Does the phenotypic adaptability of Candida auris enable its success as a multi-drug resistant human pathogen?
Lead Research Organisation:
University of Aberdeen
Department Name: Sch of Medicine, Medical Sci & Nutrition
Abstract
Candida auris has recently emerged as a major hospital-acquired pathogen and poses a global health concern. C. auris is highly drug resistant, adhesive to skin and medical devices, and difficult to eradicate from intensive care wards. Mortality rates for invasive disease approach 50%. Consequently, major health authorities, including the CDC (Centre for Disease Control) and PHE (Public Health England) at Bristol have posted alerts about C. auris.
Stress resistance and adaptation promote fungal pathogenicity and immune evasion. Therefore, we urgently need to characterise how stress adaptation in C. auris impacts host-pathogen interactions. We hypothesize that, under stress, C. auris shows a strong propensity for genomic and phenotypic adaptations, which drive its resistance and tolerance to antifungal drugs, and its ability to evade host immunity.
(1) Impact of stress adaptation upon the C. auris cell wall
We have recently surveyed cell wall ultrastructure and biofilm formation in multiple clinical isolates of C. auris covering all four geographical clades. We observed, that C. auris tends to have thicker cell walls but shorter mannan fibrils than the C. albicans SC5314 strain, when grown on YPD, and that C. auris is not a particularly efficient biofilm producer. Importantly, the clinical C. auris isolates display substantial variability in these features both within and between clades. This strongly indicates that C. auris shows plasticity in these cellular properties, which probably contributes to fungal adapation to host niches. This certainly is the case for C. albicans, where we have shown that the Candida cell wall is substantially remodelled in response to physiologically relevant environmental stresses posed by niches in the host body. To elucidate how C. auris reacts to host-relevant osmotic/cationic and oxidative stresses, we will expose a selection of C. auris strains to sorbose, H2O2, and NaCl, and monitor their ultrastructural and biochemical cell wall features using an array o0f methods. In addition, we will monitor the expression of genes encoding key cell wall proteins and regulators by qRT-PCR to ascertain whether they are induced during stress adaptation.
(2) Impact of stress adaptation upon antifungal drug tolerance and resistance in C. auris
To assess whether stress adaptation protects C. auris against antifungal treatment, we will subject the C. auris isolates to osmotic, oxidative/cationic stresses, and then expose these stress-adapted cells to MIC-testing with an array of clinically relevant antifungals. Furthermore, using an established strategy we will micro-evolve stress-resistant C. auris strains to explore the mechanistic links between epigenetic stress adaptation and genetic stress resistance in C. auris and the resultant protection against antifungal drugs. To characterise genetic mechanisms, we will test whether stress and antifungal drug resistances depend on stress regulators and ABC efflux transporters.
(3) Does stress adaptation give C. auris the upper hand during host-pathogen interactions?
To test whether stress-adapted and stress-resistant C. auris cells gain an advantage during interaction with the host immune system, we will employ ex vivo macrophages assays, and in vivo zebrafish infection models. The in vivo model will establish whether stress-adapted and stress-resistant strains are more virulent than the untreated control strain, by testing how quickly and efficiently particular strains kill zebrafish larvae via hindbrain-injection. We will also utilise fluorophore reporters on macrophages and on neutrophils in zebrafish to visualise yeast-macrophage and yeast-neutrophil interactions in vivo. Detailed insight into the host-pathogen interactions will be provided by imaging interactions between C. auris cells and murine bone marrow-derived macrophages (BMDMs) ex vivo. We will assay rates of fungal recognition, phagocytosis and killing.
Stress resistance and adaptation promote fungal pathogenicity and immune evasion. Therefore, we urgently need to characterise how stress adaptation in C. auris impacts host-pathogen interactions. We hypothesize that, under stress, C. auris shows a strong propensity for genomic and phenotypic adaptations, which drive its resistance and tolerance to antifungal drugs, and its ability to evade host immunity.
(1) Impact of stress adaptation upon the C. auris cell wall
We have recently surveyed cell wall ultrastructure and biofilm formation in multiple clinical isolates of C. auris covering all four geographical clades. We observed, that C. auris tends to have thicker cell walls but shorter mannan fibrils than the C. albicans SC5314 strain, when grown on YPD, and that C. auris is not a particularly efficient biofilm producer. Importantly, the clinical C. auris isolates display substantial variability in these features both within and between clades. This strongly indicates that C. auris shows plasticity in these cellular properties, which probably contributes to fungal adapation to host niches. This certainly is the case for C. albicans, where we have shown that the Candida cell wall is substantially remodelled in response to physiologically relevant environmental stresses posed by niches in the host body. To elucidate how C. auris reacts to host-relevant osmotic/cationic and oxidative stresses, we will expose a selection of C. auris strains to sorbose, H2O2, and NaCl, and monitor their ultrastructural and biochemical cell wall features using an array o0f methods. In addition, we will monitor the expression of genes encoding key cell wall proteins and regulators by qRT-PCR to ascertain whether they are induced during stress adaptation.
(2) Impact of stress adaptation upon antifungal drug tolerance and resistance in C. auris
To assess whether stress adaptation protects C. auris against antifungal treatment, we will subject the C. auris isolates to osmotic, oxidative/cationic stresses, and then expose these stress-adapted cells to MIC-testing with an array of clinically relevant antifungals. Furthermore, using an established strategy we will micro-evolve stress-resistant C. auris strains to explore the mechanistic links between epigenetic stress adaptation and genetic stress resistance in C. auris and the resultant protection against antifungal drugs. To characterise genetic mechanisms, we will test whether stress and antifungal drug resistances depend on stress regulators and ABC efflux transporters.
(3) Does stress adaptation give C. auris the upper hand during host-pathogen interactions?
To test whether stress-adapted and stress-resistant C. auris cells gain an advantage during interaction with the host immune system, we will employ ex vivo macrophages assays, and in vivo zebrafish infection models. The in vivo model will establish whether stress-adapted and stress-resistant strains are more virulent than the untreated control strain, by testing how quickly and efficiently particular strains kill zebrafish larvae via hindbrain-injection. We will also utilise fluorophore reporters on macrophages and on neutrophils in zebrafish to visualise yeast-macrophage and yeast-neutrophil interactions in vivo. Detailed insight into the host-pathogen interactions will be provided by imaging interactions between C. auris cells and murine bone marrow-derived macrophages (BMDMs) ex vivo. We will assay rates of fungal recognition, phagocytosis and killing.
