Login Register Cart 0
Generic placeholder image

Anti-Infective Agents

Editor-in-Chief

ISSN (Print): 2211-3525
ISSN (Online): 2211-3533

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Characterization of Therapeutic Targets from Aspergillus fumigates in response to Adjunctive Combination Therapy (Ketoconazole with EDTA)

Author(s): Sonam Ruhil, Vikash Kumar*, Monika Malik, Meenakshi Balhara and Anil Kumar Chhillar*

Volume 20, Issue 1, 2022

Published on: 24 February, 2021

Article ID: e171221191761 Pages: 18

DOI: 10.2174/2211352519666210224095411

Price: $65

Abstract

Background: The Kingdom (Fungi) comprises numerous species that are associated with numerous fungal diseases. Moreover, the fungal resistance, stagnation in the development of antifungal agents and unacceptably high mortality rate associated with some resistant fungus indicate that alternative therapeutic options should be considered.

Objective: The objective of this study was to find out new therapeutic targets of A.fumigatus in response to adjunctive combination, i.e., Ketoconazole (KTZ) plus EDTA.

Methods: A.fumigatus was cultured in the absence and presence of a sublethal dose (MIC 50) of EDTA, KTZ and a combination of KTZ plus EDTA. The cytosolic proteins were extracted by mechanical grinding of fungal cells. The protein profile was studied by using a proteomic approach and the identification of protein was done by MALDI-TOF/MS. The morphological effect of the combination on A. fumigatus was studied by Scanning Electron Microscopy (SEM) and toxic effect on erythrocytes by haemolytic assay.

Results: The combination of KTZ with EDTA was non-toxic up to 500 μg/ml by MTT assay. It inhibits the expression of the following proteins: Glutamatedehydrogenase, Phenyl alanyl t-RNA synthetase POD G, CaO19-5601, AN6454.2 (Conserved domain; MFS (Major Facilitator Superfamily), serine/threonine-protein kinase and dipeptidyl peptidase (identified by peptide mass fingerprinting). Some of these proteins are involved in hyphal development. Morphological defects on the development of fungus (like disrupted hyphal tips, phialide) were observed.

Conclusion: These targets can be used for novel drug development as some of them are involved in fungal virulence, and adjunctive combination therapy can be an optimistic approach.

Keywords: A. fumigatus, adjunctive combination, conidiophore development, hyphae morphology, SEM.

Graphical Abstract

[1]
Ramana, K.V.; Sabitha, K.; Venkata, B.P.; Sharada, C.H.V.; Ratna, R.; Ratna, M.; Sanjeev, D.R. Invasive Fungal Infections: A Comprehensive Review. American Journal of Infectious Diseases and Microbiology, 2013, 4, 64-69.
[http://dx.doi.org/10.12691/ajidm-1-4-2]
[2]
Ascioglu, S.; Rex, J.H.; de Pauw, B.; Bennett, J.E.; Bille, J.; Crokaert, F.; Denning, D.W.; Donnelly, J.P.; Edwards, J.E.; Erjavec, Z.; Fiere, D.; Lortholary, O.; Maertens, J.; Meis, J.F.; Patterson, T.F.; Ritter, J.; Selleslag, D.; Shah, P.M.; Stevens, D.A.; Walsh, T.J. Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious Diseases. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin. Infect. Dis., 2002, 34(1), 7-14.
[http://dx.doi.org/10.1086/323335] [PMID: 11731939]
[3]
Wang, H.; Ding, Y.; Li, X.; Yang, L.; Zhang, W.; Kang, W. Fatal aspergillosis in a patient with SARS who was treated with corticosteroids. N. Engl. J. Med., 2003, 349(5), 507-508.
[http://dx.doi.org/10.1056/NEJM200307313490519] [PMID: 12890854]
[4]
Pfeiffer, C.D.; Fine, J.P.; Safdar, N. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin. Infect. Dis., 2006, 42(10), 1417-1427.
[http://dx.doi.org/10.1086/503427] [PMID: 16619154]
[5]
Siddiqui, A.A.; Shah, A.A.; Bashir, S.H. Craniocerebral aspergillosis of sinonasal origin in immunocompetent patients: clinical spectrum and outcome in 25 cases. Neurosurgery, 2004, 55(3), 602-611.
[http://dx.doi.org/10.1227/01.NEU.0000134597.94269.48] [PMID: 15335427]
[6]
Odds, F.C.; Brown, A.J.; Gow, N.A. Antifungal agents: mechanisms of action. Trends Microbiol., 2003, 11(6), 272-279.
[http://dx.doi.org/10.1016/S0966-842X(03)00117-3] [PMID: 12823944]
[7]
Ruhil, S.; Balhara, M.; Dhankhar, S.; Kumar, M.; Kumar, V.; Chhillar, A.K. Advancement in infection control of opportunistic pathogen (Aspergillus spp.): adjunctive agents. Curr. Pharm. Biotechnol., 2013, 14(2), 226-232.
[PMID: 23360263]
[8]
Chhillar, A.K.; Arya, P.; Mukherjee, C.; Kumar, P.; Yadav, Y.; Sharma, A.K.; Yadav, V.; Gupta, J.; Dabur, R.; Jha, H.N.; Watterson, A.C.; Parmar, V.S.; Prasad, A.K.; Sharma, G.L. Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: novel compounds against aspergillosis. Bioorg. Med. Chem., 2006, 14(4), 973-981.
[http://dx.doi.org/10.1016/j.bmc.2005.09.014] [PMID: 16214352]
[9]
Chhillar, A.K.; Yadav, V.; Kumar, A.; Kumar, M.; Parmar, V.S.; Prasad, A.; Sharma, G.L. Differential expression of Aspergillus fumigatus protein in response to treatment with a novel antifungal compound, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate. Mycoses, 2009, 52(3), 223-227.
[http://dx.doi.org/10.1111/j.1439-0507.2008.01563.x] [PMID: 18793265]
[10]
Carberry, S.; Neville, C.M.; Kavanagh, K.A.; Doyle, S. Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity. Biochem. Biophys. Res. Commun., 2006, 341(4), 1096-1104.
[http://dx.doi.org/10.1016/j.bbrc.2006.01.078] [PMID: 16455047]
[11]
Laemmli, U.K.; Favre, M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol., 1973, 80(4), 575-599.
[http://dx.doi.org/10.1016/0022-2836(73)90198-8] [PMID: 4204102]
[12]
Blum, H.; Beier, H.; Gross, H.J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 1987, 8, 93-99.
[http://dx.doi.org/10.1002/elps.1150080203]
[13]
Ren, Q.; Chen, K.; Paulsen, I.T. TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels. Nucleic Acids Res., 2007, 35(Database issue), D274-D279.
[http://dx.doi.org/10.1093/nar/gkl925] [PMID: 17135193]
[14]
Moran, G.P.; Sanglard, D.; Donnelly, S.M.; Shanley, D.B.; Sullivan, D.J.; Coleman, D.C. Identification and expression of multidrug transporters responsible for fluconazole resistance in Candida dubliniensis. Antimicrob. Agents Chemother., 1998, 42(7), 1819-1830.
[http://dx.doi.org/10.1128/AAC.42.7.1819] [PMID: 9661028]
[15]
Goldway, M.; Teff, D.; Schmidt, R.; Oppenheim, A.B.; Koltin, Y. Multidrug resistance in Candida albicans: disruption of the BENr gene. Antimicrob. Agents Chemother., 1995, 39(2), 422-426.
[http://dx.doi.org/10.1128/AAC.39.2.422] [PMID: 7726508]
[16]
Pasrija, R.; Banerjee, D.; Prasad, R. Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport. Eukaryot. Cell, 2007, 6(3), 443-453.
[http://dx.doi.org/10.1128/EC.00315-06] [PMID: 17209122]
[17]
Sengupta, M.; Datta, A. Two membrane proteins located in the Nag regulon of Candida albicans confer multidrug resistance. Biochem. Biophys. Res. Commun., 2003, 301(4), 1099-1108.
[http://dx.doi.org/10.1016/S0006-291X(03)00094-9] [PMID: 12589826]
[18]
Park, G.; Servin, J.A.; Turner, G.E.; Altamirano, L.; Colot, H.V.; Collopy, P.; Litvinkova, L.; Li, L.; Jones, C.A.; Diala, F.G.; Dunlap, J.C.; Borkovich, K.A. Global analysis of serine-threonine protein kinase genes in Neurospora crassa. Eukaryot. Cell, 2011, 10(11), 1553-1564.
[http://dx.doi.org/10.1128/EC.05140-11] [PMID: 21965514]
[19]
Harris, S.D. Septum formation in Aspergillus nidulans. Curr. Opin. Microbiol., 2001, 4(6), 736-739.
[http://dx.doi.org/10.1016/S1369-5274(01)00276-4] [PMID: 11731327]
[20]
Colabardini, A.C.; Brown, N.A.; Savoldi, M.; Goldman, M.H.S.; Goldman, G.H. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK. PLoS One, 2013, 8(3)e57630
[http://dx.doi.org/10.1371/journal.pone.0057630] [PMID: 23472095]
[21]
Warnecke, D.; Heinz, E. Recently discovered functions of glucosylceramides in plants and fungi. Cell. Mol. Life Sci., 2003, 60(5), 919-941.
[http://dx.doi.org/10.1007/s00018-003-2243-4] [PMID: 12827281]
[22]
Luberto, C.; Toffaletti, D.L.; Wills, E.A.; Tucker, S.C.; Casadevall, J.R.; Perfect, Y.A. Hannun, and M. Del Poeta. Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of Cryptococcus neoformans. Genes Dev., 2001, 15, 201-212.
[http://dx.doi.org/10.1101/gad.856001] [PMID: 11157776]
[23]
Jimenez-Lucho, V.; Ginsburg, V.; Krivan, H.C. Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipidlactosylceramide, a possible adhesionreceptor for yeasts. Infect. Immun., 1990, 58, 2085-2090.
[http://dx.doi.org/10.1128/IAI.58.7.2085-2090.1990] [PMID: 2194958]
[24]
Heidler, S.A.; Radding, J.A. The AUR1 gene in Saccharomyces cerevisiae encodes dominant resistance to the antifungal agent aureobasidin A (LY295337). Antimicrob. Agents Chemother., 1995, 39(12), 2765-2769.
[http://dx.doi.org/10.1128/AAC.39.12.2765] [PMID: 8593016]
[25]
Heung, L.J.; Luberto, C.; Del Poeta, M. Role of sphingolipids in microbial pathogenesis. Infect. Immun., 2006, 74(1), 28-39.
[http://dx.doi.org/10.1128/IAI.74.1.28-39.2006] [PMID: 16368954]
[26]
Drubin, D.G.; Nelson, W.J. Origins of cell polarity. Cell, 1996, 84(3), 335-344.
[http://dx.doi.org/10.1016/S0092-8674(00)81278-7] [PMID: 8608587]
[27]
Harris, S.D.; Hofmann, A.F.; Tedford, H.W.; Lee, M.P. Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans. Genetics, 1999, 151(3), 1015-1025.
[PMID: 10049919]
[28]
Osherov, N.; Mathew, J.; May, G.S. Polarity-defective mutants of Aspergillus nidulans. Fungal Genet. Biol., 2000, 31(3), 181-188.
[http://dx.doi.org/10.1006/fgbi.2000.1236] [PMID: 11273680]
[29]
Willger, S.D.; Grahl, N.; Cramer, R.A., Jr Aspergillus fumigatus metabolism: clues to mechanisms of in vivo fungal growth and virulence. Med. Mycol., 2009, 47(Suppl. 1), S72-S79.
[http://dx.doi.org/10.1080/13693780802455313] [PMID: 19253141]
[30]
Rementeria, A.; López-Molina, N.; Ludwig, A.; Vivanco, A.B.; Bikandi, J.; Pontón, J.; Garaizar, J. Genes and molecules involved in Aspergillus fumigatus virulence. Rev. Iberoam. Micol., 2005, 22(1), 1-23.
[http://dx.doi.org/10.1016/S1130-1406(05)70001-2] [PMID: 15813678]
[31]
Kogan, T.V.; Jadoun, J.; Mittelman, L.; Hirschberg, K.; Osherov, N. Involvement of secreted Aspergillus fumigatus proteases in disruption of the actin fiber cytoskeleton and loss of focal adhesion sites in infected A549 lung pneumocytes. J. Infect. Dis., 2004, 189(11), 1965-1973.
[http://dx.doi.org/10.1086/420850] [PMID: 15143461]
[32]
Dunn-Coleman, N.S.; Nassiff, M.D.; Garrett, R.H. Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa. Curr. Genet., 1984, 8(6), 423-427.
[http://dx.doi.org/10.1007/BF00433908] [PMID: 24177912]
[33]
Perrine, K.G.; Marzluf, G.A. Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa. Curr. Genet., 1986, 10(9), 677-684.
[http://dx.doi.org/10.1007/BF00410916] [PMID: 2965995]
[34]
Dantzig, A.H.; Wiegmann, F.L., Jr; Nason, A. Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa. J. Bacteriol., 1979, 137(3), 1333-1339.
[http://dx.doi.org/10.1128/JB.137.3.1333-1339.1979] [PMID: 35517]
[35]
Bullwinkle, T.J.; Ibba, M. Emergence and evolution. Top. Curr. Chem., 2014, 344, 43-87.
[http://dx.doi.org/10.1007/128_2013_423] [PMID: 23478877]

Rights & Permissions Print Cite
Article Metrics
22
1
© 2024 Bentham Science Publishers | Privacy Policy