Fig 1.
Hsp90 governs thermotolerance in C. neoformans.
(a) Cells were grown in the presence of Hsp90 inhibitor RAD at various concentrations ranging from 5nM to 100 μM at the temperatures indicated above. Comparison of MIC50 values for RAD at 25°C and 37°C clearly indicates that C. neoformans growth is hypersensitive to Hsp90 inhibition at 37°C. (b) Spot agar assay without inhibitors indicate that Hsp90 inhibition is fungicidal at 37°C. Growth is not seen when cells grown in presence of RAD at 37°C were spotted and incubated at 25°C and 37°C. (c) Comparison of MIC50 values for antifungal drug Amphotericin B shows similar susceptibilities at both temperatures ruling out lower MIC value due to enhanced drug uptake at higher temperature. (d) C. neoformans clinical isolate shows 12.4-fold hypersensitivity to Hsp90 inhibition at 37°C. (e) C. neoformans clinical isolate show similar susceptibility to AmB at both temperatures tested ruling out nonspecific effect. (f) Comparison of MIC50 values for RAD in Candida albicans at 25°C and 37°C clearly indicates that C. albicans growth is not hypersensitive to Hsp90 inhibition at 37°C.
Fig 2.
CnHsp90 is a functional ATPase.
(a) Cloning of Cryptococcus Hsp90 in pRSET-A vector. Lane1, DNA ladder; lane 2, insert (2.1 kb CnHsp90). (b) Coomassie-stained gel showing purified fraction of recombinant His-tagged CnHsp90 protein obtained using Ni-NTA chromatography (molecular mass, ~86 kDa). (c) Binding affinity of cognate ligand ATP to CnHsp90 using tryptophan fluorescence quenching assay. Change in intrinsic fluorescence intensity upon ligand binding was plotted against ligand concentration. Dissociation constant, Kd, for ATP binding was found to be 497.05 μM. (d) Rate of ATP hydrolysis at 25°C and 37°C was measured by monitoring the hydrolysis of radiolabelled ATP to ADP. A Michaelis–Menten plot shows the fractional cleavage of γ-32P-labeled ATP plotted against ATP concentration. (e) Catalytic efficiency of CnHsp90, Kcat/Km, was found to be 5.07 × 10−5 min−1 μM−1 at 25°C and 6.39 × 10−5 min−1 μM−1 at 25°C and 37°C, respectively. Thus, the ATPase activity is slightly higher at 37°C.
Fig 3.
Binding of Hsp90 inhibitor 17-AAG and its effect on CnHsp90 ATPase activity.
(a) Binding affinity of competitive inhibitor 17-AAG to CnHsp90 using tryptophan fluorescence was determined. Change in intrinsic fluorescence intensity upon ligand binding was plotted against ligand concentration. Dissociation constant, Kd, for 17-AAG binding was calculated to be 12.92 μM. (b) IC50 for inhibition of ATPase activity of CnHsp90 was determined by incubating the pure protein with fixed, saturating concentration of ATP and varying concentrations of 17-AAG. The reaction was carried out at 25°C and 37°C. The percent activity remaining was plotted against concentration of 17-AAG in logarithmic scale to obtain the inhibition curve. 17-AAG mediates inhibition of CnHsp90 activity at both temperatures tested. (c) IC50 values obtained at 25°C and 37°C for CnHsp90 inhibition by 17-AAG was found to be 26.89 μM and 117.15 μM respectively. Therefore, 17-AAG inhibits CnHsp90 more potently at 37°C.
Table 1.
Comparison of biochemical properties of Hsp90 from C. neoformans and comparison with Hsp90 from other organisms [3,6,24,29].
Fig 4.
Hsp90 is associated with the fungal cell wall.
(a) Exponential phase cells were grown at 25°C and 37°C and subjected to indirect immunofluorescence without permeabilization using specific antibody raised against CnHsp90. Hsp90 was found to be localized at the cell surface. Colocalization of CFW (chitin staining) and FITC signal (Hsp90 staining) is also observed indicating cell wall localization of CnHsp90. Antibody to cytosolic protein PGK and pre-immune sera served as negative controls for the experiments. b) Association of Hsp90 at the cell surface was also probed by whole cell based ELISA wherein plates were incubated with intact yeast cells. Significant binding of CnHsp90 Ab to cell surface Hsp90 was seen at both 25°C and 37°C. Anti PGK Ab was used as a negative control. c) Immunoblot analysis of C. neoformans βME cell surface extract also detected a band corresponding to Hsp90 further confirming the presence of Hsp90 in the cell wall fraction.
Fig 5.
Cell surface association of Hsp90 depends on ER Golgi classical secretory pathway.
Indirect immunofluorescence was carried out using Anti CnHsp90 Ab after treatment with different inhibitors. In each case first panel corresponds to cells grown at 25°C and second panel represents cells grown at 37°C. (a) Indirect Immunofluorescence using specific antibody show surface localization of Hsp90 at both temperatures. (b) Cells were treated with vesicle trafficking inhibitor BFA and immunoflourescence was carried out as above. Complete loss of surface signal for Hsp90 was seen in the treated cells both at 25°C (first panel) and 37°C (second panel). (c) Disulfide blocker NEM also abrogated Hsp90 localisation at cell surface. At 25°C diffused signal is seen whereas at 37°C there is no signal thereby implicating the involvement of ER Golgi classical secretion pathway in the process. (d) Hsp90 may be involved in chaperoning a client protein which gets localised to the cell wall. To test this hypothesis, RAD was used to abrogate client association with Hsp90. Loss of cell surface localization was seen in case of RAD treated cells indicating pharmacological inhibitor can also interfere with the process of Hsp90 transport to cell surface.
Fig 6.
Hsp90 is involved in induction and maintenance of capsule in C. neoformans.
(a)Western blot analysis to probe levels of Hsp90 at 25°C and 37°C indicate similar expression levels at both temperatures tested. (b) Hsp90 is significantly upregulated under capsule inducing conditions. (c) Representative microscopic images showing compromise of Hsp90 function during or after capsule induction leads to drastic compromise in capsular size. (d) Hsp90 is involved in capsule assembly as well as maintenance around the cell wall as pre-and post-treatment with RAD leads to approximately 60% decrease in capsular volume relative to no drug and vehicle control.
Fig 7.
Hsp90 plays a crucial role in intrinsic anidulafungin tolerance of Cryptococcus neoformans.
(a) In vitro antifungal susceptibility of C. neoformans to anidulafungin was evaluated at both 25°C and 37°C. Growth in presence of anidulafungin was seen even at high concentrations of the drug indicating robust intrinsic resistance. (b) MIC assay was performed with AF and Hsp90 inhibitor RAD at the concentrations indicated and incubated for 72 hours at 25°C. Growth was not found to be sensitive to combination of both drugs at 25°C. (c) Pharmacological inhibition of Hsp90 with RAD at 37°C reduces tolerance to AF at 37°C indicating additive interaction between these two drugs.