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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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Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and HSP90 gene deleted to evaluate BER MIC50, (b) end point comparative RTPCR of genes involved in CW integrity in WT C. albicans cells in presence and absence of BER, (c) and in HSF1 conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.
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pone-0104554-g004: Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and HSP90 gene deleted to evaluate BER MIC50, (b) end point comparative RTPCR of genes involved in CW integrity in WT C. albicans cells in presence and absence of BER, (c) and in HSF1 conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.

Mentions: Taking clue from hsf1Δ/tet-HSF1, which was susceptible to CW inhibitors, we explored whether the genes involved in the CW integrity pathway were affected in BER-treated cells [36]. By employing liquid assay according to the CLSI protocol, we compared BER MICs in mutant strains for the genes involved in heat shock (HSP90), calcineurin (CNA1, CNA2, CRZ1), and MAP kinase pathways (BCK1, MKC1). As evident from the assay, calcineurin (crz1Δ/crz1Δ, cna1Δ/cna1Δ) and MAP kinase pathways (bck1Δ/bck1Δ) mutants were more susceptible to BER treatment with MIC50 of 25 µg/ml as compared to wild type (100 µg/ml) (Figure 4(a)). End point comparative RTPCR confirmed that the expression of genes involved in these pathways (including CRZ1, CNA1 and BCK1) was increased upon BER treatment (Figure 4(b)). Notably, depletion of HSF1 levels did not arrest the increased expression of these genes upon BER treatment (Figure 4(c)) in combination of DOX indicating BER effect on these genes is independent of HSF1.


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)

Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and HSP90 gene deleted to evaluate BER MIC50, (b) end point comparative RTPCR of genes involved in CW integrity in WT C. albicans cells in presence and absence of BER, (c) and in HSF1 conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104554-g004: Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and HSP90 gene deleted to evaluate BER MIC50, (b) end point comparative RTPCR of genes involved in CW integrity in WT C. albicans cells in presence and absence of BER, (c) and in HSF1 conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.
Mentions: Taking clue from hsf1Δ/tet-HSF1, which was susceptible to CW inhibitors, we explored whether the genes involved in the CW integrity pathway were affected in BER-treated cells [36]. By employing liquid assay according to the CLSI protocol, we compared BER MICs in mutant strains for the genes involved in heat shock (HSP90), calcineurin (CNA1, CNA2, CRZ1), and MAP kinase pathways (BCK1, MKC1). As evident from the assay, calcineurin (crz1Δ/crz1Δ, cna1Δ/cna1Δ) and MAP kinase pathways (bck1Δ/bck1Δ) mutants were more susceptible to BER treatment with MIC50 of 25 µg/ml as compared to wild type (100 µg/ml) (Figure 4(a)). End point comparative RTPCR confirmed that the expression of genes involved in these pathways (including CRZ1, CNA1 and BCK1) was increased upon BER treatment (Figure 4(b)). Notably, depletion of HSF1 levels did not arrest the increased expression of these genes upon BER treatment (Figure 4(c)) in combination of DOX indicating BER effect on these genes is independent of HSF1.

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