The Endoplasmic Reticulum-Mitochondrion Tether ERMES Orchestrates Fungal Immune Evasion, Illuminating Inflammasome Responses to Hyphal Signals

The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

grown overnight in repressive conditions at 30 °C. Following dilution to OD 600 =0.1 at time point 0, growth at 37 °C was monitored by measuring OD 600 over time in repressive (+ methionine and cysteine) media.

Microscopy
Fluorescence images of mitochondria were produced using 1 µM MitoTracker Red CMXRos (Life Technologies, M7512) or 1 µM MitoTracker Green FM (Life Technologies, M7514) stock diluted into the growth media to give a final concentration of 0.1 µM and used as per the manufacturer's instructions. Images were taken at 100x objective, 72 DPI resolution (1600 x 1200 pixels) using an Olympus BX60 fluorescence microscope equipped with Spot Advanced Software (http://www.spotimaging.com/software/), and images were subsequently re-sampled for inclusion into 300 DPI resolution figures. For a given experiment containing wild type and mutant cells, all bright field and fluorescence images were taken with the same exposure time and under equivalent brightness and contrast settings. Red or green filters were applied to representative images using the ImageJ software to reflect the dyes used.
For liquid filamentation experiments, strains were grown in repressive conditions at 30 °C overnight, then diluted to OD 600 =0.1 in RPMI media containing 2.5 mM methionine and 0.5 mM cysteine. Hyphal formation was assessed after 3 h of growth at 37 °C at 200 rpm. Hyphal lengths were quantified using Fiji (http://www.fiji.sc/Fiji) following image collection. To measure hyphal lengths, Fiji's freehand tool was used to draw a distance from cell edge to hyphal tip, and this distance measured using Fiji's measure tool, resulting in an output of length in pixels.
Pixel length was converted to µm based on microscope scale bar settings. Hyphae and cells that extended past the edges of images were not quantified.

Macrophage interaction assays
For the macrophage interaction assays, single colonies of the C. albicans strains were patched on plates repressive for ERMES gene expression (complete synthetic media with uridine, and with addition of 2.5 mM methionine and 0.5 mM cysteine). Strains were grown over night for 12 h at 30 °C. Cells were taken from the plates and resuspended in PBS, followed by counting before addition to macrophages. All The immortalised mCerulean-tagged ASC inflammasome reporter macrophages were cultured in RPMI with 12.5 mM HEPES and 10% FBS and seeded at a density of 2.5 x 10 5 cells/well in 24-well tissue culture plates. Macrophages were infected with C.
albicans at a MOI of 3:1 (Candida:macrophage), and live cell imaging was set up as described above, but with the following adjustments: time-lapse images were acquired with bright-field, CFP, and Y5 filters every 1 hour up to 24h, and z-stacks spaced 8.5 µm totaling 42.5 µm were taken to account for all ASC specks across macrophages in multiple planes of focus. These image slices were subsequently deconvolved using The colony forming units of C. albicans following co-incubation with macrophages were determined as previously described (2), with the exception that after extraction from macrophages, C. albicans strains were plated on plates without methionine or cysteine, i.e. permissive for growth of the mmm1 mutant.

Quantitative RT-PCR
Total RNA was extracted by the hot acid phenol method. DNase treatment of 10 µg of RNA using TURBO DNase (Ambion) was performed according to the manufacturer's instructions. One µg of DNase treated total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. qPCR reaction was done on LightCycler 480 (Roche) using the FastStart Universal SYBR Green Master Rox (Roche) reagent mix. Data was analysed with the LinReg software (3,4). Primers are listed in Table S3.

Phospholipid analysis
For experiments involving the conditional ERMES mutants, 5

Analysis of 1,3 β-glucan by flow cytometry
Surface exposed 1, 3 β-glucan on hyphal cells was stained and quantified as described in (2). Briefly, hyphal cells were stained with the mouse anti-1, 3 β-glucan antibody (Biosupplies Australia) by incubating for 30 min at room temperature, followed by washing with PBS and incubation with AlexaFluor 488-labelled goat anti-mouse IgG (Invitrogen) for a further 30 min at room temperature. After washing with PBS, stained hyphae were analyzed by flow cytometry (FACSCalibur, BD Biosciences) using the FACSDiva 5.0 software (BD Biosciences). The dot plots and different gates were generated in Weasel V3 software (WEHI, Australia).