Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs.

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.

Isolation of Legionella pneumophila by Co-culture with Local Ameba, Canada.

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.

Beauvericin Potentiates Azole Activity via Inhibition of Multidrug Efflux, Blocks Candida albicans Morphogenesis, and Is Effluxed via Yor1 and Circuitry Controlled by Zcf29.

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.

A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.