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Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Dhamgaye S, Devaux F, Vandeputte P, Khandelwal NK, Sanglard D, Mukhopadhyay G, Prasad R - PLoS ONE (2014)

Bottom Line: However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes.Additionally, unlike hsf1 strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe.Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India; Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

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HSF1 conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, HSF1 conditional mutant and HSF1 heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, HSF1 conditional mutant and HSF1 heterozygous in presence of BER.
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pone-0104554-g003: HSF1 conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, HSF1 conditional mutant and HSF1 heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, HSF1 conditional mutant and HSF1 heterozygous in presence of BER.

Mentions: HSF1 is an essential gene and it could be likely that this TF mutant that was generated by UAU transposition could still exhibit HSF1 activity due to the chosen disruption method [19], [30]. To reconfirm HSF1 impact on MDR, we employed a conditional HSF1 mutant (hsf1Δ/tet-HSF1) in which HSF1 expression is under control of the TET promoter [19]. We tested the susceptibility of the HSF1 conditional mutant in presence of TET analogue, DOX, which shuts off the expression of HSF1. The results mirrored the BER susceptibility phenotype that was observed with the mutant strain JMR044 of TF library screen (Figure 3(a)). We also verified whether conditional mutant hsf1Δ/tet-HSF1 was collaterally affecting susceptibility to other drugs. FLC, AMB, TRB, and CAS (caspofungin). Additionally, we have also evaluated the effect of combination of BER and these drugs on conditional mutant hsf1Δ/tet-HSF1. Interestingly, the conditional HSF1 mutant was highly susceptible towards different classes of antifungal drugs including FLC, AMB, TRB, and CAS (caspofungin) (Figure 3(b)). Our susceptibility assays revealed that hsf1Δ/tet-HSF1 not only displayed enhanced susceptibility to BER but also to drugs acting on membrane ergosterol (TRB, FLC, AMB) or inhibit CW synthesis (CAS) (Figure 3(b)) [31], [32]. However, combination of BER and above mentioned drugs did not seem to enhance the susceptibility of HSF1 conditional mutant (Figure 3(b)). This highlights that the effects of BER and HSF1 leading to drug susceptibility are manifested by independent circuitry. The impact of HSF1 on drug susceptibilities appeared to be independent of known attributes of MDR [33], [34]. For example, the expression of CDR1, CDR2, MDR1, ERG11, TAC1, UPC2 did not change in the HSF1 conditional mutant in presence and absence of BER (figure S2 in File S1).


Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Dhamgaye S, Devaux F, Vandeputte P, Khandelwal NK, Sanglard D, Mukhopadhyay G, Prasad R - PLoS ONE (2014)

HSF1 conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, HSF1 conditional mutant and HSF1 heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, HSF1 conditional mutant and HSF1 heterozygous in presence of BER.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126717&req=5

pone-0104554-g003: HSF1 conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, HSF1 conditional mutant and HSF1 heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, HSF1 conditional mutant and HSF1 heterozygous in presence of BER.
Mentions: HSF1 is an essential gene and it could be likely that this TF mutant that was generated by UAU transposition could still exhibit HSF1 activity due to the chosen disruption method [19], [30]. To reconfirm HSF1 impact on MDR, we employed a conditional HSF1 mutant (hsf1Δ/tet-HSF1) in which HSF1 expression is under control of the TET promoter [19]. We tested the susceptibility of the HSF1 conditional mutant in presence of TET analogue, DOX, which shuts off the expression of HSF1. The results mirrored the BER susceptibility phenotype that was observed with the mutant strain JMR044 of TF library screen (Figure 3(a)). We also verified whether conditional mutant hsf1Δ/tet-HSF1 was collaterally affecting susceptibility to other drugs. FLC, AMB, TRB, and CAS (caspofungin). Additionally, we have also evaluated the effect of combination of BER and these drugs on conditional mutant hsf1Δ/tet-HSF1. Interestingly, the conditional HSF1 mutant was highly susceptible towards different classes of antifungal drugs including FLC, AMB, TRB, and CAS (caspofungin) (Figure 3(b)). Our susceptibility assays revealed that hsf1Δ/tet-HSF1 not only displayed enhanced susceptibility to BER but also to drugs acting on membrane ergosterol (TRB, FLC, AMB) or inhibit CW synthesis (CAS) (Figure 3(b)) [31], [32]. However, combination of BER and above mentioned drugs did not seem to enhance the susceptibility of HSF1 conditional mutant (Figure 3(b)). This highlights that the effects of BER and HSF1 leading to drug susceptibility are manifested by independent circuitry. The impact of HSF1 on drug susceptibilities appeared to be independent of known attributes of MDR [33], [34]. For example, the expression of CDR1, CDR2, MDR1, ERG11, TAC1, UPC2 did not change in the HSF1 conditional mutant in presence and absence of BER (figure S2 in File S1).

Bottom Line: However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes.Additionally, unlike hsf1 strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe.Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India; Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Show MeSH
Related in: MedlinePlus