Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance

Abstract

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

Echinocandin persistence directly impacts the evolution of resistance and survival of the pathogenic fungus Candida glabrata

“…Intriguingly, compared with those of THP1-derived macrophages, primary macrophages harbored significantly lower ECR colonies, and only HP isolates produced ECR colonies ( Fig. 1E ; Table 1 ) ( 18 ). Collectively, these observations suggested that although short-term macrophage internalization equalizes the ECP level, HP isolates generally have a significantly higher burden and ECR rate upon long-term macrophage internalization.…”

“…We have previously shown that brief internalization of CBS138 by THP1 macrophages for 3 h dramatically increases the ECP level by enriching persisters ( 18 ). Therefore, we hypothesized that internalization by fully mature hMDMs would enrich the persisters in all C. glabrata isolates, and thereby, they would exhibit similar survival regardless of the ECP level upon exposure to cidal concentrations of micafungin (hereafter refer to as hMDMs).…”

“…The colonies growing on micafungin-containing plates were further subjected to sequencing of HS1 and HS2 of FKS1 and FKS2 . Notably, our analysis did not include the burden and ECR rate in cRPMI alone since we previously observed phenotypic resistance and extremely low levels of bona fide ECR colonies harboring mutations in the HS regions of the FKS1 and FKS2 genes ( 18 ). For scientific rigor, we used only fully mature macrophages derived from the monocytes of three healthy donors.…”

“…The initial findings on both concepts suggest stochastic heterogeneity, such as physiological differences, in a presumably clonal susceptible population ( 15 , 16 ). Recent studies on C. glabrata have identified mitochondrial disruption as one of the mechanisms potentially contributing to echinocandin persistence ( 17 – 19 ). However, aneuploidy has been suggested to be a mechanism potentially driving fluconazole tolerance ( 20 ).…”

“…Moreover, the tiny population of persisters, which also has downregulated respiration, challenges the applicability of tools relying on adenosine triphosphate measurements as a readout, which could be successfully used to discern susceptible and resistant isolates ( 21 ). Finally, since persisters encompass an extremely low subpopulation whose size varies depending on the drug concentration and exposure time and because they do not replicate, antifungal susceptibility testing protocols are unable to infer the persistence level ( 14 , 17 , 18 ). Therefore, devising a rapid, convenient, and inexpensive method capable of reliably predicting persister levels in real time could enhance advancements in various aspects of persister biology in mycology and microbiology in general, such as high-throughput mutant library screening aimed at identifying novel drug targets and screening drug libraries exerting anti-persister activities.…”