Acquired amphotericin B resistance and fitness trade-off compensation in Candida auris

Abstract

Candida auris is an emergent human fungal pathogen of growing concern due to common drug resistance to all major antifungal drug classes. Although resistance to amphotericin B (AMB) has been detected in 30 to 60% of clinical isolates of C. auris, mechanisms of AMB resistance remain poorly characterized. Here we present a large-scale investigation of how AMB resistance can be acquired through genetic adaptation. We typed 441 in vitro and in vivo evolved C. auris lineages from four AMB-susceptible clinical strains of different clades. We show a great diversity of acquired resistance responses with resistance magnitude- and strain-dependent fitness trade-offs. Genotyping and membrane sterol analyses of selected lineages show four major types of membrane sterol alterations. Using a novel, plasmid-based CRISPR-Cas9 allele editing method and Cas9-RNP meditated gene deletions, we show that AMB resistance can be acquired through variation in several sterol biosynthesis regulators including ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. Additionally, we show how aneuploidies in chromosomes 4 and 6 emerge during AMB resistance evolution. By leveraging fitness trade-off phenotyping and mathematical modelling of the in vivo environment during treatment, we evaluated the potential of different mechanisms to establish resistant infections and discover a mechanism of fitness trade-off compensation. Variation in CDC25 substantially enhanced the capacity to establish a resistant infection and may have played a role in facilitating the sole documented clinical case of acquired AMB resistance during treatment in C. auris. In summary, our findings show that fitness trade-off compensation along with several sterol modulating mechanisms of acquired AMB resistance represent a potential risk for AMB treatment failure in the clinic.

Alternative ergosterol biosynthetic pathways confer antifungal drug resistance in the human pathogens within theMucorspecies complex

“…It is likely that the residual Erg6 activity observed in the erg6a Δ mutants can rescue the most deleterious consequences of loss of Erg6 function, while still conferring amphotericin B resistance due to cholesta-type sterols production. A comparable scenario was recently reported in C. auris evolved isolates, where erg6 mutations conferring amphotericin B resistance emerged in vivo, occasionally accompanied by other mechanisms that compensate for fitness trade-offs (71). Similarly, the loss of Erg6a function in mucoralean pathogens may arise in vivo and lead to mucormycosis infections that do not respond to first-line antifungal therapies, and may challenge recent and exciting developments in polyene antifungal drug discovery (73).…”

“…Despite these defects, the absence of Erg6 activity can provide a selective advantage by conferring amphotericin B drug resistance, as seen in multiple Candida species and Cryptococcus neoformans (17– 21, 23). Indeed, a recently conducted large-scale study, involving a collection of over four hundred C. auris strains evolved from clinical isolates, reported that mutations in erg6 were the most frequent cause of amphotericin B resistance (71). Exploring the function of Erg6 in Mucor species presented additional challenges due to the presence of three different Erg6 paralogs.…”

Functional Redundancy inCandida aurisCell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation

“…Strain construction. For strain construction, C. auris cells were prepared as described by Carolus et al 53 . For electroporation, 40 l of competent cells was mixed with the transformation mixture and transferred to 2 mm electroporation cuvettes (Pulsestar, Westburg).…”

Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes

“…Inactivation or modification of ERG3 or ERG6 impacts drug susceptibility of many Candida species [14,20,22,[28][29][30][31][32][33]. For example, loss of Erg6 function reduces susceptibility to nystatin and polyenes in C. glabrata [28,34,35]. Loss of Erg3 function confers resistance to azoles in C. albicans, C. parapsilosis, C. dubliniensis and resistance to polyenes in C. albicans and C. lusitaniae [22,[29][30][31]36,37].…”