The Ins and Outs and ABCs of Antifungal Drug Transport: Characterizing the Role of Membrane Transporters in Pathogenic Fungi

Title from PDF of title page, viewed March 29, 2023Dissertation advisor: Theodore C. WhiteVitaIncludes bibliographical referencesDissertation (Ph.D)--Department of Cell Biology and Biophysics, Department of Molecular Biology and Biochemistry. University of Missouri--Kansas City, 2016Pathogenic fungi cause serious disease and even death in humans, animals and plants. In medicine and agriculture alike, fungal infections are widespread and represent a significant threat to global public health. The number and array of fungal species, each exhibiting diverse mechanisms of pathogenesis, makes the challenge of fungal infection prevention and treatment formidable. The current repertoire of effective antifungal treatment strategies is very limited. As a result of increased use of antifungals to treat and prevent clinical fungal infections in humans, as well as widespread use of fungicides in agriculture, fungal strains that are resistant to each of the classes of antifungals have emerged. A significant rise in the number of fungal infections in recent years, combined with an increasing amount of drug resistant fungal strains is great cause for concern and places urgency on the development of new and more effective fungal infection treatment and prevention strategies. New fungal drug targets may be discovered with a better understanding of basic fungal biological processes. New or improved fungal infection treatment strategies may stem from a more complete knowledge of fungal response to drug treatment, worldwide trends of fungal pathogenesis and development of resistance, and even fungal evolutionary relationships. The goal of this research was to characterize the most basic fungal/drug interactions, which includes the balance of uptake, retention, and efflux of antifungal drugs in the fungal cell. We analyzed a variety of environmental and cellular factors that affect antifungal drug uptake and retention in two medically and agriculturally important pathogenic fungi, Aspergillus fumigatus and Magnaporthe oryzae. We then identified and characterized a number of A. fumigatus plasma membrane ABC transporters that may contribute to antifungal drug resistance due to their role in the efflux of antifungal drugs. To analyze antifungal drug uptake and retention, we developed an assay to directly measure accumulation of radioactively-labeled azoles in A. fumigatus and M. oryzae. Our analysis of drug uptake under a variety of cellular and environmental conditions demonstrated that these filamentous fungi import azoles by a facilitated diffusion mechanism. Contrasts between the M. oryzae and A. fumigatus data revealed interesting differences that suggest variations in expression, induction, or function of efflux transporters in the two organisms. To analyze antifungal efflux, we cloned and expressed a selection of putative ABC transporter genes from the A. fumigatus genome and heterologously expressed each gene in S. cerevisiae for direct characterization of drug efflux potential. Our efflux transporter analysis showed differences in substrate specificity, drug susceptibilities, energy-dependent efflux activity, and effect of efflux-inhibitor treatment between the different transporters. These data illustrate the complexity of predicting and counteracting fungal drug treatment response, but also highlight the possibilities for identifying new drug targets.Introduction to azole drug resistance mechanisms in pathogenic fungi -- Azole drug import into the pathogenic fungus Aspergillus Fumigatus -- Azole drug import into the fungal plant pathogen Magnaporthe Oryzae -- Functional and inducible expression of A. Fumigatus putative efflux transporters in S. Cerevisiae -- Conclusions and future direction

Comparison of major depression diagnostic classification probability using the SCID, CIDI, and MINI diagnostic interviews among women in pregnancy or postpartum: An individual participant data meta‐analysis

OBJECTIVES: A previous individual participant data meta-analysis (IPDMA) identified differences in major depression classification rates between different diagnostic interviews, controlling for depressive symptoms on the basis of the Patient Health Questionnaire-9. We aimed to determine whether similar results would be seen in a different population, using studies that administered the Edinburgh Postnatal Depression Scale (EPDS) in pregnancy or postpartum. METHODS: Data accrued for an EPDS diagnostic accuracy IPDMA were analysed. Binomial generalised linear mixed models were fit to compare depression classification odds for the Mini International Neuropsychiatric Interview (MINI), Composite International Diagnostic Interview (CIDI), and Structured Clinical Interview for DSM (SCID), controlling for EPDS scores and participant characteristics. RESULTS: Among fully structured interviews, the MINI (15 studies, 2,532 participants, 342 major depression cases) classified depression more often than the CIDI (3 studies, 2,948 participants, 194 major depression cases; adjusted odds ratio [aOR] = 3.72, 95% confidence interval [CI] [1.21, 11.43]). Compared with the semistructured SCID (28 studies, 7,403 participants, 1,027 major depression cases), odds with the CIDI (interaction aOR = 0.88, 95% CI [0.85, 0.92]) and MINI (interaction aOR = 0.95, 95% CI [0.92, 0.99]) increased less as EPDS scores increased. CONCLUSION: Different interviews may not classify major depression equivalently

Accumulation of Azole Drugs in the Fungal Plant Pathogen Magnaporthe oryzae Is the Result of Facilitated Diffusion Influx

Magnaporthe oryzae is an agricultural mold that causes disease in rice, resulting in devastating crop losses. Since rice is a world-wide staple food crop, infection by M. oryzae poses a serious global food security threat. Fungicides, including azole antifungals, are used to prevent and combat M. oryzae plant infections. The target of azoles is CYP51, an enzyme localized on the endoplasmic reticulum (ER) and required for fungal ergosterol biosynthesis. However, many basic drug-pathogen interactions, such as how the azole gets past the fungal cell wall and plasma membrane, and is transported to the ER, are not understood. In addition, reduced intracellular accumulation of antifungals has consistently been observed as a drug resistance mechanism in many fungal species. Studying the basic biology of drug-pathogen interactions may elucidate uncharacterized mechanisms of drug resistance and susceptibility in M. oryzae and potentially other related fungal pathogens. We characterized intracellular accumulation of azole drugs in M. oryzae using a radioactively labeled fluconazole uptake assay to gain insight on whether azoles enter the cell by passive diffusion, active transport, or facilitated diffusion. We show that azole accumulation is not ATP-dependent, nor does it rely on a pH-dependent process. Instead there is evidence for azole drug uptake in M. oryzae by a facilitated diffusion mechanism. The uptake system is specific for azole or azole-like compounds and can be modulated depending on cell phase and growth media. In addition, we found that co-treatment of M. oryzae with ‘repurposed’ clorgyline and radio-labeled fluconazole prevented energy-dependent efflux of fluconazole, resulting in an increased intracellular concentration of fluconazole in the fungal cell

Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae

Ergosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. The effects of changes in expression of each ERG biosynthesis enzyme in Saccharomyces cerevisiae were analyzed by the use of gene deletion or plasmid-borne overexpression constructs. The strains overexpressing the ERG pathway genes were examined for changes in doubling time and responses to a variety of stress agents. In addition, ERG gene overexpression strains and ERG gene deletion strains were tested for alterations in antifungal drug susceptibility. The data show that disruptions in ergosterol biosynthesis regulation can affect a diverse set of cellular processes and can cause numerous phenotypic effects. Some of the phenotypes observed include dramatic increases in doubling times, respiratory deficiencies on glycerol media, cell wall insufficiencies on Congo red media, and disrupted ion homeostasis under iron or calcium starvation conditions. Overexpression or deletion of specific enzymes in the ERG pathway causes altered susceptibilities to a variety of classes of antifungal ergosterol inhibitors, including fluconazole, fenpropimorph, lovastatin, nystatin, amphotericin B, and terbinafine. This analysis of the effect of perturbations to the ERG pathway caused by systematic overexpression of each of the ERG pathway genes contributes significantly to the understanding of the ergosterol biosynthetic pathway and its relationship to stress response and basic biological processes. The data indicate that precise regulation of ERG genes is essential for cellular homeostasis and identify several ERG genes that could be exploited in future antifungal development efforts.A common target of antifungal drug treatment is the fungal ergosterol biosynthesis pathway. This report helps to identify ergosterol biosynthesis enzymes that have not previously been appreciated as drug targets. The effects of overexpression of each of the 25 ERG genes in S. cerevisiae were analyzed in the presence of six stress agents that target essential cellular processes (cell wall biosynthesis, protein translation, respiration, osmotic/ionic stress, and iron and calcium homeostasis), as well as six antifungal inhibitors that target ergosterol biosynthesis. The importance of identifying cell perturbations caused by gene overexpression or deletion is emphasized by the prevalence of gene expression alterations in many pathogenic and drug-resistant clinical isolates. Genes whose altered expression causes the most extensive phenotypic alterations in the presence of stressors or inhibitors have the potential to be drug targets

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae

One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. TheA. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable

Drug-mediated metabolic tipping between antibiotic resistant states in a mixed-species community (dataset)

The article associated with this dataset is located in ORE at: http://hdl.handle.net/10871/33536This is the dataset used for the Beardmore et al. (2018) article "Drug-mediated metabolic tipping between antibiotic resistant states in a mixed-species community" published in the Nature Ecology and Evolution journal

Drug-mediated metabolic tipping between antibiotic resistant states in a mixed-species community

Acknowledgements In memory of our friend and colleague Ken Haynes who sadly passed away on 19th March 2018. Author Correction | Published: 19 September 2018 Author Correction: Drug-mediated metabolic tipping between antibiotic resistant states in a mixed-species community Robert E. Beardmore, Emily Cook, Susanna Nilsson, Adam R. Smith, Anna Tillmann, Brooke D. Esquivel, Ken Haynes, Neil A. R. Gow, Alistair J. P. Brown, Theodore C. White & Ivana Gudelj Nature Ecology & Evolution (2018) https://doi.org/10.1038/s41559-018-0678-0Peer reviewedPostprin

College student sleep quality and mental and physical health are associated with food insecurity in a multi-campus study

AbstractObjective:To assess the relationship between food insecurity, sleep quality, and days with mental and physical health issues among college students.Design:An online survey was administered. Food insecurity was assessed using the ten-item Adult Food Security Survey Module. Sleep was measured using the nineteen-item Pittsburgh Sleep Quality Index (PSQI). Mental health and physical health were measured using three items from the Healthy Days Core Module. Multivariate logistic regression was conducted to assess the relationship between food insecurity, sleep quality, and days with poor mental and physical health.Setting:Twenty-two higher education institutions.Participants:College students (n 17 686) enrolled at one of twenty-two participating universities.Results:Compared with food-secure students, those classified as food insecure (43·4 %) had higher PSQI scores indicating poorer sleep quality (P \u3c 0·0001) and reported more days with poor mental (P \u3c 0·0001) and physical (P \u3c 0·0001) health as well as days when mental and physical health prevented them from completing daily activities (P \u3c 0·0001). Food-insecure students had higher adjusted odds of having poor sleep quality (adjusted OR (AOR): 1·13; 95 % CI 1·12, 1·14), days with poor physical health (AOR: 1·01; 95 % CI 1·01, 1·02), days with poor mental health (AOR: 1·03; 95 % CI 1·02, 1·03) and days when poor mental or physical health prevented them from completing daily activities (AOR: 1·03; 95 % CI 1·02, 1·04).Conclusions:College students report high food insecurity which is associated with poor mental and physical health, and sleep quality. Multi-level policy changes and campus wellness programmes are needed to prevent food insecurity and improve student health-related outcomes