Echinocandins are a new generation of novel antifungal agent that inhibit cell wall β(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca2+-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.
References
[1]
Klis FM, De Groot P, Hellingwerf K (2001) Molecular organisation of the cell wall of Candida albicans. Med Mycol 39: Suppl 11–8.
[2]
Munro CA, Gow NA (2001) Chitin synthesis in human pathogenic fungi. Med Mycol 39: Suppl 141–53.
[3]
De Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong L, et al. (2004) Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell 3: 955–965.
[4]
Roncero C (2002) The genetic complexity of chitin synthesis in fungi. Curr Genet 41: 367–378.
[5]
Heinisch JJ, Lorberg A, Schmitz HP, Jacoby JJ (1999) The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol Microbiol 32: 671–680.
[6]
Douglas CM (2001) Fungal beta(1,3)-D-glucan synthesis. Med Mycol 39: Suppl 155–66.
[7]
Popolo L, Gualtieri T, Ragni E (2001) The yeast cell-wall salvage pathway. Med Mycol 39: Suppl 1111–121.
[8]
Levin DE (2005) Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69: 262–291.
[9]
Douglas CM, D'Ippolito JA, Shei GJ, Meinz M, Onishi J, et al. (1997) Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother 41: 2471–2479.
[10]
Bowman JC, Hicks PS, Kurtz MB, Rosen H, Schmatz DM, et al. (2002) The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 46: 3001–3012.
Pfaller MA, Messer SA, Boyken L, Rice C, Tendolkar S, et al. (2003) Caspofungin activity against clinical isolates of fluconazole-resistant Candida. J Clin Microbiol 41: 5729–5731.
[13]
Gardiner RE, Souteropoulos P, Park S, Perlin DS (2005) Characterization of Aspergillus fumigatus mutants with reduced susceptibility to caspofungin. Med Mycol 43: Suppl 1299–305.
[14]
Park S, Kelly R, Kahn JN, Robles J, Hsu MJ, et al. (2005) Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob Agents Chemother 49: 3264–3273.
[15]
Balashov SV, Park S, Perlin DS (2006) Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by profiling mutations in FKS1. Antimicrob Agents Chemother 50: 2058–2063.
[16]
Katiyar S, Pfaller M, Edlind T (2006) Candida albicans and Candida glabrata clinical isolates exhibiting reduced echinocandin susceptibility. Antimicrob Agents Chemoth 50: 2892–2894.
[17]
Kahn JN, Garcia-Effron G, Hsu MJ, Park S, Marr KA, et al. (2007) Acquired echinocandin resistance in a Candida krusei isolate due to modification of glucan synthase. Antimicrob Agents Chemother 51: 1876–1878.
[18]
Ha YS, Covert SF, Momany M (2006) FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani. Eukaryot Cell 5: 1036–1042.
[19]
Garcia-Rodriguez LJ, Trilla JA, Castro C, Valdivieso MH, Duran A, et al. (2000) Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae. Febs Letters 478: 84–88.
[20]
Markovich S, Yekutiel A, Shalit I, Shadkchan Y, Osherov N (2004) Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae. Antimicrob Agents Chemother 48: 3871–3876.
[21]
Osmond BC, Specht CA, Robbins PW (1999) Chitin synthase III: Synthetic lethal mutants and “stress related” chitin synthesis that bypasses the CSD3/CHS6 localization pathway. Proc Nat Acad Sci USA 96: 11206–11210.
[22]
Lesage G, Shapiro J, Specht CA, Sdicu AM, Menard P, et al. (2005) An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae. BMC Genet 6: 8.
[23]
Liu TT, Lee RE, Barker KS, Lee RE, Wei L, et al. (2005) Genome-wide expression profiling of the response to azole, polyene, echinocandin, and pyrimidine antifungal agents in Candida albicans. Antimicrob Agents Chemother 49: 2226–2236.
[24]
Neuhof T, Seibold M, Thewes S, Laue M, Han CO, et al. (2006) Comparison of susceptibility and transcription profile of the new antifungal hassallidin A with caspofungin. Biochem Biophys Res Commun 349: 740–749.
[25]
Reinoso-Martin C, Schuller C, Schuetzer-Muehlbauer M, Kuchler K (2003) The yeast protein kinase C cell integrity pathway mediates tolerance to the antifungal drug caspofungin through activation of Slt2p mitogen-activated protein kinase signaling. Eukaryot Cell 2: 1200–1210.
[26]
Kraus PR, Fox DS, Cox GM, Heitman J (2003) The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function. Mol Microbiol 48: 1377–1387.
[27]
Wiederhold NP, Kontoyiannis DP, Prince RA, Lewis RE (2005) Attenuation of the activity of caspofungin at high concentrations against Candida albicans: possible role of cell wall integrity and calcineurin pathways. Antimicrob Agents Chemother 49: 5146–5148.
[28]
Garcia R, Bermejo C, Grau C, Perez R, Rodriguez-Pena JM, et al. (2004) The global transcriptional response to transient cell wall damage in Saccharomyces cerevisiae and its regulation by the cell integrity signaling pathway. J Biol Chem 279: 15183–15195.
[29]
Mazur P, Morin N, Baginsky W, el Sherbeini M, Clemas JA, et al. (1995) Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase. Mol Cell Biol 15: 5671–5681.
[30]
Zhao C, Jung US, Garrett-Engele P, Roe T, Cyert MS, et al. (1998) Temperature-induced expression of yeast FKS2 is under the dual control of protein kinase C and calcineurin. Mol Cell Biol 18: 1013–1022.
[31]
Munro CA, Selvaggini S, de Bruijn I, Walker L, Lenardon MD, et al. (2007) The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans. Mol Microbiol 63: 1399–1413.
[32]
Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J (2003) Calcineurin A of Candida albicans:involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 48: 959–976.
[33]
Del Poeta M, Cruz MC, Cardenas ME, Perfect JR, Heitman J (2000) Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans. Antimicrob Agents Chemother 44: 739–746.
[34]
Kontoyiannis DP, Lewis RE, Osherov N, Albert ND, May GS (2003) Combination of caspofungin with inhibitors of the calcineurin pathway attenuates growth in vitro in Aspergillus species. J Antimicrob Chemother 51: 313–316.
[35]
Steinbach WJ, Cramer RA Jr, Perfect BZ, Henn C, Nielsen K, et al. (2007) Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus. Antimicrob Agents Chemother 51: 2979–2981.
[36]
Munro CA, Winter K, Buchan A, Henry K, Becker JM, et al. (2001) Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity. Mol Microbiol 39: 1414–1426.
[37]
Gow NAR, Robbins PW, Lester JW, Brown AJP, Fonzi WA, et al. (1994) A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans. Proc Natl Acad Sci USA 91: 6216–6220.
[38]
Munro CA, Schofield DA, Gooday GW, Gow NAR (1998) Regulation of chitin synthesis during dimorphic growth of Candida albicans. Microbiology 144: 391–401.
[39]
Munro CA, Whitton RK, Hughes HB, Rella M, Selvaggini S, et al. (2003) CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth. Fungal Genet Biol 40: 146–158.
[40]
Bulawa CE, Miller DW, Henry LK, Becker JM (1995) Attenuated virulence of chitin-deficient mutants of Candida albicans. Proc Natl Acad Sci USA 92: 10570–10574.
[41]
Lenardon MD, Whitton RK, Munro CA, Marshall D, Gow NAR (2007) Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in Candida albicans. Mol Microbiol. doi: 10.1111/j.1365-2958.2007.05990.x.
[42]
Bulik DA, Olczak M, Lucero HA, Osmond BC, Robbins PW, et al. (2003) Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryot Cell 2: 886–900.
[43]
Sudoh M, Yamazaki T, Masubuchi K, Taniguchi M, Shimma N, et al. (2000) Identification of a novel inhibitor specific to the fungal chitin synthase; inhibition of chitin synthase 1 arrests the cell growth, but that of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans. J Biol Chem 275: 32901–32905.
[44]
Rogers TR, Johnson EM, Munro CA (2007) Echinocandin antifungal drug resistance. J Invasive Fung Infect 1: 99–105.
[45]
Hernandez S, Lopez-Ribot JL, Najvar LK, McCarthy DI, Bocanegra R, et al. (2004) Caspofungin resistance in Candida albicans: correlating clinical outcome with laboratory susceptibility testing of three isogenic isolates serially obtained from a patient with progressive Candida esophagitis. Antimicrob Agents Chemother 48: 1382–1383.
[46]
Prabhu RM, Orenstein R (2004) Failure of caspofungin to treat brain abscesses secondary to Candida albicans prosthetic valve endocarditis. Clin Infect Dis 39: 1253–1254.
[47]
Laverdiere M, Lalonde RG, Baril JG, Sheppard DC, Park S, et al. (2006) Progressive loss of echinocandin activity following prolonged use for treatment of Candida albicans oesophagitis. J Antimicrob Chemother 57: 705–708.
[48]
Miller CD, Lomaestro BW, Park S, Perlin DS (2006) Progressive esophagitis caused by Candida albicans with reduced susceptibility to caspofungin. Pharmacotherapy 26: 877–880.
[49]
Krogh-Madsen M, Arendrup MC, Heslet L, Knudsen JD (2006) Amphotericin B and caspofungin resistance in Candida glabrata isolates recovered from a critically ill patient. Clin Infect Dis 42: 938–944.
[50]
Moudgal V, Little T, Boikov D, Vazquez JA (2005) Multiechinocandin- and multiazole-resistant Candida parapsilosis isolates serially obtained during therapy for prosthetic valve endocarditis. Antimicrob Agents Chemother 49: 767–769.
[51]
Cheung C, Guo Y, Gialanella P, Feldmesser M (2006) Development of candidemia on caspofungin therapy: a case report. Infection 34: 345–348.
[52]
Pelletier R, Alarie I, Lagace R, Walsh TJ (2005) Emergence of disseminated candidiasis caused by Candida krusei during treatment with caspofungin: case report and review of literature. Med Mycol 43: 559–564.
[53]
Hakki M, Staab JF, Marr KA (2006) Emergence of a Candida krusei isolate with reduced susceptibility to caspofungin during therapy. Antimicrob Agents Chemother 50: 2522–2524.
[54]
Stevens DA, Ichinomiya M, Koshi Y, Horiuchi H (2006) Escape of Candida from caspofungin inhibition at concentrations above the MIC (paradoxical effect) accomplished by increased cell wall chitin; evidence for beta-1,6-glucan synthesis inhibition by caspofungin. Antimicrob Agents Chemother 50: 3160–3161.
[55]
National Committee for Clinical Laboratory Standards (2002) Reference method for broth dilution testing of yeasts. Approved standard, second edition, M27-A2. Wayne, PA: National Committee for Clinical Laboratory Standards.
[56]
Uhl MA, Johnson AD (2001) Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans. Microbiology 147: 1189–1195.
[57]
Wilson RB, Davis D, Mitchell AP (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181: 1868–1874.
[58]
Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134: 717–728.
[59]
Mio T, Yabe T, Sudoh M, Satoh Y, Nakajima T, et al. (1996) Role of three chitin synthase genes in the growth of Candida albicans. J Bacteriol 178: 2416–2419.
[60]
Navarro-Garcia F, Sanchez M, Pla J, Nombela C (1995) Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity. Mol Cell Biol 15: 2197–2206.
[61]
Alonso-Monge R, Navarro-Garcia F, Molero G, Diez-Orejas R, Gustin M, et al. (1999) Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 181: 3058–3068.
