Hsp21potentiates antifungal drug tolerance in Candida albicans

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Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections

Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans

This is the final version. Available from Nature Research via the DOI in this record.RNA-sequencing data sets are available at ArrayExpress (www.ebi.ac.uk) under accession code E-MTAB-4075. ChIP-seq data sets are available at the NCBI SRA database (http://www.ncbi.nlm.nih.gov) under accession code SRP071687. The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding author upon request.Fever is a universal response to infection, and opportunistic pathogens such as Candida albicans have evolved complex circuitry to sense and respond to heat. Here we harness RNA-seq and ChIP-seq to discover that the heat shock transcription factor, Hsf1, binds distinct motifs in nucleosome-depleted promoter regions to regulate heat shock genes and genes involved in virulence in C. albicans. Consequently, heat shock increases C. albicans host cell adhesion, damage and virulence. Hsf1 activation depends upon the molecular chaperone Hsp90 under basal and heat shock conditions, but the effects are opposite and in part controlled at the level of Hsf1 expression and DNA binding. Finally, we demonstrate that Hsp90 regulates global transcription programs by modulating nucleosome levels at promoters of stress-responsive genes. Thus, we describe a mechanism by which C. albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence.Wellcome TrustCanadian Institutes of Health ResearchCanadian Institutes of Health ResearchBiotechnology and Biological Sciences Research Council (BBSRC)European Research Council (ERC)Science and Technology Development Fund of Macau S.A.R (FDCT)Research and Development Administrative Office of the University of MacauNational Institutes of Health (NIH

Mapping the Hsp90 Genetic Interaction Network in Candida albicans Reveals Environmental Contingency and Rewired Circuitry

The molecular chaperone Hsp90 regulates the folding of diverse signal transducers in all eukaryotes, profoundly affecting cellular circuitry. In fungi, Hsp90 influences development, drug resistance, and evolution. Hsp90 interacts with ∼10% of the proteome in the model yeast Saccharomyces cerevisiae, while only two interactions have been identified in Candida albicans, the leading fungal pathogen of humans. Utilizing a chemical genomic approach, we mapped the C. albicans Hsp90 interaction network under diverse stress conditions. The chaperone network is environmentally contingent, and most of the 226 genetic interactors are important for growth only under specific conditions, suggesting that they operate downstream of Hsp90, as with the MAPK Hog1. Few interactors are important for growth in many environments, and these are poised to operate upstream of Hsp90, as with the protein kinase CK2 and the transcription factor Ahr1. We establish environmental contingency in the first chaperone network of a fungal pathogen, novel effectors upstream and downstream of Hsp90, and network rewiring over evolutionary time

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

Planar Graph Coloring with Forbidden Subgraphs: Why Trees and Paths Are Dangerous

We consider the problem of coloring a planar graph with the minimum number of colors such that each color class avoids one or more forbidden graphs as subgraphs. We perform a detailed study of the computational complexity of this problem. We present a complete picture for the case with a single forbidden connected (induced or non-induced) subgraph. The 2-coloring problem is NP-hard if the forbidden subgraph is a tree with at least two edges, and it is polynomially solvable in all other cases. The 3-coloring problem is NP-hard if the forbidden subgraph is a path, and it is polynomially solvable in all other cases. We also derive results for several forbidden sets of cycles

Temporal trends in vent fluid iron and sulfide chemistry following the 2005/2006 eruption at East Pacific Rise, 9°50′N

The chemistry of vent fluids that emanate to the seafloor undergoes dramatic changes after volcanic eruptions. Data on these changes are still limited, but the best studied example is the East Pacific Rise (EPR) at 9°50′N, where the temporal evolution of the vent fluid chemistry after the 1991/1992 eruption was documented. The area underwent another eruption sequence during late 2005/early 2006, and here we show that a similar evolution is recurring in the iron and sulfide contents of the high-temperature fluids sampled in June 2006, January 2007, and June 2008. The vents have had increasing dissolved iron and decreasing acid-volatile sulfide (free sulfide plus FeS) concentrations with 1 order of magnitude variation. In addition, chromium reducible sulfide (mainly pyrite) also had fivefold decreasing concentrations over the 3 years. Our results confirm a pattern that was noted only once before for 9°50′N EPR and emphasize the dramatic yearly variability in the concentrations of iron-sulfur species emanating from vents

Hospital clusters of invasive Group B Streptococcal disease: A systematic review.

OBJECTIVES: To characterize outbreaks of invasive Group B Streptococcal (iGBS) disease in hospitals. METHODS: Systematic review using electronic databases to identify studies describing iGBS outbreaks/clusters or cross-infection/acquisition in healthcare settings where 'cluster' was defined as ≥2 linked cases. PROSPERO CRD42018096297. RESULTS: Twenty-five references were included describing 30 hospital clusters (26 neonatal, 4 adult) in 11 countries from 1966 to 2019. Cross-infection between unrelated neonates was reported in 19 clusters involving an early-onset (<7 days of life; n = 3), late-onset (7-90 days; n = 13) index case or colonized infant (n = 3) followed by one or more late-onset cases (median serial interval 9 days (IQR 3-17, range 0-50 days, n = 45)); linkage was determined by phage typing in 3 clusters, PFGE/MLST/PCR in 8, WGS in 4, non-molecular methods in 4. Postulated routes of transmission in neonatal clusters were via clinical personnel and equipment, particularly during periods of crowding and high patient-to-nurse ratio. Of 4 adult clusters, one was attributed to droplet spread between respiratory cases, one to handling of haemodialysis catheters and two unspecified. CONCLUSIONS: Long intervals between cases were identified in most of the clusters, a characteristic which potentially hinders detection of GBS hospital outbreaks without enhanced surveillance supported by genomics

Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target