Biology subjects

Cunningham, K. W.

Publications and source records attributed to Cunningham, K. W..

9 recordsLinked to original sources

bioRxiv2026

A Natural qE- and qZ-Deficient Alga Retains Functional Plant-Like and Chlorophycean Violaxanthin De-Epoxidases

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

plant biology↗

bioRxiv2026

A Mechanism Driving Echinocandin Heteroresistance in Candida glabrata

While pathogenic fungi can acquire resistance to the current arsenal of antifungals through genetic mutations, heteroresistance has emerged as an important new cause of therapeutic failures. Heteroresistance is generally thought to arise in small subpopulations that display phenotypic resistance to antifungals without genetic mutations. This study blurs that line by showing gain-of-resistance mutations in FKS2, which encodes a target of echinocandins and fungerps, cause large amounts of heteroresistance in Candida glabrata through heterogeneous expression of the gene even in clonal cell populations. Heteroresistance decreased when stress-responsive transcription factors (Crz1, Rlm1) were eliminated and was nearly abolished when the upstream regulators (calcineurin, Slt2) were mutated or inhibited. Identical gain-of-resistance mutations in FKS1, a paralog of FKS2, showed much less heteroresistance due to its constitutive expression coupled with variable levels of antagonism by wild-type FKS2. A genome-wide screen using Tn-seq revealed additional regulators of heteroresistance and resistance including IRA1, an inhibitor of the Ras1-PKA signaling pathway that senses glucose availability. IRA1 increased expression of FKS2 and decreased expression of FKS1, which increased heteroresistance and decreased resistance, respectively, when these genes carried resistance mutations. Similar principles may govern heteroresistance in other fungal pathogens such as Candida parapsilosis, which naturally carries resistance mutations in FKS1 and frequently exhibits heteroresistance to echinocandins, and Candidozyma auris, which easily acquires such mutations.

microbiology↗

bioRxiv2025

Echinocandin tolerance and persistence in vitro are regulated by calcineurin signaling in Candida glabrata

Upon exposure to echinocandins, growing yeast cells begin to accumulate cell wall damage and eventually die, resulting in therapeutic effects. While resistance to echinocandins is well studied, tolerance and persistence mechanisms that may also contribute to clinical failures and relapses remain understudied. In time-kill assays with micafungin in vitro, the opportunistic pathogen Candida glabrata exhibited biphasic kinetics of cell death. Modeling with exponential decay equations distinguished a fast-dying major population from a slow-dying minor population, indicative of persistence. A genome-wide forward-genetic screen revealed dozens of genes that appeared to regulate persistence and/or tolerance, but not resistance. Several of those genes encoded calcineurin and its upstream regulators. Using individual gene knockout mutants and FK506, we show that calcineurin signaling increases the lifespans of most C. glabrata cells through a process that is largely independent of Crz1, one of its downstream effectors. The formation of long-lived persister-like cells (i.e. persistence) was strongly dependent on calcineurin signaling, independent of Crz1. Pre-activation of calcineurin using genetic or chemical stressors, such as manogepix, strongly increased tolerance and persistence in C. glabrata, suggesting antagonism of echinocandin efficacy by this new antifungal. Calcineurin signaling was also necessary for induction of tolerance and persistence in Candida albicans. The findings suggest that short-term administration of FK506 during the earliest stages of echinocandin treatment may improve clinical outcomes while possibly avoiding long-term immunosuppression. IMPORTANCETreatment of fungal infections is often unsuccessful. Potential causes of antifungal failure include tolerance and persistence, which are poorly understood processes used by fungal pathogens to survive our assaults. This study utilizes detailed experimental protocols and genome-wide screens to discover how Candida glabrata induces tolerance and persistence to a major class of antifungals. The findings suggest that a clinical immunosuppressant may be repurposed to combat tolerance and persistence in this pathogenic yeast as well as Candida albicans and perhaps others.

microbiology↗

bioRxiv2024

Tn-seq of the Candida glabrata reference strain CBS138 reveals epigenetic plasticity, structural variation, and intrinsic mechanisms of resistance to micafungin

C. glabrata is an opportunistic pathogen that can resist common antifungals and rapidly acquire multidrug resistance. A large amount of genetic variation exists between isolates, which complicates generalizations. Portable Tn-seq methods can efficiently provide genome-wide information on strain differences and genetic mechanisms. Using the Hermes transposon, the CBS138 reference strain and a commonly studied derivative termed 2001 were subjected to Tn-seq in control conditions and after exposure to varying doses of the clinical antifungal micafungin. The approach revealed large differences between these strains, including a 131 kb tandem duplication and a variety of fitness differences. Additionally, both strains exhibited up to 1000-fold increased transposon accessibility in subtelomeric regions relative to the BG2 strain, indicative of open subtelomeric chromatin in these isolates and large epigenetic variation within the species. Unexpectedly, the Pdr1 transcription factor conferred resistance to micafungin through targets other than CDR1. Other micafungin resistance pathways were also revealed including mannosyltransferase activity and biosynthesis of the lipid precursor sphingosine, the drugging of which by SDZ 90-215 or myriocin enhanced the potency of micafungin in vitro. These findings provide insights into complexity of the C. glabrata species as well as strategies for improving antifungal efficacy. SummaryCandida glabrata is an emerging pathogen with large genetic diversity and genome plasticity. The type strain CBS138 and a laboratory derivative were mutagenized with the Hermes transposon and profiled using Tn-seq. Numerous genes that regulate innate and acquired resistance to an important clinical antifungal were uncovered, including a pleiotropic drug resistance gene (PDR1) and a duplication of part of one chromosome. Compounds that target PDR1 and other genes may augment the potency of existing antifungals.

microbiology↗

bioRxiv2023

Calcineurin-dependent contributions to fitness in the opportunistic pathogen Candida glabrata

The protein phosphatase calcineurin is vital for virulence of the opportunistic fungal pathogen Candida glabrata. The host-induced stresses that activate calcineurin signaling are unknown, as are the targets of calcineurin relevant to virulence. To potentially shed light on these processes, millions of transposon insertion mutants throughout the genome of C. glabrata were profiled en masse for fitness defects in the presence of FK506, a specific inhibitor of calcineurin. 87 specific gene deficiencies depended on calcineurin signaling for full viability in vitro both in wild type and pdr1{Delta} null strains lacking pleiotropic drug resistance. Three genes involved in cell wall biosynthesis (FKS1, DCW1, FLC1) possess co-essential paralogs whose expression depended on calcineurin and Crz1 in response to micafungin, a clinical antifungal that interferes with cell wall biogenesis. Interestingly, 80% of the FK506-sensitive mutants were deficient in different aspects of vesicular trafficking, such as endocytosis, exocytosis, sorting, and biogenesis of secretory proteins in the ER. In response to the experimental antifungal manogepix that blocks GPI-anchor biosynthesis in the ER, calcineurin signaling increased and strongly prevented cell death independent of Crz1, one of its major targets. Comparisons between manogepix, micafungin, and the ER-stressing tunicamycin reveal a correlation between the degree of calcineurin signaling and the degree of cell survival. These findings suggest that calcineurin plays major roles in mitigating stresses of vesicular trafficking. Such stresses may arise during host infection and in response antifungal therapies.

microbiology↗

bioRxiv2023

Redefining Pleiotropic Drug Resistance in a Pathogenic Yeast: Pdr1 Functions as a Sensor of Cellular Stresses in Candida glabrata

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. ImportanceCandida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1 - a key determinant of fluconazole resistance - is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

genetics↗

bioRxiv2021

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

Unicellular eukaryotes are suggested to undergo self-inflicted destruction. However, molecular details are sparse by comparison to the mechanisms of cell death known for human cells and animal models. Here we report a molecular pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization and cell death. Following exposure to heat-ramp conditions, a model of environmental stress, we observed that yeast cell death occurs over several hours, suggesting an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 adaptor complex. AP-3 promotes stress-induced cell death through its Arf1-GTPase-dependent vesicle trafficking function, which is required to transport and install proteins on the vacuole/lysosome membrane, including a death-promoting protein kinase Yck3. Time-lapse microscopy revealed a sequence of events where AP-3-dependent vacuole permeability occurs hours before the loss of plasma membrane integrity. An AP-3-dependent cell death pathway appears to be conserved in the human pathogen Cryptococcus neoformans.

cell biology↗

bioRxiv2020

Identification of essential genes and fluconazole resistance genes in Candida glabrata by profiling of Hermes transposon insertions.

Within the budding yeasts, the opportunistic pathogen Candida glabrata and other members of the Nakaseomyces clade have developed virulence traits independently from the CTG clade that includes Candida albicans. To begin exploring the genetic basis of C. glabrata virulence and its innate resistance to antifungals, we launched the Hermes transposon from a plasmid and obtained more than 500,000 different semi-random insertions throughout the genome. Using machine learning, we identify up to 1278 protein-encoding genes (25% of total) that cannot tolerate transposon insertions and are thus essential for C. glabrata fitness in vitro. Interestingly, genes involved in mRNA splicing were less likely to be essential in C. glabrata than their orthologs in S. cerevisiae, whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. Insertions in several known genes (e.g. PDR1, CDR1, PDR16, PDR17, UPC2A, DAP1) caused hypersensitivity to the first-line antifungal fluconazole, and we identify 12 additional genes that also contribute to innate fluconazole resistance (KGD1, KGD2, YHR045W, etc). Insertions in 200 other genes conferred significant resistance to fluconazole, two-thirds of which function in mitochondria and likely down-regulate Pdr1 expression or function. These findings show the utility of transposon insertion profiling in genome-wide forward-genetic investigations of fungal pathogens. IMPORTANCEPathogenic yeasts cause mucosal and systemic infections in millions of people each year. The innate resistance of Candida glabrata to fluconazole and its ability to acquire resistance to 2 other antifungals are contributing to its rise in incidence. Our understanding of C. glabrata biology has been hampered by inefficient genetic and genomic tools. This study addresses those deficiencies by developing powerful transposon mutagenesis strategies for the first time in this pathogen. We identify nearly all essential genes of C. glabrata that could be targeted for development of new antifungals. We generate large pools of random insertion mutants that can be easily monitored en masse with deep sequencing, thus enabling identification of genes involved in any number of biological processes. We identify dozens of new genes that increase or decrease innate resistance of clinical isolate BG2 to fluconazole and provide resources for further exploration of C. glabrata genetics and genomics.

genetics↗

bioRxiv2019

Comparing the utility of in vivo transposon mutagenesis approaches in yeast species to infer gene essentiality

ABSTRACTIn vivo transposon mutagenesis, coupled with deep sequencing, enables large-scale genome-wide mutant screens for genes essential in different growth conditions. We analyzed six large-scale studies performed on haploid strains of three yeast species (Saccharomyces cerevisiae, Schizosaccaromyces pombe, and Candida albicans), each mutagenized with two of three different heterologous transposons (AcDs, Hermes, and PiggyBac). Using a machine-learning approach, we evaluated the ability of the data to predict gene essentiality. Important data features included sufficient numbers and distribution of independent insertion events. All transposons showed some bias in insertion site preference because of jackpot events, and preferences for specific insertion sequences and short-distance vs long-distance insertions. For PiggyBac, a stringent target sequence limited the ability to predict essentiality in genes with few or no target sequences. The machine learning approach also robustly predicted gene function in less well-studied species by leveraging cross-species orthologs. Finally, comparisons of isogenic diploid versus haploid S. cerevisiae isolates identified several genes that are haplo-insufficient, while most essential genes, as expected, were recessive. We provide recommendations for the choice of transposons and the inference of gene essentiality in genome-wide studies of eukaryotic haploid microbes such as yeasts, including species that have been less amenable to classical genetic studies.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioinformatics↗