Figure 1.
Upregulation of CHS expression in response to caspofungin is dependent on the PKC, HOG and Ca2+-calcineurin pathways.
Response of CHS promoter-lacZ reporters in the absence (empty bars) and presence (solid bars) of caspofungin in signal transduction mutants. Error bars are S.D. (n = 9 from three separate experiments). Asterisks indicate significant differences (p<0.05) compared to the untreated control in the same genetic background. # indicates significantly different (p<0.05) to the wild type cells in the same growth conditions. The fold inductions for LacZ activity upon caspofungin exposure are shown in Table S2.
Figure 2.
Activation of chitin synthesis by caspofungin.
Chitin synthase activity (A) and chitin content (B) of various chitin synthase, signal transduction and fks1 point mutants (fks1/FKS1 = NR2; fks1/fks1 = NR3) in the absence (empty bars) and presence (solid bars) of 0.032 µg/ml caspofungin. Isolation of NR2 and NR3 are described in Douglas et al (1997) [9]. Asterisks indicate significant differences (p<0.05) compared to the untreated control for each strain. # indicates a significant difference (p<0.05) to the wild type cells in the same growth conditions. Chitin synthase assays were performed in triplicate (average ±SD, n = 3). Cell wall chitin assays were performed five times on three biologically independent samples (average ±SD, n = 15). Time course (min) of Mkc1 phosphorylation in wild type cells in response to 0.032 µg/ml caspofungin (C). Western analysis of Chs3p levels in cells exposed to 0.032 µg/ml caspofungin, strain NGY477 carries Chs3p C-terminally tagged with YFP and BWP17 is the untagged parental strain (Table S1) (D).
Figure 3.
Pre-growing cells in CaCl2 and CFW reduces their susceptibility to caspofungin.
Plate dilution sensitivity tests of wild type (SC5314), a fks1 homozygous point mutant (NR3) and a range of signalling and chitin synthase single and double mutants on YPD agar containing CFW, caspofungin or CFW and caspofungin. Rows marked with * indicate pre-growth of the inoculum in YPD containing both CaCl2 and CFW to raise the chitin content of the cells. Cell numbers per spot are from 5000, 500, 50 to 5 cells, from left to right.
Figure 4.
Pre-growing cells in CaCl2 and CFW increases their caspofungin MIC.
The MIC of C. albicans strain SC5314 was measured in RPMI 1640 and YPD medium supplemented with caspofungin. The effect of growing the inoculum on YPD or YPD with CaCl2 and CFW was tested; cells pre-grown in CaCl2 and CFW had an increased MIC for caspofungin.
Figure 5.
Cells pre-grown in glucosamine have reduced susceptibility to caspofungin.
Pre-growing strains that were hypersensitive to caspofungin (chs3Δ, cna1Δ, mkc1Δ) in YPD plus 23 mM glucosamine reduced their susceptibility to 0.032 µg/ml caspofungin. Cell numbers per spot are from 5000, 500, 50 to 5 cells, from left to right.
Figure 6.
Transient elevation of chitin in cells with reduced susceptibility to caspofungin.
CaCl2 and CFW pre-treated cells grown on medium containing 16 µg/ml caspofungin formed rich zones of growth within a lawn of surrounding cells (A, top panel). Inocula from the richer zones of the colony contain resistant cells that grew well on 16 µg/ml caspofungin (A, left hand panel), while the surrounding cells grow poorly to give few resistant colonies (A, right hand panel). DIC images and fluorescence micrographs of CFW and DAPI-stained cells (B, D) and propidium iodide stained cells (C). In (B) cells from the 16 µg/ml caspofungin-resistant, richer zones of the colony have a higher chitin content, i.e. stain more strongly with CFW, than caspofungin-sensitive cells and the inoculum. Caspofungin-sensitive cells are non-viable and take up propidium iodide whereas caspofungin-insensitive cells are viable and do not (C). Increased CFW staining of resistant cells was lost upon sub-culture under non-selective conditions, in the absence of caspofungin (D). The average relative chitin content of yeast cells, measured by intensity of CFW fluorescence, is shown for cells in untreated controls and after pretreatment with 0.32 µg/ml caspofungin or CaCl2 and CFW and for cells pre-treated with CaCl2 and CFW then grown in 0.32 µg/ml caspofungin (E). In (E) statistical differences are shown compared to untreated control (*P<0.001) and compared to Ca/CFW pre-treated cultures (# P<0.001) (by t-test). Under-non-selective conditions, the relative chitin content of resistant cells decreased progressively to that of the wild type (W.T.) as shown by the loss of CFW fluorescence (F). Error bars are S.D. (n = 6 from 3 separate experiments). Scale bars are 2 µm.
Figure 7.
The role of chitin synthase isoenzymes in elevation of chitin levels in response to caspofungin.
DIC (top panels) and fluorescent images (bottom panels) of wild type and chsΔ mutant strains grown in the presence and absence of 0.032 µg/ml caspofungin with and without pre-treatment of the inoculum with CaCl2 and CFW. Scale bars are 2 µm. Enlarged images are presented on the bottom panel showing the chitin distribution in cells induced by CaCl2 and CFW in the chs3Δ (20) and chs1Δchs3Δ (22) mutants, with the latter showing induced synthesis of salvage septa. In (23) increased chitin formation is shown as induced by CaCl2 and CFW and subsequent culture in caspofungin.
Figure 8.
The salvage septum of C. albicans in the chs1Δchs3Δ double mutant (under repressing conditions for the conditional MRP1p-CHS1 mutant).
(A, B) The chained septum-less phenotype of the chs1Δchs3Δ mutant (A) is abrogated by pre-growth on YPD with CaCl2 and CFW to stimulate chitin synthesis prior to growth of the mutant cells under repressing conditions (B,C). (C) Shows CFW-fluorescence image of cells shown in (B). TEM images of proximal offset thickened septa and (D,E). Scale bars are 2 µm for (A–D) and 0.2 µm for (E). Growth of the inoculum in YPD with CaCl2 and CFW allows the chs1Δchs3Δ mutant to grow in the presence of caspofungin while treatment of these cells with 10 µM nikkomycin Z leads to inhibition of growth and cell lysis (F).
Figure 9.
Synergistic inhibition of growth of a wild type strain (SC5314) and an fks1 homozygous point mutant (NR3) by 16 µg/ml caspofungin and 100 µg/ml CFW or 10 µM nikkomycin Z as chitin synthase inhibitors is further demonstrated in plate sensitivity tests.