Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to antifungal compounds.

Candida albicans, an opportunistic human pathogen, poses a significant threat to human health and is associated with significant socio-economic burden. Current antifungal treatments fail, at least in part, because C. albicans can initiate a strong drug tolerance response that allows some cells to grow at drug concentrations above their minimal inhibitory concentration. To better characterize this cytoprotective tolerance program at the molecular single-cell level, we used a nanoliter droplet-based transcriptomics platform to profile thousands of individual fungal cells and establish their subpopulation characteristics in the absence and presence of antifungal drugs. Profiles of untreated cells exhibit heterogeneous expression that correlates with cell cycle stage with distinct metabolic and stress responses. At 2 days post-fluconazole exposure (a time when tolerance is measurable), surviving cells bifurcate into two major subpopulations: one characterized by the upregulation of genes encoding ribosomal proteins, rRNA processing machinery, and mitochondrial cellular respiration capacity, termed the Ribo-dominant (Rd) state; and the other enriched for genes encoding stress responses and related processes, termed the Stress-dominant (Sd) state. This bifurcation persists at 3 and 6 days post-treatment. We provide evidence that the ribosome assembly stress response (RASTR) is activated in these subpopulations and may facilitate cell survival.

Many drugs currently used to treat fungal diseases are becoming less effective. This is partly due to the rise of antifungal resistance, where certain fungal cells acquire mutations that enable them to thrive and proliferate despite the medication. Antifungal tolerance also contributes to this problem, wherein certain cells can continue to grow and multiply, while other ­ genetically identical ones ­ cannot. This variability is partly due to differences in gene expression within the cells. The specific nature of these differences has remained elusive, mainly because their study requires the use of expensive and challenging single-cell technologies. To address this challenge, Dumeaux et al. adapted an existing technique to perform single-cell transcriptomics in the pathogenic yeast Candida albicans. Their approach was cost effective and made it possible to examine the gene expression in thousands of individual cells within a population that had either been treated with antifungal drugs or were left untreated. After two to three days following exposure to the antifungal treatment, C. albicans cells commonly exhibited one of two states: one subgroup, the 'Ribo-dominant' cells, predominantly expressed genes for ribosomal proteins, while the other group, the 'Stress-dominant' cells, upregulated their expression of stress-response genes. This suggests that drug tolerance may be related to different gene expression patterns in growing cell subpopulations compared with non-growing subpopulations. The findings also indicate that the so-called 'ribosome assembly stress response' known to help baker's yeast cells to survive, might also aid C. albicans in surviving exposure to antifungal treatments. The innovative use of single-cell transcriptomics in this study could be applied to other species of fungi to study differences in cell communication under diverse growth conditions. Moreover, the unique gene expression patterns in C. albicans identified by Dumeaux et al. may help to design new antifungal treatments that target pathways linked to drug resistance.

Mutations, protein homeostasis, and epigenetic control of genome integrity.

From bacteria to humans, ancient stress responses enable organisms to contend with damage to both the genome and the proteome. These pathways have long been viewed as fundamentally separate responses. Yet recent discoveries from multiple fields have revealed surprising links between the two. Many DNA-damaging agents also target proteins, and mutagenesis induced by DNA damage produces variant proteins that are prone to misfolding, degradation, and aggregation. Likewise, recent studies have observed pervasive engagement of a p53-mediated response, and other factors linked to maintenance of genomic integrity, in response to misfolded protein stress. Perhaps most remarkably, protein aggregation and self-assembly has now been observed in multiple proteins that regulate the DNA damage response. The importance of these connections is highlighted by disease models of both cancer and neurodegeneration, in which compromised DNA repair machinery leads to profound defects in protein quality control, and vice versa.

Elucidating drug resistance in human fungal pathogens.

Fungal pathogens cause life-threatening infections in immunocompetent and immunocompromised individuals. Millions of people die each year due to fungal infections, comparable to the mortality attributable to tuberculosis or malaria. The three most prevalent fungal pathogens are Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Fungi are eukaryotes like their human host, making it challenging to identify fungal-specific therapeutics. There is a limited repertoire of antifungals in clinical use, and drug resistance and host toxicity compromise the clinical utility. The three classes of antifungals for treatment of invasive infections are the polyenes, azoles and echinocandins. Understanding mechanisms of resistance to these antifungals has been accelerated by global and targeted approaches, which have revealed that antifungal drug resistance is a complex phenomenon involving multiple mechanisms. Development of novel strategies to block the emergence of drug resistance and render resistant pathogens responsive to antifungals will be critical to treating life-threatening fungal infections.

Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata.

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.

Loss of elongation factor P disrupts bacterial outer membrane integrity.

Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.

PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica.

We report an interaction between poxA, encoding a paralog of lysyl tRNA-synthetase, and the closely linked yjeK gene, encoding a putative 2,3-beta-lysine aminomutase, that is critical for virulence and stress resistance in Salmonella enterica. Salmonella poxA and yjeK mutants share extensive phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered expression of multiple proteins, including several encoded on the SPI-1 pathogenicity island. PoxA mediates posttranslational modification of bacterial elongation factor P (EF-P), analogous to the modification of the eukaryotic EF-P homolog, eIF5A, with hypusine. The modification of EF-P is a mechanism of regulation whereby PoxA acts as an aminoacyl-tRNA synthetase that attaches an amino acid to a protein resembling tRNA rather than to a tRNA.