er stress due to an accumulation of unfolded or misfolded proteins activates the unfolded protein response ( upr ) signaling pathway in eukaryotic cells and has been shown to be involved in c. albicans morphogenesis . upr signaling activates a variety of downstream transcripts , including the bzip transcription factor hac1 that impacts c.  albicans hyphal growth in response to er stress . tunicamycin ( tm ) and dithiothreitol ( dtt ) induce the upr by inhibiting n - linked glycosylation of secreted proteins and preventing disulfide bond formation , respectively . we found that c.  albicans and c.  dubliniensis calcineurin mutants are hypersensitive to tunicamycin and dtt compared with the wild - type ( fig .  2 ) . furthermore , the role of crz1 in er stress is different in c.  albicans compared with c.  dubliniensis ( fig .  2 ) . while c.  dubliniensis crz1/crz1 , crz2/crz2 , and crz1/crz1 crz2/crz2 double mutants grew similarly to wild - type in the presence of tm , the c.  albicans crz1/crz1 mutant exhibited attenuated growth that was intermediate between wild - type and calcineurin mutants ( figs .  1 
 and 
 2 ) , indicating a divergent function of crz1 in er stress responses in both candida species . interestingly , we found that the c.  dubliniensis wild - type strainis hypersensitive to the er stress inducer dtt compared with the c.  albicans wild - type strain ( fig . 2 ) , indicating that the tolerance to protein misfolding has been reduced in c.  dubliniensis compared with c.  albicans . calcineurin functions are conserved whereas crz1 has different roles during er stress in c.  albicans and c.  dubliniensis . c.  dubliniensis and c.  albicans calcineurin mutants were hypersensitive to the er stress inducers tunicamycin ( tm ) and dithiothreitol ( dtt ) . the c. albicans crz1/crz1 mutants , but not c. dubliniensis crz1/crz1 mutants , exhibit tm sensitivity intermediate between wild - type and calcineurin mutants . cells were grown overnight in ypd medium at 24c , washed twice in dh2o , 5-fold serially diluted , and spotted onto ypd medium containing tm ( 1 g / ml ) or dtt ( 45 mm ) . another crucial factor for hyphal growth and virulence of c.  albicans is the ability to respond to environmental ph changes . both calcineurin and crz1 have been demonstrated to play a role in c.  albicans tolerance to changes in environmental ph through the rim101/pacc ph - sensing pathway . previoius studies have shown that rim101 acts in parallel with calcineurin and crz1 to allow for adaption to alkaline ph in c.  albicans . furthermore , crz1 , crz2 , and calcineurin are needed for adaptation to acidic ph in c.  albicans . c.  dubliniensis forms true hyphae less efficiently than c.  albicans in response to ph shifts in vitro . here , we show that c.  dubliniensis cna1/cna1 and cnb1/cnb1 mutants are hypersensitive to acidic and alkaline phs compared with wild - type ( fig . 3 ) , whereas c.  albicans calcineurin mutants did not show attenuated growth at acidic ph . this result differs from previous studies where calcineurin was shown to be required for growth at acidic ph and may be attributable to the use of different ph buffering systems or experimental procedures . taken together , we conclude that in c.  dubliniensis calcineurin plays a greater role in acidic ph homeostasis compared with its role in c.  albicans ( figs .  1 
 and 
 3 ) . interestingly , while c.  dubliniensis and c.  albicans crz1/crz1 mutants grew similarly to wild - type at acidic ph , they exhibited attenuated growth that was intermediate between wild - type and calcineurin mutants at alkaline ph . however , c.  dubliniensis crz2/crz2 mutants did not have growth defects in acidic or alkaline ph ( fig .  3 ) . these results suggest that alkaline ph homeostasis is controlled by the crz1-dependent calcineurin pathway in c. dubliniensis and c. albicans , whereas acidic ph homeostasis is governed by a crz1-independent calcineurin pathway in c. dubliniensis . calcineurin is essential for growth at alkaline conditions ( ph = 9 ) in c. dubliniensis and c. albicans . while c. dubliniensis calcineurin mutants had attenuated growth at acidic ph , the c. albicans calcineurin mutants did not . cells were grown overnight in ypd medium at 24c , washed twice in dh2o , 5-fold serially diluted , and spotted onto ypd medium containing 150 mm hepes buffered at ph 2 or 7 . for ph 9 medium , ypd was buffered with 150 mm ph 14 hepes ( 85 ml of ypd plus 15 ml of 1 m hepes at ph 14 ) . the phs of solid media were confirmed with ph indicator strips ( sigma - aldrich , z134147 ) . to determine if the calcineurin inhibitor fk506 exhibits synergism with caspofungin against the echinocandin - resistant strain dpl278 , we performed disk diffusion and checkerboard assays following the procedures described by the clinical and laboratory standards institute ( clsi ) protocol m27-a3 . the dpl278 strain is resistant to caspofungin but susceptible to azoles ( fig .  4 
 and 
 table 1 ) . we further found that the resistance to caspofungin of the dpl278 strain was reversed by supplementation with fk506 ( fig . meanwhile , we demonstrated that fk506 exhibits synergism with caspofungin , fluconazole , or posaconazole against c.  dubliniensis wild - type and echinocandin - resistant strain [ fractional inhibitory concentrations ( fic ) < 0.5 , table 1 ] , suggesting that these drug combinations have therapeutic potential for c.  dubliniensis infections . figure  4 . calcineurin inhibitor exhibits synergism with caspofungin against wild - type and echinocandin - resistant c. dubliniensis strain . disk diffusion assays were used to determine echinocandin and azole susceptibility of a wild - type ( cd36 ) and an echinocandin - resistant strain ( dpl278 ) . cells were grown overnight at 30c , and cells at a density of 0.1 od600 ( in 100 l ) were spread on the surface of ypd medium in the absence or presence of fk506 ( 1 g / ml ) . the disk ( 6 mm ) containing 5 l of caspofungin ( cas , 5 g ) , posaconazole ( psc , 1 g ) , or fluconazole ( flc , 10 g ) was placed on the surface of the plates , and incubated at 30c for 48 h.   one wild - type c. dubliniensis strain ( cd36 ) and one echinocandin - resistant strain ( dpl278 ) were grown overnight with shaking at 30c and washed twice in dh2o . the od600 was taken of the cultures with a spectrophotometer and diluted to 0.01 od600/ml in rpmi-1640 medium ( sigma r6504 , 8.4 g in 1 l dh2o buffered to ph = 7 with sodium hydroxide pellets ) . minimum inhibitory concentrations ( mic ) of each drug alone and fractional inhibitory concentrations of the drugs in combination were determined using the broth microdilution method according to the clinical and laboratory standards institute ( clsi ) protocol m27-a3 . final concentrations of fluconazole ( flc ) and caspofungin ( cas ) ranged from 16 to 0.0312 g / ml . fk506 concentrations ranged from 4.0 to 0.063 g / ml while posaconazole ( psc ) concentrations ranged from 2.0 to 0.0039 g / ml .   fic index = ( miccombined drug 1/micalone drug 1 ) + ( miccombined drug 2/micalone drug 2 ) fic  0.5 ( synergy ) , > 0.5 but < 1.0 ( additive ) , > 1.0 but  2.0 ( no interaction ) , > 2.0 ( antagonism ) .