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1.
Emerg Infect Dis ; 25(11): 2104-2107, 2019 11.
Article in English | MEDLINE | ID: mdl-31625866

ABSTRACT

Legionellosis was diagnosed in an immunocompromised 3-year-old girl in Canada. We traced the source of the bacterium through co-culture with an ameba collected from a hot tub in her home. We identified Legionella pneumophila serogroup 6, sequence type 185, and used whole-genome sequencing to confirm the environmental and clinical isolates were of common origin.


Subject(s)
Amoeba/microbiology , Legionella pneumophila/isolation & purification , Legionnaires' Disease/epidemiology , Legionnaires' Disease/microbiology , Canada/epidemiology , Coculture Techniques , Disease Outbreaks , Genome, Bacterial , Humans , Legionella pneumophila/classification , Legionella pneumophila/genetics , Phylogeny , Public Health Surveillance , Whole Genome Sequencing
2.
PLoS Genet ; 14(4): e1007319, 2018 04.
Article in English | MEDLINE | ID: mdl-29702647

ABSTRACT

Invasive fungal infections caused by the pathogen Candida albicans have transitioned from a rare curiosity to a major cause of human mortality. This is in part due to the emergence of resistance to the limited number of antifungals available to treat fungal infections. Azoles function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. Loss-of-function mutations in the ergosterol biosynthetic gene ERG3 mitigate azole toxicity and enable resistance that depends upon fungal stress responses. Here, we performed a genome-wide synthetic genetic array screen in Saccharomyces cerevisiae to map ERG3 genetic interactors and uncover novel circuitry important for azole resistance. We identified nine genes that enabled erg3-mediated azole resistance in the model yeast and found that only two of these genes had a conserved impact on resistance in C. albicans. Further, we screened a C. albicans homozygous deletion mutant library and identified 13 genes for which deletion enhances azole susceptibility. Two of the genes, RGD1 and PEP8, were also important for azole resistance acquired by diverse mechanisms. We discovered that loss of function of retrograde transport protein Pep8 overwhelms the functional capacity of the stress response regulator calcineurin, thereby abrogating azole resistance. To identify the mechanism through which the GTPase activator protein Rgd1 enables azole resistance, we selected for mutations that restore resistance in strains lacking Rgd1. Whole genome sequencing uncovered parallel adaptive mechanisms involving amplification of both chromosome 7 and a large segment of chromosome 3. Overexpression of a transporter gene on the right portion of chromosome 3, NPR2, was sufficient to enable azole resistance in the absence of Rgd1. Thus, we establish a novel mechanism of adaptation to drug-induced stress, define genetic circuitry underpinning azole resistance, and illustrate divergence in resistance circuitry over evolutionary time.


Subject(s)
Azoles/pharmacology , Candida albicans/drug effects , Drug Resistance, Fungal/drug effects , Saccharomyces cerevisiae/drug effects , Antifungal Agents/pharmacology , Candida albicans/genetics , Candida albicans/physiology , Drug Resistance, Fungal/genetics , GTPase-Activating Proteins/genetics , Host-Pathogen Interactions/drug effects , Humans , Microbial Sensitivity Tests , Mutation , Mycoses/microbiology , Oxidoreductases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Vesicular Transport Proteins/genetics , Whole Genome Sequencing/methods
3.
Antimicrob Agents Chemother ; 60(12): 7468-7480, 2016 12.
Article in English | MEDLINE | ID: mdl-27736764

ABSTRACT

Invasive fungal infections are a leading cause of human mortality. Effective treatment is hindered by the rapid emergence of resistance to the limited number of antifungal drugs, demanding new strategies to treat life-threatening fungal infections. Here, we explore a powerful strategy to enhance antifungal efficacy against leading human fungal pathogens by using the natural product beauvericin. We found that beauvericin potentiates the activity of azole antifungals against azole-resistant Candida isolates via inhibition of multidrug efflux and that beauvericin itself is effluxed via Yor1. As observed in Saccharomyces cerevisiae, we determined that beauvericin inhibits TOR signaling in Candida albicans To further characterize beauvericin activity in C. albicans, we leveraged genome sequencing of beauvericin-resistant mutants. Resistance was conferred by mutations in transcription factor genes TAC1, a key regulator of multidrug efflux, and ZCF29, which was uncharacterized. Transcriptional profiling and chromatin immunoprecipitation coupled to microarray analyses revealed that Zcf29 binds to and regulates the expression of multidrug transporter genes. Beyond drug resistance, we also discovered that beauvericin blocks the C. albicans morphogenetic transition from yeast to filamentous growth in response to diverse cues. We found that beauvericin represses the expression of many filament-specific genes, including the transcription factor BRG1 Thus, we illuminate novel circuitry regulating multidrug efflux and establish that simultaneously targeting drug resistance and morphogenesis provides a promising strategy to combat life-threatening fungal infections.


Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Antifungal Agents/pharmacology , Candida albicans/drug effects , Depsipeptides/pharmacology , Drug Resistance, Fungal/genetics , Fungal Proteins/antagonists & inhibitors , Gene Expression Regulation, Fungal , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Azoles/pharmacology , Base Sequence , Biological Products/pharmacology , Candida albicans/genetics , Candida albicans/growth & development , Candida albicans/metabolism , Drug Synergism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Profiling , Microarray Analysis , Mutation , Signal Transduction , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism
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