Monday Article #58: Role of phosphorylation for chromatin remodelling in antifungal gene expression
Is ATPase activity in the Snf2 catalytic subunit of SWI/SNF chromatin remodelling complex is vital for Pleiotropic Drug Resistance gene expression? - Let’s find out!
Candida species (spp.) has been an underlying public health issue and has posed a serious clinical problem to mankind since its discovery in 400 BC. Candida infections are usually the cause of thrushes. These thrush symptoms are often described as irritations or burns . Infections by Candida species are often superficial such as oral and vaginal candidiasis . There is a high infection rate as evidently suggested by an annual estimate of 400,000 Candida cases reported, with a relatively concerning mortality rate of 38-49% [3-6]. There have been proven and effective therapies to treat Candida infections such as azoles and the fungicidal echinocandins drug . However, efforts to combat Candida spp. have been futile, as there is an increasing emergence of multi-drug resistant Candida strains. This is seen especially in pathogenic Candida glabrata and the emerging Candida auris strain .
This article aims to tackle the issue of multi-drug resistant strains by observing the function of multidrug transporters and its regulation in the model organism Saccharomyces cerevisiae. The strain of interest, C. glabrata, is closely related to the model organism S. cerevisiae .
An overview of multidrug transporters and control of pleiotropic drug resistance
Multidrug transporters are glycoproteins located in the cell membrane that actively transport small lipophilic molecules from one side of the cell membrane to the other, usually from inside to the outside of a cell. Common examples of multidrug transporters include - ATP-binding cassette (ABC) transporters, which can either be influx and efflux transporters [9,10]. An ABC transporter is a multisubunit protein consisting of two transmembrane domains and two ATP-binding domains .
In normal conditions, there are usually fewer multidrug transporters, and thus the yeast cell is only able to efflux some antifungal drugs. The remaining drugs, such as azoles, will interfere with an enzyme responsible for fungal cell membrane formation and will eventually cause cell death. Multidrug resistance is caused by overexpression of multidrug transporters - where more transporters extrude the antifungal drugs, whereby the yeast cell will be drug-free. In fungal cells, this resistance is known as the pleiotropic drug resistance (PDR) [12,13]. The gene regulation of PDR5; a homolog of CDR1 which is responsible for conferring drug resistance in Candida strains, is under investigation in this study to improve our understanding on the specific mechanism as it is currently not confidently understood .
SWI/SNF chromatin remodeler regulates the PDR5 gene, encoding for Pdr5 multidrug transporters. The Kubota group at the University of Aberdeen recently found that SWI/SNF localises at the PDR5 gene promoter and upregulates its expression . Specifically, transcription factors Pdr1 and Pdr3 recruit the SWI/SNF remodelling complex, activating transcription and causing this upregulation [16,17]. The remodelling complex is ATP-dependent and modulates nucleosome positions so RNA polymerase is able to gain access to the PDR5 gene for transcription . However, the molecular mechanism involving SWI/SNF in the regulation of PDR5 is unknown.
An approach to determine whether ATPase activity in the catalytic subunit ofthe SWI/SNF chromatin remodelling complex is crucial for the development ofpleiotropic drug resistance.
This study aims to suggest how SWI/SNF might control expression of PDR5 in S. cerevisiae. This is done by using gene manipulation techniques to manipulate the ATPase activity of SWI/SNF to evaluate the importance of the ATPase activity in regards to PDR5 expression18. The ATPase mutant was designed by altering the lysine codon to that of alanine, giving rise to the snf2-K798A mutant. This point mutation was introduced into the ATPase domain of Snf2, the catalytic subunit of the remodelling complex SWI/SNF. Transformation reactions were performed to create this mutant strain. PCR and agarose gel electrophoresis were carried out to confirm which DNA regions have been successfully amplified. The successful fragments were then sequenced and the sequencing data was analysed using the Clustal Omega software. Finally, a drug sensitivity assay was performed to examine the sensitivity of the wild-type, snf2Δ, snf2-K798A and pdr5Δ strains to 3.2 µM ketoconazole antifungal.
The drug sensitivity assay showed that pdr5Δ was extremely sensitive to the azole antifungal because of its inability to express the PDR5 genes encoding for multidrug transporters. Similar results were observed in both snf2Δ and snf2-K798A, suggesting that the SNF2 catalytic subunit and ATPase activity are indeed vital for expression of PDR5 since a lack of cell growth was observed. PDR5 was not upregulated and therefore, yeast cells are sensitive to ketoconazole (Figure 1).
Based on findings from this study, it is evident that ATPase activity is crucial for upregulation of the PDR5 gene as ATP is required as an energy source for modulation of nucleosome position, permitting access to RNA polymerase for PDR5 gene transcription.
However, there were some instances of growth even when the strains were treated with ketoconazole. This could suggest that the PDR5 gene is still constitutively expressed although ATP is absent. This would be a future area of investigation; we would need to confirm whether it is a constitutive expression of PDR5 and if it is; it is unknown what governs this expression.
To further investigate the role of PDR5 and its regulation, we could examine mRNA levels of the PDR5. This could be done through molecular biology techniques such as Northern blotting, ChIP assays and RT-qPCR using the PDR5 mRNA obtained from transcription. This would potentially help to confirm how much expression there is with the induction of the K798A mutation and that multidrug transporters are still being encoded for despite the absence of ATPase activity.
Candida infections and resistance to antifungals are increasing daily. Therefore, it is of utmost importance that further investigations have to be carried out in regards to improving our understanding on pleiotropic drug resistance. Every effort would also prevent extensively drug resistant candida strains from arising, which would further lengthen our ensuing battle with pathogenic Candida species.
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