2. stricted to sessile cells within the biofilms and may not be retained
by the cells once they disperse out of the biofilm community.
Through evolution, C. albicans has developed mechanisms
that allow this opportunistic pathogen to cope with a variety of
environmental stresses and adapt to diverse niches within the host
(14, 17). Thus, we hypothesize that the induced resistance to
caspofungin seen subsequent to fluconazole treatment might have
been due to a cellular stress response subsequent to the exposure
of biofilm cells to fluconazole. In fact, these cellular stress re-
sponses have been involved in the development of resistance to
both azoles and echinocandins, mostly controlled by the Hsp90
molecular chaperone (14, 17–19) and, more recently, we have
reported on the important role of Hsp90 in mediating fungal bio-
film resistance (20). Therefore, our original investigations into the
mechanism(s) of induced caspofungin resistance subsequent to
fluconazole treatment focused first on a potential role of Hsp90.
Briefly, we used a tetracycline-regulatable HSP90 strain where
Hsp90 levels can be regulated by doxycycline (DOX) in growth
medium (21). In the absence of DOX (when HSP90 is being ex-
pressed), the tet-HSP90 strain behaved similarly to the wild-type
strains of C. albicans in that the biofilms were completely resistant
to fluconazole and sensitive to caspofungin (Fig. 1B). Also, simi-
larly to control biofilms, under these conditions the tet-HSP90
biofilms displayed elevated caspofungin resistance after prior ex-
posure to fluconazole (Fig. 1B). However, when DOX was added
into the biofilm growth medium, leading to a depletion of Hsp90
levels, we found that the induced resistance to the echinocandin
was virtually abolished (Fig. 1C). Overall, these results highlight
that C. albicans Hsp90 plays an important role in the induced
resistance to caspofungin displayed by cells within the biofilms
subsequent to fluconazole pretreatment.
Hsp90 helps C. albicans cells to survive the lethal effect of the
antifungal drugs by stabilizing key regulators of cellular signaling
(14, 17, 21). Thus, to further dissect the mechanism(s) by which
Hsp90 regulates the induced resistance in the sequential flucona-
zole-caspofungin treatment, we utilized strains lacking specific
downstream effectors of Hsp90. In particular, both calcineurin, a
Ca2ϩ
-calmodulin-activated protein phosphatase, and Mkc1, the
terminal mitogen-activated protein kinase (MAPK) in the protein
kinase C (PKC) signaling cascade, represent major Hsp90 client
proteins involved in antifungal drug resistance in C. albicans, in-
cluding during the biofilm mode of growth (14, 20, 22). To this
end, we examined the C. albicans mutant strain lacking the cata-
lytic subunit of calcineurin (⌬cna1 cna1) (19) in our sequential
checkerboard antifungal drug assay. As expected, the biofilms
formed by the calcineurin mutant were more sensitive to flucona-
zole with an SMIC50 of 32 g/ml (Fig. 2A). These biofilms were
also hypersensitive to caspofungin. We found that pretreatment of
the calcineurin mutants with fluconazole did not translate into
subsequent caspofungin resistance (Fig. 2A). In fact, despite the
fluconazole exposure, these biofilms were still hypersensitive to
caspofungin.
As mentioned before, Mkc1 is an important determinant of the
PKC-cell wall integrity pathway (23). Since the primary target of
caspofungin is -1,3-glucan in the cell wall (24), we questioned if any
compensatorymechanismsinthecellwallmayalsoinfluencebiofilm
resistance to caspofungin after fluconazole treatment. However, as
shown in Fig. 2B and in stark contrast to results obtained using the
calcineurin mutant, fluconazole pretreatment still resulted in in-
ducedresistancetocaspofunginofbiofilmsformedbythemkc1mkc1
mutant strain (25). These results clearly indicate that Mkc1, the ter-
minal MAPK in the PKC signaling cascade, is dispensable for the
induction of caspofungin resistance after exposure to fluconazole.
FIG 1 (A) Biofilms of C. albicans SC5314 were grown in 96-well microtiter
plates in RPMI medium at 37°C. After 24 h, cells were washed with phosphate-
buffered saline (PBS) to remove nonadherent cells and fresh medium was
added with various concentrations of antifungal drugs in a checkerboard for-
mat. First, fluconazole (FLC) was added in all but the first column of the wells,
which was replenished with RPMI medium. After 24 h of fluconazole pretreat-
ment, biofilms were washed once with PBS and then treated with different
concentrations of caspofungin (CAS) for an additional 24 h. Metabolic activity
of the biofilm cells was measured using the XTT assay. (B and C) Genetic
compromise of HSP90 results in sensitivity to caspofungin despite fluconazole
pretreatment. HSP90 levels were regulated in the C. albicans tetracycline-reg-
ulatable (⌬hsp90 tet HSP90) strain by the presence and absence of DOX. In the
absence of DOX (when HSP90 was being expressed), the strain showed a re-
sponse similar to that of the SC5314 wild-type strain with induction of caspo-
fungin resistance. However, in the presence of DOX (lacking HSP90 expres-
sion), the biofilms were sensitive to caspofungin subsequent to fluconazole
exposure. Color gradients are representative of biofilm viability such that light
colors depict maximum metabolic activity (low inhibition) and the darkest
color represents maximum inhibition of biofilm viability.
TABLE 1 Preexposure to azole antifungal agents induces subsequent
resistance to echinocandin derivatives in C. albicans biofilms, as
measured by SMIC80 using the XTT reduction assaya
Sequential treatment
SMIC80 (g/ml)a
No azole
(echinocandin
only)
Low
azole
concn
Medium
azole
concn
High
azole
concn
Fluconazole-caspofungin 0.25 0.25 Ͼ2 Ͼ2
Fluconazole-micafungin 0125 0.125 Ͼ2 Ͼ2
Fluconazole-anidulafungin 0.125 0.125 Ͼ2 Ͼ2
Voriconazole-caspofungin 0.25 0.5 Ͼ2 Ͼ2
a
The low, medium, and high azole concentrations were 1, 64 and 512 g/ml for
fluconazole and 0.25, 16, and 128 g/ml for voriconazole, respectively.
Sarkar et al.
1184 aac.asm.org Antimicrobial Agents and Chemotherapy
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3. Overall, this report reveals that, in a sequential antifungal drug
therapy regimen in vitro, prior treatment of C. albicans biofilms
with an azole leads to a significant decrease in the efficacy of echi-
nocandin agents, almost completely abolishing their otherwise ex-
cellent in vitro antibiofilm activity. This activity is dependent on
the concentration of azole used, in that biofilms pretreated with
higher concentrations of azole derivatives demonstrated higher
resistance to echinocandins under both static and flow conditions.
This phenomenon is related to the induction of cell stress re-
sponses mediated by Hsp90 and its client protein calcineurin but
not by Mkc1. If confirmed in vivo, these observations may have
profound implications for the clinical management of patients
with candidiasis, further advocating for the use of echinocandins
as first-line therapy when a biofilm etiology is suspected.
ACKNOWLEDGMENTS
This work was supported by a grant from Merck & Co., Inc.
We thank Leah E. Cowen for C. albicans mutant strains and Rick
Kirkpatrick and the Fungus Testing Lab at UTHSCSA for isolates of non-
albicans Candida spp.
The content of this work is solely our responsibility and does not
necessarily represent the official views of the founder.
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