CandidOmics: Integrative omics study to probe Candida auris

This project will investigate the emerging fungal pathogen of Candida auris in regard to its pathogenicity and the molecular mechanisms underlying antifungal multidrug resistance (AMDR). Discovered in 2009, C. auris has now spread in over 20 countries on 5 continents in less than a decade. It can cause deep-seated infections disseminating via the bloodstream, as well as infections of the respiratory tract, urinary tract, bone, and soft tissues. Importantly, C. auris can thrive on abiotic surfaces such as catheters, posing a severe risk for individuals with impaired host immune system or other health conditions prevalent in the ageing population. It poses a global health threat due to its high transmissibility, AMDR and the notorious misdiagnosis in clinical diagnostics. Its pronounced drug resistance to all antifungal drugs enables C. auris to persist, colonize and re-infect patients. The rapid spreading in hospital settings is further enhanced by its propensity to strongly adhere to skin tissues. Due to this high transmissibility by skin-to-skin contact, it is the first fungal pathogen to be categorized by the Centers for Disease Control and Prevention (CDC) as a public health threat. A major bottleneck to combat C. auris infection is our lack of molecular understanding about its pathogenesis and virulence. To address this issue, this project will exploit an integrative multi-omics approach to unravel the molecular basis of C. auris pathogenesis and its AMDR mechanisms. We will apply state-of-the-art technologies in genomics and proteomics to decipher C. auris virulence, interaction with host immunity using primary immune cell cultures and in vivo mouse models. We will study the genes and regulators required for AMDR, especially for the drugs caspofungin and amphotericin B. The results will yield a better understanding of pathogenicity mechanisms of C. auris, and reveal the molecular bases of AMDR. Furthermore, data from this project will be relevant with regard to C. auris biomarker discovery and identification of new therapeutic targets for antifungal drug discovery. In an era of infectious disease outbreaks, advancing the molecular knowledge about infectious microbes are of fundamental importance to be better prepared in case of pandemics. Therefore, data from this project will serve as an invaluable asset to discover new therapeutic options for infectious human fungal pathogens, particularly those caused by Candida species, and more importantly, disclose the molecular underpinnings of C. auris infections.

Candida auris is an emerging multidrug-resistant fungal pathogen recognized as an urgent threat by the US Centers for Disease Control and categorized as critical priority by the World Health Organization. Despite its clinical importance our grasp of C. auris pathogenesis and mechanisms of drug resistance remains elusive due to limited molecular data. This poses a fundamental challenge to our understanding of the pathogen and devise intervention strategies. The primary aim of this project was to decipher the pathogenesis of C. auris and unravel its drug resistance mechanisms particularly against Amphotericin B (AmB). To this end, we have utilized an integrative quantitative proteomics strategy to address the host-pathogen interaction by extracting molecular data from C. auris during; i) in-vitro co-culture with macrophages from mice and, ii) in-vivo assay by intraperitoneal injection (i.p.) of C. auris cells into mice and subsequent re-isolation of fungal cells. Integrated omics data analysis revealed several virulence factors including proteins such as ADH2 and HSP21. To validate their function in virulence, C. auris mutants lacking genes ADH2 and HSP21 were generated by a fusion-PCR strategy. These mutants were subsequently injected in mice and tested for viability, which revealed their high sensitivity to killing by the host in-vivo in contrast to wild-type C. auris. To elucidate drug resistance mechanism of C. auris, fungal cells were treated with AmB and subjected to proteomics analysis. Additionally, a novel drug-repurposing strategy was developed utilizing the proteome database of C. auris to obtain a list of FDA-approved drugs for anti-auris activity. The screening resulted in the identification of the drug Tavaborole (C7H6BFO2) which showed promising antifungal effect against all five clades of C. auris with IC50 in M-range. To exploit the novel function of Tavaborole, a comparative proteomics analysis was performed between fungal cells treated with Tavaborole and AmB. Upon AmB treatment, stress response molecules (DDR48, orf19.7085, orf19.4503, orf19.813), molecules of glycerol biosynthesis (RHR2) and efflux drug transporter (SGE1) were identified. The resistance mechanism against AmB seems to be the overexpression of stress molecules to counter the oxidative stress induced by the drug, expression of efflux drug transporters and synthesis of osmolytes such as glycerol to counter the osmolytic effect of AmB. Tavaborole inhibits Leucyl-tRNA synthetase which catalyzes ligation of the amino acid L-leucine to tRNA. Proteomics data from candida challenged with Tavaborole, revealed the overexpression of proteins associated with leucine biosynthesis (LEU1, LEU4) and GLT1 responsible for glutamate biosynthesis suggesting an intricate control of amino acid biosynthesis in response to Tavaborole. Additionally, electron microscopy was performed on C. auris cells treated with AmB and Tavaborole. Results revealed the build-up of large intracellular space in both AmB and Tavaborole treated candida cells when compared to untreated controls.