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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

BER treatment results in dysfunctional mitochondria (a) growth of C. albicans cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in C. albicans WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, HSF1 conditional mutant and HSF1 heterozygous strains in presence and absence of BER.
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pone-0104554-g005: BER treatment results in dysfunctional mitochondria (a) growth of C. albicans cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in C. albicans WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, HSF1 conditional mutant and HSF1 heterozygous strains in presence and absence of BER.

Mentions: To assess functional mitochondria, we compared the growth of BER treated cells in non-fermentative and in fermentative carbon sources and compared it with the growth of untreated cells. We observed that, even at non-lethal BER concentration (below MIC50; 75 µg/ml), cells were unable to grow when non-fermentable glycerol was provided as sole carbon source (Figure 5(a)). We addressed the mitochondrial activity by employing MTR-FM probe [25]. MTR-FM probe is known to passively diffuse across yeast cell membrane and to accumulate in active mitochondria, which can be visualized by enhanced fluorescence. Using flow cytometry depicted in Figure 5(b) BER-treated cells showed poor fluorescence intensity as compared to untreated cells. Together, poor growth in non-fermentable carbon source and decrease in MTR-FM fluorescence reinforced dysfunctional mitochondria following BER treatment. Notably, HSF1 conditional mutant did not show any impact on mitochondrial functional status (Figure 5 (c))


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)

BER treatment results in dysfunctional mitochondria (a) growth of C. albicans cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in C. albicans WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, HSF1 conditional mutant and HSF1 heterozygous strains in presence and absence of BER.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104554-g005: BER treatment results in dysfunctional mitochondria (a) growth of C. albicans cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in C. albicans WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, HSF1 conditional mutant and HSF1 heterozygous strains in presence and absence of BER.
Mentions: To assess functional mitochondria, we compared the growth of BER treated cells in non-fermentative and in fermentative carbon sources and compared it with the growth of untreated cells. We observed that, even at non-lethal BER concentration (below MIC50; 75 µg/ml), cells were unable to grow when non-fermentable glycerol was provided as sole carbon source (Figure 5(a)). We addressed the mitochondrial activity by employing MTR-FM probe [25]. MTR-FM probe is known to passively diffuse across yeast cell membrane and to accumulate in active mitochondria, which can be visualized by enhanced fluorescence. Using flow cytometry depicted in Figure 5(b) BER-treated cells showed poor fluorescence intensity as compared to untreated cells. Together, poor growth in non-fermentable carbon source and decrease in MTR-FM fluorescence reinforced dysfunctional mitochondria following BER treatment. Notably, HSF1 conditional mutant did not show any impact on mitochondrial functional status (Figure 5 (c))

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