Genetically identical microbes can display striking levels of heterogeneity. This phenomenon is best described by evolutionary biologists as a strategy for bet-hedging in response to shifting environmental conditions. Population heterogeneity increases the likelihood that some cells adapt to and survive stressful environments, thereby increasing the overall fitness of the population.
Microbial heterogeneity is often overlooked in molecular studies as most approaches analyze whole populations rather than single cells. While it is no longer surprising that seemingly identical populations are highly heterogeneous, the challenge is to decipher how cell-to-cell diversity translates into meaningful biological functions. For human pathogens, cellular heterogeneity can have important functional consequences, as seen for the bacterium Pseudomonas aeruginosa. Here, division of labor within biofilm communities can lead to cooperative interactions between different cell subtypes and enhance virulence.
Population heterogeneity is also an important strategy that pathogens can utilize to escape antimicrobial treatment. This is now evidenced by our work in Candida albicans (https://www.nature.com/articles/s41467-018-04926-x), a prevalent fungal pathogen that can occupy diverse niches in the human body, either as a commensal or as an invasive pathogen. Antifungal resistance has been described for all drugs used to treat infections by this species. Frontline therapies include azoles, a class of drugs which target the fungal cell membrane and inhibit cell growth.
We now show that population heterogeneity of infecting isolates can enable azole escape – in many strains a subpopulation of C. albicans cells can still grow, albeit slowly, in the presence of high drug concentrations. Importantly, these cells are not drug resistant per se, and the size of the subpopulation does not change with drug concentration. We define this as antifungal tolerance and show that it arises due to decreased intracellular drug levels and signaling via cellular stress response pathways. We hypothesize that tolerance might buy time for a subset of cells, during which mutations that confer true drug resistance can arise. Strikingly, C. albicans clinical isolates, as well as other pathogenic Candida species, harbor variable levels of tolerant subpopulations. We found that isolates with high azole tolerance are associated with infections that persist in the bloodstream even with antifungal treatment.
Curiously, antifungal tolerance has been previously noted by researchers as trailing growth, but remained largely ignored in the literature. In fact, clinical assays measuring azole susceptibility have been optimized to avoid trailing growth, as it can confound measurements of drug resistance. The current study now proposes that tolerance should not be ignored and could serve as a useful predictor for distinguishing infections likely to respond to azole therapy from those likely to be recalcitrant to these drugs. It also highlights that combination therapies (azoles together with secondary drugs) can successfully target highly tolerant subpopulations and improve infection outcomes. It remains to be determined what mechanisms enable Candida albicans population heterogeneity, and what other roles heterogeneity plays during host-pathogen interactions.