PhD SCHOLAR, MONASH UNIVERSITY
One drug. One target. Multiple pathways.
The human body houses millions of microbes, both friendly and hostile. The harmful ones are kept in check by a healthy population of the friendly microbes and the host immune response. But when these lines of control fail, a war is declared; the immune system versus pathogen battle. Accounting for a mortality rate of up to ~75% and 4,00,000 life-threatening infections per year, Candida albicans is the worst foe. This otherwise docile fungus “switches” into an infectious form that can invade and colonize almost every body organ, resulting in fatal candidiasis. Attempts to target this switch has met with little success since it involves large numbers of molecular players.
Switching requires turning on the expression of thousands of genes, which is no easy task because the DNA in the cell is stored like a tangled ball of yarn, that needs to be unwound to read the genetic information contained within. Cells achieve this remarkable feat by deploying molecular machines known as chromatin remodelers, that physically open up and prime the DNA for expression. My research focusses on one such remodeler, a master regulator whose absence brings all the vital processes of a cell to a standstill, and the pathogen eventually succumbing to death. While we are getting closer to target this complex for designing antifungals, we aren’t there yet.
By combining various “omics” approaches, my work aims to identify the key players and pathways associated with the complex. In addition to finding the missing links, I attempt to understand the consequences of crippling its action, at the host and pathogen levels. Although the remodeler could serve as a potent target, the presence of a similar, closely related complex in humans, remains a big challenge. A drug disarming the Candida target could become a double-edged sword. My goal is to tackle this problem by characterizing a Candida-specific subunit of this multi-membered complex.
Development of resistance to the limited clinical arsenal has been a longstanding dilemma for treating infectious diseases; Candida albicans is no exception. This is mainly because the bugs can easily tweak the only pathway targeted by a drug and render it useless. By exploiting the powers of a hub controlling multiple biological circuits, I hope to bring about a change in the conventional ‘one drug-one pathway’ paradigm.
Abstract: The human pathogen Candida albicans is able to undergo a reversible switch between two distinct cell types called white and opaque, which are considered different transcriptional states of cells harbouring identical genomes. The present model of switching regulation includes the bistable expression of a master switch gene that is controlled by multiple transcriptional feedback loops. Here, we show that chromatin-modifying enzymes constitute an additional important regulatory layer of morphogenetic switching. We identify eight chromatin modifiers as switching modulators. Extensive epistasis analysis maps them into at least two independent signalling pathways overlaying the known transcriptional network. Interestingly, we identify the conserved Set3/Hos2 histone deacetylase complex as a key regulator relying on the methylation status of histone H3 lysine 4 for switching modulation. Furthermore, we demonstrate that opaque to white switching is facilitated by the presence of adenine in vitro, but adenine has no effect on switching once the Set3/Hos2 complex is disrupted. Our observations postulate that chromatin modifications may serve as a means to integrate environmental or host stimuli through the underlying transcriptional circuits to determine cell fate in C. albicans.
Pub.: 27 Jun '09, Pinned: 31 Dec '17
Abstract: Fungal septicemia is an increasingly common complication of immunocompromised patients worldwide. Candida species are the leading cause of invasive mycoses with Candida glabrata being the second most frequently isolated Candida species from Intensive Care Unit patients. Despite its clinical importance, very little is known about the mechanisms that C. glabrata employs to survive the antimicrobial and immune response of the mammalian host. Here, to decipher the interaction of C. glabrata with the host immune cells, we have screened a library of 18,350 C. glabrata Tn7 insertion mutants for reduced survival in human THP-1 macrophages via signature-tagged mutagenesis approach. A total of 56 genes, belonging to diverse biological processes including chromatin organization and golgi vesicle transport, were identified which are required for survival and/or replication of C. glabrata in macrophages. We report for the first time that C. glabrata wild-type cells respond to the intracellular milieu of macrophage by modifying their chromatin structure and chromatin resistance to micrococcal nuclease digestion, altered epigenetic signature, decreased protein acetylation and increased cellular lysine deacetylase activity are the hall-marks of macrophage-internalized C. glabrata cells. Consistent with this, mutants defective in chromatin organization (Cgrsc3-aΔ, Cgrsc3-bΔ, Cgrsc3-aΔbΔ, Cgrtt109Δ) and DNA damage repair (Cgrtt107Δ, Cgsgs1Δ) showed attenuated virulence in the murine model of disseminated candidiasis. Further, genome-wide transcriptional profiling analysis on THP-1 macrophage-internalized yeasts revealed deregulation of energy metabolism in Cgrsc3-aΔ and Cgrtt109Δ mutants. Collectively, our findings establish chromatin remodeling as a central regulator of survival strategies which facilitates a reprogramming of cellular energy metabolism in macrophage-internalized C. glabrata cells and provide protection against DNA damage.
Pub.: 24 Aug '12, Pinned: 31 Dec '17
Abstract: Recent cumulative data show that various transcription factors are recruited to the chromatin in an iron-responsive manner to affect diverse cellular functions in the pathogenic fungus Candida albicans. Here we identified groups of iron-responsive genes in C. albicans by chromatin remodelling analysis at gene promoters, using micrococcal nuclease (MNase) digestion followed by deep sequencing. Chromatin in the promoter regions of iron uptake and utilization genes showed repressed and active configuration, respectively, under iron-replete conditions. GO Term enrichment analysis of genes with differentially remodelled chromatin, in respective promoter locales, suggested that many genes involved in adhesion are also iron-responsive. C. albicans was observed to be more self-adherent (twofold increase) and formed higher biofilm mass (77% increase) in the presence of iron. Furthermore, we identified various known and novel adhesion-related genes with iron-dependent active chromatin profiles that are indicative of potential upregulation under iron-replete conditions. Transcription factor Cph1 that is activated upon Cek1 phosphorylation also showed an active chromatin profile under iron-replete conditions and cells showed iron-responsive Cek1 MAPK phosphorylation in the presence of iron. Thus, iron affects diverse biological functions by modulating chromatin profiles of large gene sets and by signalling through Cek1 MAPK in C. albicans.
Pub.: 04 Jun '14, Pinned: 31 Dec '17
Abstract: Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.
Pub.: 19 May '17, Pinned: 31 Dec '17
Abstract: Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1, a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We've identified the Swi/Snf chromatin remodeling complex as a key co-activator for Mrr1, which is required to maintain basal and induced 'open' chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2, the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1(GOF) mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1(GOF) mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1(GOF) strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple co-activators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity restoring small molecules more complicated.
Pub.: 16 Aug '17, Pinned: 31 Dec '17
Abstract: The ability of dimorphic transition between yeast and hyphal forms in Candida albicans is one of the vital determinants for its pathogenicity and virulence. We isolated C. albicans SWI1 as a suppressor of the invasive growth defect in a Saccharomyces cerevisiae mutant. Expression of C. albicans SWI1 in S. cerevisiae partially complemented the growth defect of a swi1 mutant in the utilization of glycerol. Swi1 is in a complex with Snf2 in C. albicans, and both proteins are localized in the nucleus independent of the growth form. Deleting SWI1 or SNF2 in C. albicans prevented true hyphal formation and resulted in constitutive pseudohypha-like growth in all media examined. Furthermore, swi1/swi1 mutant was defective in hypha-specific gene expression and avirulent in a mouse model of systemic infection. These data strongly suggest the conserved Swi/Snf complex in C. albicans is required for hyphal development and pathogenicity.
Pub.: 02 May '06, Pinned: 31 Dec '17