Kawar, N. | bioRxiv Science

bioRxiv2024

Step-wise evolution of azole resistance through copy number variation followed by KSR1 loss of heterozygosity in Candida albicans.

Antimicrobial drug resistance poses a global health threat, requiring a deeper understanding of the evolutionary processes that lead to its emergence in pathogens. Complex evolutionary dynamics involve multiple mutations that can result in cooperative or competitive (clonal interference) effects. Candida albicans, a major fungal pathogen, displays high rates of copy number variation (CNV) and loss of heterozygosity (LOH). CNV and LOH events involve large numbers of genes and could synergize during evolutionary adaptation. Understanding the contributions of CNV and LOH to antifungal drug adaptation is challenging, especially in the context of whole-population genome sequencing. Here, we document the sequential evolution of fluconazole tolerance and then resistance in a C. albicans isolate involving an initial CNV on chromosome 4, followed by an LOH on chromosome R that involves KSR1. Similar LOH events involving KSR1, which encodes a reductase involved in sphingolipid biosynthesis, were also detected in independently evolved fluconazole resistant isolates. We dissect the specific KSR1 codons that affect fluconazole resistance and tolerance. The combination of the chromosome 4 CNV and KSR1 LOH results in a >500-fold increase in azole resistance, illustrating a compelling example of rapid, yet step-wise, interplay between CNV and LOH in drug resistance evolution.

microbiology↗

bioRxiv2022

Antifungal tolerance and resistance emerge at distinct drug concentrations and rely upon different aneuploid chromosomes

Antifungal drug tolerance is a response distinct from resistance, in which cells grow slowly above the minimum inhibitory drug concentration (MIC). Here we found that the majority (69.2%) of 133 Candida albicans clinical isolates, including standard lab strain SC5314, exhibited temperature-enhanced tolerance at 37{degrees}C and 39{degrees}C, and were not tolerant at 30{degrees}C. Other isolates were either always tolerant (23.3%) or never tolerant (7.5%) at these three temperatures, suggesting that tolerance requires different physiological processes in different isolates. At supra-MIC fluconazole concentrations (8-128 g/ml), tolerant colonies emerged rapidly at a frequency of ~10-3. In liquid passages over a broader range of fluconazole concentrations (0.25-128 g/ml), tolerance emerged rapidly (within one passage) at supra-MIC concentrations. By contrast, resistance appeared at sub-MIC concentrations after 5 or more passages. Of 155 adaptors that evolved higher tolerance, all carried one of several recurrent aneuploid chromosomes, often including chromosome R, alone or in combination with other chromosomes. Furthermore, loss of these recurrent aneuploidies was associated with a loss of acquired tolerance, indicating that specific aneuploidies confer fluconazole tolerance. Thus, genetic background and physiology, and the degree of drug stress (above or below the MIC) influence the evolutionary trajectories and dynamics with which antifungal drug resistance or tolerance emerges. ImportanceAntifungal drug tolerance differs from drug resistance: tolerant cells grow slowly in drug, while resistant cells usually grow well, due to mutations in a few known genes. More than half of Candida albicans clinical isolates have higher tolerance at body temperature than they do at the lower temperatures used for most lab experiments. This implies that different isolates achieve drug tolerance via several cellular processes. When we evolved different strains at a range of high drug concentrations above inhibitory levels, tolerance emerged rapidly and at high frequency (one in 1000 cells) while resistance only appeared later at very low drug concentrations. An extra copy of all or part of chromosome R was associated with tolerance, while point mutations or different aneuploidies were seen with resistance. Thus, genetic background and physiology, temperature, and drug concentration all influence how drug tolerance or resistance evolves.

evolutionary biology↗