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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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Related in: MedlinePlus

Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT C. albicans cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.
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pone-0104554-g006: Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT C. albicans cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.

Mentions: Mitochondrial dysfunction on BER treatment led us to determine the status of ROS in presence of BER. To measure the changes in ROS generation we exploited a fluorescent molecule chloromethyl-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which can readily enter into the cells and is sensitive to redox changes [24]. It is deacetylated by endogenous esterase into dichlorofluorescein, which fluoresces on oxidation by ROS. Wild type cells treated with a MIC50 concentration of BER (100 µg/ml) were allowed to incubate in presence of 10 µM of CM-H2DCFDA. As shown in Figure 6, fluorescence increased after BER treatment indicating generation ROS. The increase in ROS following BER treatment could be partially reversed by the addition of antioxidant such as AA [37]. This implied that BER treatment results in production of ROS, which could contribute to the antifungal effect exerted by BER.


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)

Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT C. albicans cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104554-g006: Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT C. albicans cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.
Mentions: Mitochondrial dysfunction on BER treatment led us to determine the status of ROS in presence of BER. To measure the changes in ROS generation we exploited a fluorescent molecule chloromethyl-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which can readily enter into the cells and is sensitive to redox changes [24]. It is deacetylated by endogenous esterase into dichlorofluorescein, which fluoresces on oxidation by ROS. Wild type cells treated with a MIC50 concentration of BER (100 µg/ml) were allowed to incubate in presence of 10 µM of CM-H2DCFDA. As shown in Figure 6, fluorescence increased after BER treatment indicating generation ROS. The increase in ROS following BER treatment could be partially reversed by the addition of antioxidant such as AA [37]. This implied that BER treatment results in production of ROS, which could contribute to the antifungal effect exerted by BER.

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