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The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes. [ABSTRACT FROM AUTHOR]

Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-β response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/β receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-β release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-β and the β-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-β release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-β signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae zinc cluster regulator War! mediates an essential transcriptional and adaptive response to weak organic acid stress. Here we investigate the mechanism of Wan activation upon weak acid stress. We identified several gain-of-function WAR1 alleles mapping to the central Wan region. These mutations constitutively increase levels of the plasma membrane ABC transporter Pdr12, the main Wan target mediating stress adaptation. Functional analysis of War1 reveals that the central region and its C-terminal activation domain are required for function. Notably, the native DNA- binding and dimerization domains appear dispensable for Wan activity, because they can be replaced by a LexA DNA-binding domain. Chromatin immunoprecipitation demonstrates elevated promoter affinity of activated War1, because its PDR12 promoter association increases upon stress. Hyperactive WAR1 alleles have constitutively high PDR12 promoter association. Furthermore, fluorescence resonance energy transfer of functional CFP-War1-YFP proteins also demonstrates conformational changes of stress-activated War1 in vivo. Our results suggest a mechanism whereby War1 activation is accompanied by conformational changes enhancing promoter association, thus initiating the adaptation process. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2A C ) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr 307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr 307 but instead are hampered by PP2A C methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr 307 antibodies are sensitive to PP2A C methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr 307 using targeted mass spectrometry after transient overexpression of PP2A C and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr 307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined., Competing Interests: Declaration of Interests E.O. serves as a consultant to Millipore Corporation. Monoclonal antibody 4B10 against Tyr(307)-phosphorylated PP2A(C) subunit is licensed to the biotech companies BioLegend/Covance, BioTechne/Novus, EMD Millipore (discontinued), and SCBT. Monoclonal antibody 1D7 against nonmethylated PP2A(C) subunit is licensed to BioLegend/Covance. Monoclonal antibody 4A6 against the myc epitope tag is licensed to EMD Millipore. Monoclonal antibody 2A3 against pan-actin is licensed to the biotech companies EMD Millipore and SCBT. The authors confirm that they will adhere to all the policies of Cell Reports on sharing data and materials. The Medical University of Vienna on behalf of E.O. is filing a patent on the PP2A methyl-C subunit-specific monoclonal antibody 7C10., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exposure of Saccharomyces cerevisiae to weak organic acids such as sorbate, propionate, or benzoate rapidly induces the plasma membrane ABC transporter Pdr12, requiring the Zn(II)(2)Cys(6) zinc-finger transcription factor War1. Weak acid stress rapidly triggers War1 phosphorylation but its role for War1 function is not clear yet. Here, we provide new insights into sorbate-induced phosphorylation of War1. A War1 zinc-finger mutant is still hyperphosphorylated in response to sorbate stress, indicating that War1 phosphorylation occurs independently of DNA recruitment. To map and identify phosphoresidues, War1 purified from stressed and unstressed cells was subjected to semiquantitative phosphopeptide mass spectrometry analysis. Remarkably, we show that weak acid stress causes a dramatic hyperphosphorylation of several already prephosphorylated residues. WAR1 alleles harboring combinations of mutations identified phosphoresidues were generated, some of which display altered gel mobility. Certain mutational combinations almost completely abolish stress-induced gel-shift, suggesting alternative phosphorylation. Surprisingly, PDR12 expression levels are similar in these mutants, demonstrating that War1 phosphorylation is not required for PDR12 induction. Strikingly, absence of hyperphosphorylation in response to stress leads to a faster stress adaptation, suggesting that phosphorylation might play a role in stabilizing War1 activity on the promoter elements, hence changing the dynamics and kinetics of the stress response.