Abstract
Infectious diseases are a major challenge for modern medicine, especially those caused by emerging multidrug resistant pathogens. In particular, limited treatment options make invasive fungal infections challenging. Compared to the numerous classes of antibiotics marketed for the treatment of bacterial infections, only five antifungal drug classes are currently used in the clinic. This is partly because similarities between fungal and mammalian cells narrow the range of targets available for antifungal treatment. There is also an increasing incidence of antifungal resistance due to extended antifungal prophylaxis and antifungal overuse. It is therefore important to develop novel treatment strategies that improve therapeutic outcomes. This requires better understanding of the biology of individual fungal pathogens and the development of robust screens to identify new drug candidates. Candida auris is an emerging pathogenic yeast that causes severe infections, especially in intensive care and healthcare settings. Since its discovery in 2009, it has been found to cause nosocomial outbreaks across the globe. These have been difficult to eliminate because C. auris persists on many surfaces and most clinical isolates are highly resistant to one or more of the current antifungal drugs. This highlights the need to discover new drugs for the treatment of C. auris infections.
The objectives of this work were to (i) Functionally express selected C. auris genes involved in resistance to azoles in a Saccharomyces cerevisiae model, (ii) Validate this platform by quantitatively assessing the resultant phenotypes at the physiological and biochemical levels, (iii) Use the platform to screen for compounds that circumvent drug efflux-mediated resistance (iv) Use combinations successfully tested in the model platform against clinical isolates of C. auris.
The C. auris Erg11 protein (CauErg11), its mutants Y132F and K143R, and the efflux pumps CauMdr1 and CauCdr1 were successfully constitutively overexpressed in a S. cerevisiae expression host that is hypersensitive to azole drugs. The resultant phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Strains overexpressing the Cdr1 protein were pan-azole resistant, additionally conferring resistance to long-tailed azoles such as Itraconazole and Posaconazole. Type II binding spectra showed tight azole binding to the affinity purified recombinant CauErg11 protein. Use of Nile Red as a drug efflux substrate confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 also exhibited an ATPase activity that was diagnostically inhibited by Oligomycin.
Clorgyline and six of its analogs were screened using a C. auris clade I strain resistant to the azole drugs Voriconazole and Posaconazole. Two of the analogs (M19 and M25) appeared to inhibit growth of a clade I C. auris strain in the presence of the azole drugs. In a secondary screen, M19 and M25 were then found to act synergistically with azoles against recombinant S. cerevisiae strains overexpressing C. auris efflux pumps. Nile Red assays with the recombinant strains showed M19 and M25 inhibited the activity of CauCdr1 and CauMdr1 efflux pumps identified from phenotypic analysis as variously contributing to azole resistance in C. auris clades I, III, and IV. Clorgyline, M19 and M25 also uncoupled the Oligomycin-sensitive ATPase activity of Cdr1 from C. albicans and C. auris. Checkerboard assays quantitatively confirmed the synergistic effects of M19 and M25 in combination with azoles against multidrug resistant clinical isolates of C. auris clade I. The synergy of M19 and M25 with azoles appeared specific for C. auris but not C. albicans cells. As the susceptibility to Posaconazole is essentially unaffected by the CauErg11 Y132F and K143R mutations and because the efflux activity in clade I clinical isolates is dominated by CauCdr1, the combination of Posaconazole with M19 or M25 provides the basis for an experimental therapy that may be expanded to include other C. auris clades.
The S. cerevisiae platform developed in this work, which functionally express key proteins involved in C. auris drug resistance, not only provides insight into the nature of drug resistance in this pathogen but will also enable screens for novel azoles using phenotype- and ultimately structure-based approaches. Further, the experimental combination therapy developed in this work is a promising starting point to combat multidrug resistance in the emerging fungal pathogen C. auris. Despite a paucity of clinical guidelines and limited data available on the efficacy of treating fungal infections with combination therapy targeting both the azole drug target and drug efflux, the development of this approach could be used to extend the longevity of azole drugs and minimize pressure on current antifungals. Our finding suggest that the strategic use of such combination therapy could be a key to combatting multidrug resistant pathogens. However, more research is needed to understand the mode of action and broader efficacy of compounds M19 and M25.