and B. S. Klein, “Blastomyces dermatitidis Yeast Cells Inhibit Nitric Oxide Production by Alveolar Macrophage Inducible Nitric Oxide Synthase,” Infection and Immunity, Vol. 79, No. 6, 2011, pp. 2385-2395. doi:10.1128/IAI.01249-10
[11]	J. E. Cutler, G. S. Deepe Jr and B. S. Klein, “Advances in Combating Fungal Diseases: Vaccines on the Threshold,” Nature Reviews Microbiology, Vol. 5, No. 1, 2007, pp. 13- 28. doi:10.1038/nrmicro1537
[12]	J. A. McKinnell and P. G. Pappas, “Blastomycosis: New Insights into Diagnosis, Prevention, and Treatment,” Clinics in Chest Medicine, Vol. 30, No. 2, 2009, pp. 227-239. doi:10.1016/j.ccm.2009.02.003
[13]	National Center for Biotechnology Information, “Blastomycosis,” US National Library of Medicine, 2010, http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001163/
[14]	K. E. Brick and W. A. Agger, “Successful Treatment of Brainstem Blastomycosis with Fluconazole,” Clinical Medicine & Research, Vol. 9, 2011, pp. 1-10.
[15]	H. V. Bossche, “Biochemical Targets for Antifungal Azole Derivatives: Hypothesis on the Mode of Action,” Current Topics in Medical Mycology, Vol. 1, 1985, pp. 313-351. doi:10.1007/978-1-4613-9547-8_12
[16]	J. W. M. van der Linden, R. R. Jansen, D. Bresters, C. E. Visser, S. E. Geerlings, E. J. Kuijper, W. J. G. Melchers and P. E. Verweij, “Azole-Resistant Central Nervous System Aspergillosis,” Clinical Infectious Diseases, Vol. 48, No. 8, 2009, pp. 1111-1113. doi:10.1086/597465
[17]	E. Snelders, H. A. van der Lee, J. Kuipers, A. J. Riji, J. Varga, R. A. Samson, E. Mellado, A. R. Donders, W. J. Melchers and P. E. Verweij, “Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism,” PLoS Medicine, Vol. 5, No. 11, 2008, p. 219. doi:10.1371/journal.pmed.0050219
[18]	G. W. Hudler, “Magical Mushrooms, Mischievous Molds,” Princeton University Press, Princeton, 1998.
[19]	A. W. Schuettelkopf, L. Gros, D. E Blair, J. A. Frearson, D. M. F. van Aalten and I. Gilbert, “Acetazolamide-Based Fungal Chitinase Inhibitors,” Bioorganic & Medicinal Chemistry, Vol. 18, No. 23, 2010, pp. 8334-8340. doi:10.1016/j.bmc.2010.09.062
[20]	M. J. Kuranda and P. W. Robbins, “Chitinase Is Required for Cell Separation during Growth of Saccharomyces cerevisiae,” Journal of Biological Chemistry, Vol. 266, No. 29, 1991, pp. 19758-19767.
[21]	B. L. Cantarel, P. M. Coutinho, C. Rancurel, T. Bernard, V. Lombard and B. Henrissat, “The Carbohydrate-Active EnZymes Database (CRZy): An Expert Resource for Glycogenomics,” Nucleic Acids Research, Vol. 37, No. 1, 2009, pp. 233-238. doi:10.1093/nar/gkn663
[22]	T. Roemer, L. G. Vallier and M. Snyder, “Selection of Polarized Growth Sites in Yeast,” Trends in Cell Biology, Vol. 6, No. 11, 1996, pp. 434-441. doi:10.1016/S0962-8924(96)10039-8
[23]	L. Ni and M. Snyder, “A Genomic Study of the Bipolar Bud Site Selection Pattern in Saccharomyces cerevisiae,” Molecular Biology of the Cell, Vol. 12, 2001, pp. 2147-2170.
[24]	J. Xiong, “Essential Bioinformatics,” Cambridge University Press, Cambridge, 2006
[25]	A. Sali and T. L. Blundell, “Comparative Protein Modeling by Satisfaction of Spatial Restraints,” Journal of Molecular Biology, Vol. 234, No. 3, 1993, pp. 779-815. doi:10.1006/jmbi.1993.1626
[26]	Howard Hughs Medical Institute, “Hmmer: Biosequence Analysis Using Profile Hidden Markov Models,” 2011. http://hmmer.janelia.org/
[27]	Bechman Institute for Advance Science and Technology, “Theoretical and Computational Biophysics Group: VMD,” 2011. http://www.ks.uiuc.edu/Research/vmd/
[28]	Jmol, “Jmol: An Open-Source Java Viewer for Chemical Structures in 3D,” http://www.jmol.org/
[29]	G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew and A. J. Olson, “Automated Docking Using a Lamarckian Genetic Algorithm and Empirical Binding Free Energy Function,” Journal of Computational Chemistry, Vol. 19, No. 14, 1998, pp. 1639-1662. dio:10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B
[30]	A. W. Schüttelkopf and D. M. F. van Aalten, “PRODRG: A Tool for High-Throughput Crystallography of Protein-Ligand Complexes,” Acta Crystallographica, Vol. 60, Pt. 8, 2004, pp. 1355-1363.
[31]	S. M. Johnson, and G. M. Scalarone, “Preparation and ELISA Evaluation of Blastomyces dermatitidis Yeast Phase Lysate Antigens,” Diagnostic Microbiology and Infectious Disease, Vol. 11, No. 2, 1988, pp. 81-86. dio:10.1016/0732-8893(88)90076-4
[32]	Broad Institute, “Blastomyces dermatitidis Database,” http://www.broadinstitute.org/annotation/genome/blastomyces_dermatitidis/MultiHome.html
[33]	G. Davies and B. Henrissat, “Structure and Mechanisms of Glycosyl Hydrolases,” Structure, Vol. 3, No. 9, 1995, pp. 853- 859. dio:10.1016/S0969-2126(01)00220-9
[34]	A. Perrakis, I. Tews, Z. Dauter, A. B. Oppenheim, I. Chet, K. S. Wilson and C. E. Vorgias, “Crystal Structure of a Bacterial Chitinase at 2.3 A Resolution,” Structure, Vol. 2, No. 12, 1994, pp. 1169-1180. dio:10.1016/S0969-2126(94)00119-7
[35]	C. Songsiriritthiqul, S. Pantooms, A. H. Aquda, R. C. Robinson and W. Suginta, “Crystal Structures of Vibrio Harveyi Chitinase A Complexed with Chitooligo-saccharides: Implications for the Catalytic Mechanism,” Journal of Structural Biology, Vol. 162, No. 3, 2008, pp. 491-499. dio:10.1016/j.jsb.2008.03.008