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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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TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of HSF1 (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.
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pone-0104554-g002: TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of HSF1 (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.

Mentions: To understand the mechanism behind susceptibility to BER, we carried out a screen of an in-house library of TF mutant strains of C. albicans[15]. There are ∼275 genes that contains DBD in C. albicans these genes were deleted individually as explained in materials and methods section. All the strains that appear reproducibly (in triplicate) susceptible were subjected to a final screen as shown in Figure 2(a). Among all the TFs, HSF1, a heat shock factor mutant (JMR044) was most susceptible to BER treatment (MIC50 25 µg/ml) in comparison with WT strain (DAY286) (MIC50 100 µg/ml) (figure S1 in File S1). End point comparative RTPCR revealed a slight increase in the expression of HSF1 after BER treatment in C. albicans, thus implying a direct effect of BER on its expression (Figure 2(b)).


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)

TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of HSF1 (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104554-g002: TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of HSF1 (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.
Mentions: To understand the mechanism behind susceptibility to BER, we carried out a screen of an in-house library of TF mutant strains of C. albicans[15]. There are ∼275 genes that contains DBD in C. albicans these genes were deleted individually as explained in materials and methods section. All the strains that appear reproducibly (in triplicate) susceptible were subjected to a final screen as shown in Figure 2(a). Among all the TFs, HSF1, a heat shock factor mutant (JMR044) was most susceptible to BER treatment (MIC50 25 µg/ml) in comparison with WT strain (DAY286) (MIC50 100 µg/ml) (figure S1 in File S1). End point comparative RTPCR revealed a slight increase in the expression of HSF1 after BER treatment in C. albicans, thus implying a direct effect of BER on its expression (Figure 2(b)).

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