Our findings show that the phenomena described can apply to the in vivo situation, i.e. during azole maintenance therapy in the host, but transcriptional analyses using different growth conditions of H99 cells, mimicking stress
conditions encountered during a human meningeal infection, may reveal new fields to pursue for anticryptococcal therapy. Acknowledgements This work was supported by grants from the Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Lazzaro Spallanzani (Strategic Research Program 2006) to GF, from the Università Cattolica del S. Cuore (Fondi Ateneo Linea D1-2009) to MS, and from the Swiss Research National Foundation 31003A_127378 to DS. Electronic supplementary material Additional file 1: Table A1 KU-57788 in vivo Primers and fluorescent probes used in qRT-PCR. Contains Table A1 selleckchem showing the qRT-PCR primers and probes. (DOC 58 KB)
Additional file 2: Figure A1 Cell wall integrity assays with H99 C. neoformans cells left untreated (H99) or exposed to FLC (H99F) at a sub-MIC concentration of 10 mg/l for 90 min at 37°C. Cells were grown at the same temperature for 48 h on YEPD supplemented with calcofluor white (CFW), Congo red, sodium dodecyl sulphate (SDS) and caffeine. Aliquots of cells were applied onto the agar surface with 10-fold serial dilutions. Contains Figure A1 showing the results of cell wall inhibitors susceptibility assays for H99 cells pre-treated with FLC at 37°C. (DOC 122 KB) Additional Proteases inhibitor file 3: Figure A2 Survival of C. neoformans after oxidative treatment. Exponentially growing cells were left untreated (H99) or exposed to 10 mg/l FLC (H99F) for 90 min at 37°C and then challenged with 20 mM H2O2 for 2 h. Aliquots were harvested at given time points and cell viability performed as described in Methods. Plotted values are means of three experiments. Contains Figure A2 showing the results of H2O2 susceptibility PLEK2 assays for H99 cells pre-treated with FLC at 37°C. (DOC 262 KB)
References 1. Perfect JR, Casadevall A: Cryptococcosis. Infect Dis Clin North Am 2002, 16:837–874.PubMedCrossRef 2. Bicanic T, Harrison TS: Cryptococcal meningitis. Br Med Bull 2005, 72:99–118.PubMedCrossRef 3. Doering TL: How sweet it is! Cell wall biogenesis and polysaccharide capsule formation in Cryptococcus neoformans . Annu Rev Microbiol 2009, 63:223–247.PubMedCrossRef 4. Silveira FP, Husain S: Fungal infections in solid organ transplantation. Med Mycol 2007, 45:305–320.PubMedCrossRef 5. Thakur R, Sarma S, Kushwaha S: Prevalence of HIV-associated cryptococcal meningitis and utility of microbiological determinants for its diagnosis in a tertiary care center. Indian J Pathol Microbiol 2008, 51:212–214.PubMedCrossRef 6.