Advance online publication:

This section includes articles accepted for publication in Microbial Cell, which have not been released in a regular issue, yet. Please note that the PDF versions of advance publication articles are generally paginated starting with page 1. This does not correspond to the final pagination upon release of the issue it will appear in.

 

Sulforaphane alters the acidification of the yeast vacuole

Alexander Wilcox, Michael Murphy, Douglass Tucker, David Laprade, Breton Roussel, Christopher Chin, Victoria Hallisey, Noah Kozub, Abraham Brass and Nicanor Austriaco

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Sulforaphane (SFN) is a compound [1-isothiocyanato-4-(methylsulfinyl)-butane] found in broccoli and other cruciferous vegetables that is currently of interest because of its potential as a chemopreventive and a chemotherapeutic drug. Recent studies in a diverse range of cellular and animal models have shown that SFN is involved in multiple intracellular pathways that regulate xenobiotic metabolism, inflammation, cell death, cell cycle progression, and epigenetic regulation. In order to better understand the mechanisms of action behind SFN-induced cell death, we undertook an unbiased genome wide screen with the yeast knockout (YKO) library to identify SFN sensitive (SFNS) mutants. The mutants were enriched with knockouts in genes linked to vacuolar function suggesting a link between this organelle and SFN’s mechanism of action in yeast. Our subsequent work revealed that SFN increases the vacuolar pH of yeast cells and that varying the vacuolar pH can alter the sensitivity of yeast cells to the drug. In fact, several mutations that lower the vacuolar pH in yeast actually made the cells resistant to SFN (SFNR). Finally, we show that human lung cancer cells with more acidic compartments are also SFNR suggesting that SFN’s mechanism of action identified in yeast may carry over to higher eukaryotic cells.

PDF | Published online: 20/03/2020 | In press

Regulation of anti-microbial autophagy by factors of the complement system

Christophe Viret, Aurore Rozières, Rémi Duclaux-Loras, Gilles Boschetti, Stéphane Nancey and Mathias Faure

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The complement system is a major component of innate immunity that participates in the defense of the host against a myriad of pathogenic microorganisms. Activation of complement allows for both local inflammatory response and physical elimination of microbes through phagocytosis or lysis. The system is highly efficient and is therefore finely regulated. In addition to these well-established properties, recent works have revealed that components of the complement system can be involved in a variety of other functions including in autophagy, the conserved mechanism that allows for the targeting and degradation of cytosolic materials by the lysosomal pathway after confining them into specialized organelles called autophagosomes. Besides impacting cell death, development or metabolism, the complement factors-autophagy connection can greatly modulate the cell autonomous, anti-microbial activity of autophagy: xenophagy. Both surface receptor-ligand interactions and intracellular interactions are involved in the modulation of the autophagic response to intracellular microbes by complement factors. Here, we review works that relate to the recently discovered connections between factors of the complement system and the functioning of autophagy in the context of host-pathogen relationship.

PDF | Published online: 19/03/2020 | In press

Broad-spectrum antifungal activities and mechanism of drimane sesquiterpenoids

Edruce Edouarzin, Connor Horn, Anuja Paudyal, Cunli Zhang, Jianyu Lu, Zongbo Tong, Guri Giaever, Corey Nislow, Raja Veerapandian, Duy H. Hua and Govindsamy Vediyappan

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Eight drimane sesquiterpenoids including (-)-drimenol and (+)-albicanol were synthesized from (+)-sclareolide and evaluated for their antifungal activities. Three compounds, (-)-drimenol, (+)-albicanol, and (1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyl-decahydronaphthalene-1-carbaldehyde (4) showed strong activity against C. albicans. (-)-Drimenol, the strongest inhibitor of the three, (at concentrations of 8 – 64 µg/ml, causing 100% death of various fungi), acts not only against C. albicans in a fungicidal manner, but also inhibits other fungi such as Aspergillus, Cryptococcus, Pneumocystis, Blastomyces, Saksenaea and fluconazole resistant strains of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. auris. These observations suggest that drimenol is a broad-spectrum antifungal agent. At a high concentration (100 μg/ml) drimenol caused rupture of the fungal cell wall/membrane. In a nematode model of C. albicans infection, drimenol rescued the worms from C. albicans-mediated death, indicating drimenol is tolerable and bioactive in metazoans. Genome-wide fitness profiling assays of both S. cerevisiae (nonessential homozygous and essential heterozygous) and C. albicans (Tn-insertion mutants) collections revealed putative genes and pathways affected by drimenol. Using a C. albicans mutant spot assay, the Crk1 kinase associated gene products, Ret2, Cdc37, and orf19.759, orf19.1672, and orf19.4382 were revealed to be involved in drimenol’s mechanism of action. The three orfs identified in this study are novel and appear to be linked with Crk1 function. Further, computational modeling results suggest possible modifications of the structure of drimenol, including the A ring, for improving the antifungal activity.

PDF | Published online: 12/03/2020 | In press

More than flipping the lid: Cdc50 contributes to echinocandin resistance by regulating calcium homeostasis in Cryptococcus neoformans

Chengjun Cao and Chaoyang Xue

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Echinocandins are the newest fungicidal drug class approved for clinical use against common invasive mycoses. Yet, they are ineffective against cryptococcosis, predominantly caused by Cryptococcus neoformans. The underlying mechanisms of innate echinocandin resistance in C. neoformans remain unclear. We know that Cdc50, the β-subunit of the lipid translocase (flippase), mediates echinocandin resistance, as loss of the CDC50 gene sensitizes C. neoformans to caspofungin, a member of the echinocandins class. We sought to elucidate how Cdc50 facilitates caspofungin resistance by performing a forward genetic screen for cdc50Δ suppressor mutations that are caspofungin resistant. We identified a novel mechanosensitive calcium channel protein Crm1 that correlates with Cdc50 function (Cao et al., 2019). In addition to regulating phospholipid translocation, Cdc50 also interacts with Crm1 to regulate intracellular calcium homeostasis and calcium/calcineurin signaling that likely drives caspofungin resistance in C. neoformans. Our study revealed a novel dual function of Cdc50 that connects lipid flippase with calcium signaling. These unexpected findings provide new insights into the mechanisms of echinocandin resistance in C. neoformans that may drive future drug design.

PDF | Published online: 20/02/2020 | In press

Stable and destabilized GFP reporters to monitor calcineurin activity in Saccharomyces cerevisiae

Jutta Diessl, Arpita Nandy, Christina Schug, Lukas Habernig and Sabrina Büttner

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The protein phosphatase calcineurin is activated in response to rising intracellular Ca2+ levels and impacts fundamental cellular processes in organisms ranging from yeast to humans. In fungi, calcineurin orchestrates cellular adaptation to diverse environmental challenges and is essential for virulence of pathogenic species. To enable rapid and large-scale assessment of calcineurin activity in living, unperturbed yeast cells, we have generated stable and destabilized GFP transcriptional reporters under the control of a calcineurin-dependent response element (CDRE). Using the reporters, we show that the rapid dynamics of calcineurin activation and deactivation can be followed by flow cytometry and fluorescence microscopy. This system is compatible with live/dead staining that excludes confounding dead cells from the analysis. The reporters provide technology to monitor calcineurin dynamics during stress and ageing and may serve as a drug-screening platform to identify novel antifungal compounds that selectively target calcineurin.

PDF | Published online: 05/02/2020 | In press

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