Characterizing molecular mechanisms of heteroresistance in Candida glabrata

Noa Werthimer 1 Ronen Ben Ami 2 Judith Berman 1
1Molecular Microbiology and biotechnology, Tel Aviv University, Tel Aviv, Israel
2Medical School, Tel Aviv University, Tel Aviv, Israel

Heteroresistance (HetR) is the ability of a small subpopulation to grow within a drug-susceptible majority population at high drug concentrations. We recently described HetR in Candida glabrata, the second most common cause of Candida infections, which is increasing in prevalence. HetR is an important phenomenon in clinical C. glabrata isolates and it appears to contribute to the recurrence of infections. Quantitative PAP (population analysis profiling) assays and more practical penetrance analysis both indicated that levels of HetR are found in C. glabrata as a continuum ranging from 1/1000 to 1/100,000 cells in the population. The dynamics of growth of different subpopulations within a genetically identical isolate was measured using ScanLag, which showed that HetR levels correlate with final colony size. Low magnification MSM 400 time-lapse microscopy (MSM) revealed that highly HetR isolates included at least two distinct subpopulations of cells that grew faster and slower in the presence of high drug concentrations, while the majority of the population did not exhibit detectable growth. These two sub-populations expressed different resistance genes, especially efflux pump genes CDR1 and PDH1, to different degrees as determined by RT-PCR. HetR is epigenetic: the rare colonies that appear on drug reproducibly gave rise to mixed populations of progeny with some forming colonies as did the parent (COL) – and others forming a lawn of cells that grow very slowly in the presence or absence of drug (tiny dense- TD). RNA-seq of the different progeny showed that when comparing COL to TD, TD cells repress genes in the ergosterol biosynthetic pathway, which is the target of the fluconazole, while COL express more genes involved in respiration -We propose that HetR is due to epigenetic or transient mechanisms that facilitate the ability of small numbers of cells to grow in drug.