BACKGROUND
candida species are commensal microorganisms of vertebrate hosts that can cause infections ranging from non-life-threatening to invasive illnesses.
although candidaemia is the most common manifestation of invasive candidiasis, extensive visceral invasion with candida can occur in all organs.
the eyes, brain, liver, spleen, and kidneys are the most commonly affected  <cit> .
candidiasis is the fourth most common cause of nosocomial bloodstream infections in brazil and the u.s.a., with a mortality rate of approximately 40%  <cit> .
a progressive increase in the number and severity of candidiasis over the past two decades has been observed worldwide, especially in immunocompromised patients and also in patients hospitalised with serious underlying diseases, during immunosuppressive therapy, or parenteral nutrition, as well as among patients exposed to invasive medical procedures.
yeasts of candida albicans are the most frequently implicated in cases of invasive candidiasis infections.
however, nowadays candida non-albicans  species such as candida glabrata, candida krusei, and candida parapsilosis have increased in importance and number among fungal infections  <cit> .
currently, the mainstay of chemotherapy employed for the treatment of fungal infections comprises drugs that affect the function or biosynthesis of membrane sterols  <cit> .
the polyenes  were the first antifungal class used to treat invasive fungal infections.
the primary mechanism of amphotericin b is its binding to the signature 24-alkyl sterols present in fungal cell membranes, leading to a perturbation of the membrane selective permeability and, consequently, loss of the cellular content.
despite the specific fungicidal effect of polyenes, they display significant toxicity to mammalian cells  <cit> .
another important antifungal class comprises the azoles, such as ketoconazole, fluconazole , itraconazole , posaconazole, and voriconazole, which are the compounds most frequently used today, and whose specific target is the cytochrome p-450-dependent c14α-demethylase, a key enzyme of the ergosterol biosynthesis pathway  <cit> .
although azoles are one of the main classes of drugs used in the treatment of fungal infections, these drugs present several problems such as their fungistatic rather than fungicidal activity, variable drug bioavailability, lack of intravenous preparations, large number of drug-drug interactions, development of resistance, and potential cross-resistance between different azoles  <cit> .
during the last two decades, some studies have described a new class of antifungals called azasterols, which are inhibitors of the Δ24-sterol methyltransferase , another key enzyme of the ergosterol biosynthesis pathway, which is absent in the mammalian host cells  <cit> .
this enzyme catalyses the s-adenosylmethionine-mediated incorporation of methyl groups at position  <dig> in sterols, which is an essential step for the biosynthesis of fungal sterols  <cit> .
20-piperidin-2-yl-5α-pregnan-3β-20-diol  and  <dig> -epiminolanosterol  are steroid compounds with a nitrogen atom in the side chain , and are known inhibitors of 24-smt in fungi  <cit> , trypanosoma cruzi  <cit> , and leishmania amazonensis  <cit> .
antifungal activities of these inhibitors were also described against pneumocytis carinii  <cit>  and paracoccidioides brasiliensis  <cit> .
the purpose of the present study was to  examine the susceptibilities of a collection of  <dig> yeasts of the genus candida to aza and eil;  determine the fungicidal activities of these compounds; and  detect the main morphology and ultrastructural alterations of the yeasts after drug treatment.
RESULTS
antifungal susceptibility of candida isolates
the mics obtained for the atcc strains to standard drugs  and to the experimental compounds  are listed in table  <dig>  interestingly, c. krusei  has aza mic <dig> of  <dig> μg.ml- <dig> and mic <dig> of  <dig> μg.ml- <dig>  on the other hand, eil did not inhibit the growth of the flc- and itc-resistant strains.
all clinical isolates were susceptible to amb, with the median mic <dig> values ranging from  <dig>  to  <dig>  μg.ml- <dig> and the mic <dig> from  <dig>  to  <dig>  μg.ml- <dig> .
however, three isolates  showed mic <dig> values higher than  <dig> μg.ml- <dig>  susceptibility to flc was observed in 92% of the isolates, although 26% showed a trailing effect.
clear resistance to flc was detected in three isolates .
45% of the strains showed mic <dig> of  <dig> – <dig>  μg.ml- <dig> and 37% showed mic <dig> of  <dig> – <dig> μg.ml- <dig>  on the other hand, 75% of the isolates were susceptible to itc, and 16% showed a trailing effect.
resistance to itc was detected in  <dig> isolates .
most of the isolates had mic <dig> and mic <dig> for itc lower than  <dig>  μg.ml- <dig> .
only c. krusei isolates were less susceptible to all standard drugs, showing a mic <dig> of  <dig>  μg.ml- <dig> for amb, >  <dig> μg.ml- <dig> for flc, and  <dig> μg.ml- <dig> for itc .
ttrailing effect, rresistant
the values are expressed in μg.ml- <dig>
-not determinate; +mic results are medians; *trailing effect to flc ; **trailing effect to itc .
when the mic values for 24-smti  were analysed, we observed important antifungal activity for almost all candida spp.
isolates .
for aza, mic <dig> and mic <dig> values lower than  <dig> μg.ml- <dig> were observed in 86% and 52% of the isolates, respectively.
for eil, mic <dig> and mic <dig> values lower than  <dig> μg.ml- <dig> were observed in 92% and 63% of the isolates, respectively.
c. zeylanoides and c. lipolytica  showed mic50– <dig> values of ≤  <dig>  μg.ml- <dig> for both inhibitors, whilst c. krusei was resistant to eil, with mic50– <dig> values of  <dig> μg.ml- <dig> .
however, both c. krusei and c. lipolytica were resistant to aza  .
finally, c. guilliermondii isolates, flc- and itc-resistant, were susceptible to aza, with mic50– <dig> values of  <dig>  –  <dig>  μg.ml- <dig>
+mic results are medians.
correlations between mic values
positive correlations of the mic <dig> values were observed between aza and amb , aza and eil , and flc and itc .
in addition, positive correlations were observed between the mic <dig> values of the flc and itc .
on the other hand, no significant correlations were observed between the mic values for azoles and 24-smti.
some clinical isolates with a trailing effect for flc  and itc  also showed residual growth at higher concentrations of aza  of 58%  and 54%  of the isolates, respectively.
residual growth was not observed in the isolates after treatment with eil.
minimum fungicidal concentration  of aza and eil
the mfcs obtained for half of our isolates were higher than  <dig> μg.ml- <dig>  revealing a predominant fungistatic activity of the smti.
interestingly,  <dig> cna isolates  showed mfcs lower than  <dig> μg.ml- <dig>  indicating a remarkable fungicidal activity, especially for aza .
on the other hand, aza killed a negligible number of the c. albicans isolates at concentrations lower than  <dig> μg.ml- <dig>
* data is expressed in percentual of isolates.
ultrastructural effects
the general morphology of untreated c. albicans was observed using scanning  and transmission  electron microscopy.
the shape of c. albicans varies from spherical  to oval cells when viewed by scanning electron microscopy .
transmission electron microscopy revealed the presence of normal cell walls with a thickness of  <dig> ±  <dig> nm , including a thin electron-dense outer layer with delicate fibrillar structures clearly visible .
a continuous cytoplasmatic membrane lining a homogeneous and electron-dense cytoplasm containing ribosomes, nucleus , and nucleoli  could also be observed .
treatment of c. albicans with mic <dig> of aza  and eil  induced significant morphological changes, which ranged from discrete alterations to total destruction of the fungal cells.
a common alteration observed after the treatment with aza and eil was a significant increase in cell size, from  <dig> μm to  <dig> μm in diameter .
the number of altered cells was counted, and the morphological alterations appeared in  <dig> % and  <dig> % of the cells after treatment with aza and eil, respectively.
among the most frequently observed ultrastructural alterations were:  presence of small buds ;  irregular cell-wall surfaces ;  loss of cell-wall integrity, with an apparent shedding of cell components ; and  a two- to three-fold increment of the cell wall thickness was observed after treatment with aza and eil, respectively .
the cytoplasmic membrane of treated cells also showed several changes, such as the presence of evaginations, discontinuity, detachment from the cell wall ; budding of small vesicles that could migrate through the periplasmatic region , accumulate in the cytoplasm , or remain close to the cell membrane .
vesicle sizes ranged from 40– <dig> nm for aza and 40– <dig> nm for eil.
mitochondrial swelling and electron dense vacuoles accumulation was also observed .
cna cells treated with mic <dig> of 24-smti showed similar ultrastructural changes .
presence of lipid bodies
treatment with mic <dig> of aza and eil induced an accumulation of lipid bodies in the cytoplasm, which can be characterised by the presence of small dots labelled with nile red , which were absent in the untreated yeasts .
these lipid bodies seen by fluorescence microscopy can be correlated with the small, electron-dense vacuoles seen by transmission electron microscopy .
effect of 24-smt inhibitors on the cell cycle
dapi staining used to label the dna revealed that the treatment of c. albicans with aza and eil induced important alterations in the cell cycle .
to quantitatively assess these alterations, different stages of the untreated yeasts were considered, such as:  cells containing one nucleus,  cells with a bud and one nucleus, and  cells with a bud and two nuclei .
after treatment with the mic50s of aza and eil, different alterations in the nucleus were observed, and these were classified as:  cells with more than one nucleus,  cells showing abnormal chromatin condensation, and  cells without a nucleus.
counting the number of abnormal cells revealed that approximately 66% of the yeasts showed abnormal chromatin condensation, whereas  <dig> % of aza-treated and  <dig> % of eil-treated cells contained more than one nucleus, and approximately 6% of the cells treated with both compounds had no nucleus .
cytotoxicity evaluation
cytotoxicity of 24-smti was evaluated against mammalian cells  using the sulforhodamine b viability assay.
for both aza and eil the cc <dig> was  <dig> μg.ml- <dig>  which corresponds to a mean selectivity index of  <dig> for aza and  <dig> for eil.
discussion
although c. albicans is the predominant species in candidiasis, cna species have increased in frequency in recent years.
the reasons for the emergence of cna species are not fully understood, but some medical conditions may frequently run the risk of developing candidaemia due to the cna species: c. parapsilopsis has been associated with vascular catheters and parenteral nutrition; c. tropicalis with cancer and neutropenia; and c. krusei and c. glabrata with previous treatments with flc and itc  <cit> .
previous studies have described a high susceptibility of c. albicans isolates to azoles and amb, whereas cna isolates are usually less susceptible and may be intrinsically resistant to flc and itc  <cit> .
as reported by other investigators  <cit> , none of our candida isolates showed mic ≥  <dig> μg.ml- <dig> for amb.
mic values found for itc and flu were similar to those previously reported by different groups  <cit> .
however, in the present study, flc-resistant candida strains were only observed among cna species .
however, itc-resistance was found in c. albicans  and cna isolates .
the observation of a high positive correlation between mic values of flc and itc , in this study, suggests that cross-resistance may be occurring.
however, no correlation was observed between mic values of the azoles and 24-smti, indicating lack of possible cross-resistance.
the general finding for our candida spp.
isolates was that they were mostly susceptible to aza and eil, because the mic50s were lower than  <dig> μg.ml- <dig> for 73% and 88% of the isolates after treatment with aza and eil, respectively.
interestingly, some flc- and itc-resistant strains were susceptible to 24-smti.
however, residual growth of candida after treatment with aza was similar to that observed for flc and itc.
no residual growth was observed after treatment with eil.
the fungicidal action of 24-smti was more prominent against cna species than against c. albicans isolates.
a concentration of  <dig>  μg.ml- <dig> of 24-smti was enough to kill 100% of c. lusitanae, c. zeylanoides, and c. rugosa, and 50% of c. glabrata.
in contrast, this same concentration killed only  <dig> % and  <dig> % of c. albicans isolates, considering aza and eil respectively.
previous studies have shown that azasterol derivatives have antifungal activity against a variety of species  <cit> .
15-azasterol, in concentrations ranging from  <dig>  μg.ml- <dig> to  <dig>  μg.ml- <dig>  inhibits the growth of saccharomyces cerevisae and c. albicans, with a concomitant accumulation of sterol intermediate molecules  <cit> .
the range of mic and mfc values for 15-azasterol analogues against these fungal species varied from  <dig>  to  <dig>  μg.ml- <dig> and  <dig>  to  <dig>  μg.ml- <dig>  respectively  <cit>  and are similar to the values obtained in the present study.
other azasterol derivatives have been shown to inhibit s. cerevisae 24-smt, leading to the accumulation of zymosterol  <cit> .
recent work demonstrated that aza displays antifungal activity against paracoccidioides brasiliensis  <cit>  and pneumocystis carinii  <cit> .
concentrations of  <dig> μm  inhibited 100% of the growth in p. brasiliensis, and the treatment of p. carinii with the ic <dig> of  <dig>  μm  led to growth arrest and accumulation of 24-desakyl sterols, indicating an inhibition of 24-smt  <cit> .
in addition, previous studies have also shown an anti-protozoan activity of aza and eil on t. cruzi epimastigotes and intracellular amastigotes  <cit> , l. amazonensis promastigotes and intracellular amastigotes  <cit> , toxoplasma gondii  <cit> , and giardia lamblia  <cit> , with mics in the low μm to sub-μm range.
for protozoans, eil was reported to be more active than aza.
in contrast, we found in this study that aza was more active than eil against candida spp.
isolates.
treatment of c. albicans yeasts with aza and eil caused dramatic changes in their cellular and sub-cellular structure.
the main alterations included changes in the cell wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm, with the presence of an electron-lucent zone between them, cell collapse and release of cellular content, mitochondrial swelling, and abnormalities in the nuclear structure.
these findings are similar to those previously reported after treatment of candida spp.
with different azoles  <cit> .
borges and co-workers  <cit>  reported that exposure of candida albicans to itc leads to primary alterations at the cell periphery and the appearance of vacuoles in the cytoplasm, which may be lipid inclusions.
these changes were usually accompanied by an increase in the cell volume and impaired cell division.
in addition, studies by hazen and co-workers  <cit>  revealed that candida treated with flc shows a distinct retraction of the membrane from the cell wall.
on the other hand, c. albicans treated with low concentrations of amb shows chromatin condensation and margination, separation of the nuclear envelope, and nuclear fragmentation  <cit> .
high concentrations of amb induce cellular changes characteristic of necrosis, showing many large vacuoles  <cit> .
additionally, bahmed and co-workers  <cit>  demonstrated an increase in cell wall thickness of candida yeasts, which may be related to alterations in the cell wall composition induced by the treatment with amb.
in addition, similar to our findings, the appearance of multivesicular bodies and myelin-like structures were reported after treatment of leishmania  <cit>  and t. cruzi  <cit>  with aza and eil.
staining with nile red revealed the presence of lipid accumulation in the cytoplasm after treatment with 24-smti, confirming that these compounds induce a perturbation in lipid biosynthesis.
similar observations have recently been made as the result of treatment of leishmania amazonensis with 24-smti, which induced several abnormalities in the lipid content, with the accumulation of steroid intermediate molecules  <cit> .
in addition, staining of dna with dapi indicates a profound alteration in the cell cycle after treatment with aza and eil.
candida yeasts produced unfertile buds that remained closely associated with the mother cell, and appeared with or without various nuclei.
the nucleus may also have an altered shape and/or with abnormal chromatin condensation that might be associated with apoptosis cell death, as previously described after treatment of c. albicans with amb  <cit> ; and also after treatment of tritrichomonas foetus with hydrogen peroxide  <cit> .
the presence of many cells with more than one nucleus may also indicate that ergosterol biosynthesis inhibitors are interfering with cytokinesis.
in fact, it was previously found that ergosterol levels modulate the activity of protein kinases such as pp60v-src and also the levels of camp, both of which are directly related to the control of the cell cycle  <cit> .
in addition, some studies have shown that drugs such as griseofulvin and nocodazole, which interfere with the assembly of cytoskeleton components, induce alterations in the cell cycle and apoptosis cell death  <cit> .
thus, there are two possible explanations for the alterations in the cell cycle:  the depletion of ergosterol and other lipids, which are essential for the maintenance of the cytoplasmatic membrane structure and are also important key regulators of the cell cycle; and/or  alterations in the cytoskeleton structure, which can affect the cytokinesis process.
the results obtained here suggest that aza and eil are probably interfering with sterol biosynthesis in candida spp., as previously described for c. albicans  <cit> , p. carinii  <cit> , t. cruzi  <cit> , and l. amazonensis  <cit> .
on the other hand, we cannot exclude the possibility that these compounds may be acting in other pathways, inducing some secondary effects that could be related to the accumulation of other lipids or, as demonstrated in crithidia deanei, that aza can interfere with phospholipid biosynthesis  <cit> .
further studies are necessary to characterise the correlation between the depletion of ergosterol and the cell cycle in c. albicans.
CONCLUSIONS
the results presented herein demonstrate the potential usefulness of the 24-smt inhibitors aza and eil as antifungal agents, including azole-resistant candida strains.
the specific in vitro and in vivo antifungal and antiprotozoal activity of azasterols has been known for years, and in most cases has been linked to their specific inhibition of 24-smt, an enzyme absent in mammals  <cit> .
however, other studies have found that these compounds are also active against parasitic protozoa that lack endogenous sterol biosynthesis, such as t. gondii  <cit>  and trypanosoma brucei  <cit> , indicating that they may have other biochemical targets.
taken together, these results indicate azasterols as useful leads for novel antifungal agents, but optimisation of their selectivity, adme, pk, and toxicological properties is required for their further advancement as drug candidates.

 candida species are commensal microorganisms of vertebrate hosts that can cause infections ranging from non-life-threatening to invasive illnesses. although candidaemia is the most common manifestation of invasive candidiasis, extensive visceral invasion with candida can occur in all organs. the eyes, brain, liver, spleen, and kidneys are the most commonly affected. candidiasis is the fourth most common cause of nosocomial bloodstream infections in brazil and the u.s.a., with a mortality rate of approximately 40%. a progressive increase in the number and severity of candidiasis over the past two decades has been observed worldwide, especially in immunocompromised patients and also in patients hospitalised with serious underlying diseases, during immunosuppressive therapy, or parenteral nutrition, as well as among patients exposed to invasive medical procedures. yeasts of candida albicans are the most frequently implicated in cases of invasive candidiasis infections. however, nowadays candida non-albicans species such as candida glabrata, candida krusei, and candida parapsilosis have increased in importance and number among fungal infections. currently, the mainstay of chemotherapy employed for the treatment of fungal infections comprises drugs that affect the function or biosynthesis of membrane sterols. the polyenes were the first antifungal class used to treat invasive fungal infections. the primary mechanism of amphotericin b is its binding to the signature 24-alkyl sterols present in fungal cell membranes, leading to a perturbation of the membrane selective permeability and, consequently, loss of the cellular content. despite the specific fungicidal effect of polyenes, they display significant toxicity to mammalian cells. another important antifungal class comprises the azoles, such as ketoconazole, fluconazole, itraconazole, posaconazole, and voriconazole, which are the compounds most frequently used today, and whose specific target is the cytochrome p-450-dependent c14α-demethylase, a key enzyme of the ergosterol biosynthesis pathway. although azoles are one of the main classes of drugs used in the treatment of fungal infections, these drugs present several problems such as their fungistatic rather than fungicidal activity, variable drug bioavailability, lack of intravenous preparations, large number of drug-drug interactions, development of resistance, and potential cross-resistance between different azoles. during the last two decades, some studies have described a new class of antifungals called azasterols, which are inhibitors of the Δ24-sterol methyltransferase, another key enzyme of the ergosterol biosynthesis pathway, which is absent in the mammalian host cells. this enzyme catalyses the s-adenosylmethionine-mediated incorporation of methyl groups at position in sterols, which is an essential step for the biosynthesis of fungal sterols. 20-piperidin-2-yl-5α-pregnan-3β-20-diol and -epiminolanosterol are steroid compounds with a nitrogen atom in the side chain, and are known inhibitors of 24-smt in fungi, trypanosoma cruzi, and leishmania amazonensis. antifungal activities of these inhibitors were also described against pneumocytis carinii and paracoccidioides brasiliensis. the purpose of the present study was to examine the susceptibilities of a collection of yeasts of the genus candida to aza and eil; determine the fungicidal activities of these compounds; and detect the main morphology and ultrastructural alterations of the yeasts after drug treatment.  antifungal susceptibility of candida isolates the mics obtained for the atcc strains to standard drugs and to the experimental compounds are listed in table interestingly, c. krusei has aza mic of μg.ml- and mic of μg.ml- on the other hand, eil did not inhibit the growth of the flc- and itc-resistant strains. all clinical isolates were susceptible to amb, with the median mic values ranging from to μg.ml- and the mic from to μg.ml-. however, three isolates showed mic values higher than μg.ml- susceptibility to flc was observed in 92% of the isolates, although 26% showed a trailing effect. clear resistance to flc was detected in three isolates. 45% of the strains showed mic of – μg.ml- and 37% showed mic of – μg.ml- on the other hand, 75% of the isolates were susceptible to itc, and 16% showed a trailing effect. resistance to itc was detected in isolates. most of the isolates had mic and mic for itc lower than μg.ml-. only c. krusei isolates were less susceptible to all standard drugs, showing a mic of μg.ml- for amb, > μg.ml- for flc, and μg.ml- for itc. ttrailing effect, rresistant the values are expressed in μg.ml- -not determinate; +mic results are medians; *trailing effect to flc; **trailing effect to itc. when the mic values for 24-smti were analysed, we observed important antifungal activity for almost all candida spp. isolates. for aza, mic and mic values lower than μg.ml- were observed in 86% and 52% of the isolates, respectively. for eil, mic and mic values lower than μg.ml- were observed in 92% and 63% of the isolates, respectively. c. zeylanoides and c. lipolytica showed mic50– values of ≤ μg.ml- for both inhibitors, whilst c. krusei was resistant to eil, with mic50– values of μg.ml-. however, both c. krusei and c. lipolytica were resistant to aza. finally, c. guilliermondii isolates, flc- and itc-resistant, were susceptible to aza, with mic50– values of – μg.ml- +mic results are medians. correlations between mic values positive correlations of the mic values were observed between aza and amb, aza and eil, and flc and itc. in addition, positive correlations were observed between the mic values of the flc and itc. on the other hand, no significant correlations were observed between the mic values for azoles and 24-smti. some clinical isolates with a trailing effect for flc and itc also showed residual growth at higher concentrations of aza of 58% and 54% of the isolates, respectively. residual growth was not observed in the isolates after treatment with eil. minimum fungicidal concentration of aza and eil the mfcs obtained for half of our isolates were higher than μg.ml- revealing a predominant fungistatic activity of the smti. interestingly, cna isolates showed mfcs lower than μg.ml- indicating a remarkable fungicidal activity, especially for aza. on the other hand, aza killed a negligible number of the c. albicans isolates at concentrations lower than μg.ml- * data is expressed in percentual of isolates. ultrastructural effects the general morphology of untreated c. albicans was observed using scanning and transmission electron microscopy. the shape of c. albicans varies from spherical to oval cells when viewed by scanning electron microscopy. transmission electron microscopy revealed the presence of normal cell walls with a thickness of ± nm, including a thin electron-dense outer layer with delicate fibrillar structures clearly visible. a continuous cytoplasmatic membrane lining a homogeneous and electron-dense cytoplasm containing ribosomes, nucleus, and nucleoli could also be observed. treatment of c. albicans with mic of aza and eil induced significant morphological changes, which ranged from discrete alterations to total destruction of the fungal cells. a common alteration observed after the treatment with aza and eil was a significant increase in cell size, from μm to μm in diameter. the number of altered cells was counted, and the morphological alterations appeared in % and % of the cells after treatment with aza and eil, respectively. among the most frequently observed ultrastructural alterations were: presence of small buds; irregular cell-wall surfaces; loss of cell-wall integrity, with an apparent shedding of cell components; and a two- to three-fold increment of the cell wall thickness was observed after treatment with aza and eil, respectively. the cytoplasmic membrane of treated cells also showed several changes, such as the presence of evaginations, discontinuity, detachment from the cell wall; budding of small vesicles that could migrate through the periplasmatic region, accumulate in the cytoplasm, or remain close to the cell membrane. vesicle sizes ranged from 40– nm for aza and 40– nm for eil. mitochondrial swelling and electron dense vacuoles accumulation was also observed. cna cells treated with mic of 24-smti showed similar ultrastructural changes. presence of lipid bodies treatment with mic of aza and eil induced an accumulation of lipid bodies in the cytoplasm, which can be characterised by the presence of small dots labelled with nile red, which were absent in the untreated yeasts. these lipid bodies seen by fluorescence microscopy can be correlated with the small, electron-dense vacuoles seen by transmission electron microscopy. effect of 24-smt inhibitors on the cell cycle dapi staining used to label the dna revealed that the treatment of c. albicans with aza and eil induced important alterations in the cell cycle. to quantitatively assess these alterations, different stages of the untreated yeasts were considered, such as: cells containing one nucleus, cells with a bud and one nucleus, and cells with a bud and two nuclei. after treatment with the mic50s of aza and eil, different alterations in the nucleus were observed, and these were classified as: cells with more than one nucleus, cells showing abnormal chromatin condensation, and cells without a nucleus. counting the number of abnormal cells revealed that approximately 66% of the yeasts showed abnormal chromatin condensation, whereas % of aza-treated and % of eil-treated cells contained more than one nucleus, and approximately 6% of the cells treated with both compounds had no nucleus. cytotoxicity evaluation cytotoxicity of 24-smti was evaluated against mammalian cells using the sulforhodamine b viability assay. for both aza and eil the cc was μg.ml- which corresponds to a mean selectivity index of for aza and for eil. discussion although c. albicans is the predominant species in candidiasis, cna species have increased in frequency in recent years. the reasons for the emergence of cna species are not fully understood, but some medical conditions may frequently run the risk of developing candidaemia due to the cna species: c. parapsilopsis has been associated with vascular catheters and parenteral nutrition; c. tropicalis with cancer and neutropenia; and c. krusei and c. glabrata with previous treatments with flc and itc. previous studies have described a high susceptibility of c. albicans isolates to azoles and amb, whereas cna isolates are usually less susceptible and may be intrinsically resistant to flc and itc. as reported by other investigators, none of our candida isolates showed mic ≥ μg.ml- for amb. mic values found for itc and flu were similar to those previously reported by different groups. however, in the present study, flc-resistant candida strains were only observed among cna species. however, itc-resistance was found in c. albicans and cna isolates. the observation of a high positive correlation between mic values of flc and itc, in this study, suggests that cross-resistance may be occurring. however, no correlation was observed between mic values of the azoles and 24-smti, indicating lack of possible cross-resistance. the general finding for our candida spp. isolates was that they were mostly susceptible to aza and eil, because the mic50s were lower than μg.ml- for 73% and 88% of the isolates after treatment with aza and eil, respectively. interestingly, some flc- and itc-resistant strains were susceptible to 24-smti. however, residual growth of candida after treatment with aza was similar to that observed for flc and itc. no residual growth was observed after treatment with eil. the fungicidal action of 24-smti was more prominent against cna species than against c. albicans isolates. a concentration of μg.ml- of 24-smti was enough to kill 100% of c. lusitanae, c. zeylanoides, and c. rugosa, and 50% of c. glabrata. in contrast, this same concentration killed only % and % of c. albicans isolates, considering aza and eil respectively. previous studies have shown that azasterol derivatives have antifungal activity against a variety of species. 15-azasterol, in concentrations ranging from μg.ml- to μg.ml- inhibits the growth of saccharomyces cerevisae and c. albicans, with a concomitant accumulation of sterol intermediate molecules. the range of mic and mfc values for 15-azasterol analogues against these fungal species varied from to μg.ml- and to μg.ml- respectively and are similar to the values obtained in the present study. other azasterol derivatives have been shown to inhibit s. cerevisae 24-smt, leading to the accumulation of zymosterol. recent work demonstrated that aza displays antifungal activity against paracoccidioides brasiliensis and pneumocystis carinii. concentrations of μm inhibited 100% of the growth in p. brasiliensis, and the treatment of p. carinii with the ic of μm led to growth arrest and accumulation of 24-desakyl sterols, indicating an inhibition of 24-smt. in addition, previous studies have also shown an anti-protozoan activity of aza and eil on t. cruzi epimastigotes and intracellular amastigotes, l. amazonensis promastigotes and intracellular amastigotes, toxoplasma gondii, and giardia lamblia, with mics in the low μm to sub-μm range. for protozoans, eil was reported to be more active than aza. in contrast, we found in this study that aza was more active than eil against candida spp. isolates. treatment of c. albicans yeasts with aza and eil caused dramatic changes in their cellular and sub-cellular structure. the main alterations included changes in the cell wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm, with the presence of an electron-lucent zone between them, cell collapse and release of cellular content, mitochondrial swelling, and abnormalities in the nuclear structure. these findings are similar to those previously reported after treatment of candida spp. with different azoles. borges and co-workers reported that exposure of candida albicans to itc leads to primary alterations at the cell periphery and the appearance of vacuoles in the cytoplasm, which may be lipid inclusions. these changes were usually accompanied by an increase in the cell volume and impaired cell division. in addition, studies by hazen and co-workers revealed that candida treated with flc shows a distinct retraction of the membrane from the cell wall. on the other hand, c. albicans treated with low concentrations of amb shows chromatin condensation and margination, separation of the nuclear envelope, and nuclear fragmentation. high concentrations of amb induce cellular changes characteristic of necrosis, showing many large vacuoles. additionally, bahmed and co-workers demonstrated an increase in cell wall thickness of candida yeasts, which may be related to alterations in the cell wall composition induced by the treatment with amb. in addition, similar to our findings, the appearance of multivesicular bodies and myelin-like structures were reported after treatment of leishmania and t. cruzi with aza and eil. staining with nile red revealed the presence of lipid accumulation in the cytoplasm after treatment with 24-smti, confirming that these compounds induce a perturbation in lipid biosynthesis. similar observations have recently been made as the result of treatment of leishmania amazonensis with 24-smti, which induced several abnormalities in the lipid content, with the accumulation of steroid intermediate molecules. in addition, staining of dna with dapi indicates a profound alteration in the cell cycle after treatment with aza and eil. candida yeasts produced unfertile buds that remained closely associated with the mother cell, and appeared with or without various nuclei. the nucleus may also have an altered shape and/or with abnormal chromatin condensation that might be associated with apoptosis cell death, as previously described after treatment of c. albicans with amb; and also after treatment of tritrichomonas foetus with hydrogen peroxide. the presence of many cells with more than one nucleus may also indicate that ergosterol biosynthesis inhibitors are interfering with cytokinesis. in fact, it was previously found that ergosterol levels modulate the activity of protein kinases such as pp60v-src and also the levels of camp, both of which are directly related to the control of the cell cycle. in addition, some studies have shown that drugs such as griseofulvin and nocodazole, which interfere with the assembly of cytoskeleton components, induce alterations in the cell cycle and apoptosis cell death. thus, there are two possible explanations for the alterations in the cell cycle: the depletion of ergosterol and other lipids, which are essential for the maintenance of the cytoplasmatic membrane structure and are also important key regulators of the cell cycle; and/or alterations in the cytoskeleton structure, which can affect the cytokinesis process. the results obtained here suggest that aza and eil are probably interfering with sterol biosynthesis in candida spp., as previously described for c. albicans, p. carinii, t. cruzi, and l. amazonensis. on the other hand, we cannot exclude the possibility that these compounds may be acting in other pathways, inducing some secondary effects that could be related to the accumulation of other lipids or, as demonstrated in crithidia deanei, that aza can interfere with phospholipid biosynthesis. further studies are necessary to characterise the correlation between the depletion of ergosterol and the cell cycle in c. albicans.  the results presented herein demonstrate the potential usefulness of the 24-smt inhibitors aza and eil as antifungal agents, including azole-resistant candida strains. the specific in vitro and in vivo antifungal and antiprotozoal activity of azasterols has been known for years, and in most cases has been linked to their specific inhibition of 24-smt, an enzyme absent in mammals. however, other studies have found that these compounds are also active against parasitic protozoa that lack endogenous sterol biosynthesis, such as t. gondii and trypanosoma brucei, indicating that they may have other biochemical targets. taken together, these results indicate azasterols as useful leads for novel antifungal agents, but optimisation of their selectivity, adme, pk, and toxicological properties is required for their further advancement as drug candidates. 
although candida species are commensal microorganisms, they can cause many invasive fungal infections. taken together, these results suggest that inhibition of 24-smt may be a novel approach to control candida spp. the purpose of this study was to evaluate the antifungal activity of inhibitors of Δ24-sterol methyltransferase, 20-piperidin-2-yl-5α-pregnan-3β-20-diol, and -epiminolanosterol, against clinical isolates of candida spp., analysing the ultrastructural changes. all strains used in this study were susceptible to amphotericin b; however, some isolates were fluconazole- and itraconazole-resistant. treatment with sub-inhibitory concentrations of aza and eil induced several ultrastructural alterations, including changes in the cell-wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, mitochondrial swelling, and the presence of electron-dense vacuoles. most of the azole-resistant isolates were candida non-albicans species, but several of them, such as c. guilliermondii, c. zeylanoides, and c. lipolytica, were susceptible to 24-smti, indicating a lack of cross-resistance. the fungicidal activity of 24-smti was particularly high against cna isolates. the selectivity of 24-smti for fungal cells versus mammalian cells was assessed by the sulforhodamine b viability assay. aza and eil were found to be potent growth inhibitors of candida spp. fluorescence microscopy analyses indicated an accumulation of lipid bodies and alterations in the cell cycle of the yeasts.
 although candida species are commensal microorganisms, they can cause many invasive fungal infections. in addition, antifungal resistance can contribute to failure of treatment. the purpose of this study was to evaluate the antifungal activity of inhibitors of Δ24-sterol methyltransferase, 20-piperidin-2-yl-5α-pregnan-3β-20-diol, and -epiminolanosterol, against clinical isolates of candida spp., analysing the ultrastructural changes.  aza and eil were found to be potent growth inhibitors of candida spp. isolates. the median mic was μg.ml- for aza and μg.ml- for eil, and the mic was μg.ml- for both compounds. all strains used in this study were susceptible to amphotericin b; however, some isolates were fluconazole- and itraconazole-resistant. most of the azole-resistant isolates were candida non-albicans species, but several of them, such as c. guilliermondii, c. zeylanoides, and c. lipolytica, were susceptible to 24-smti, indicating a lack of cross-resistance. reference strain c. krusei was consistently susceptible to aza, although not to eil. the fungicidal activity of 24-smti was particularly high against cna isolates. treatment with sub-inhibitory concentrations of aza and eil induced several ultrastructural alterations, including changes in the cell-wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, mitochondrial swelling, and the presence of electron-dense vacuoles. fluorescence microscopy analyses indicated an accumulation of lipid bodies and alterations in the cell cycle of the yeasts. the selectivity of 24-smti for fungal cells versus mammalian cells was assessed by the sulforhodamine b viability assay.  taken together, these results suggest that inhibition of 24-smt may be a novel approach to control candida spp. infections, including those caused by azole-resistant strains. 