The C. albicans strains used in the study are described in Table 1. The gem1Δ/Δ mutant was constructed in the BWP17 background using PCR and homologous recombination using the URA3 and ARG4 markers to replace the two copies of the GEM1 open reading frame. Histidine prototrophy was restored by integrating the HIS1-containing integrative pDDB78 vector, into the endogenous HIS1 locus. The same vector was used to construct the complemented strain, by introducing a wild type GEM1 gene under the control of its own promoter. The conditional mdm12↓ was constructed by deleting one copy of the gene utilizing the ARG4 marker and placing the other under the control of the MET3 promoter, utilizing the URA3 marker. A 3xHA tag was introduced at the C-terminus of Cek1 in wild type and gem1Δ/Δ strains by homologous recombination of the 3HA-HIS1 cassette from the plasmid pFA-HA-HIS1 (Lavoie et al., 2008). Primers used in this study are listed in Table S1. Strains were grown at 30°C, 200 rpm unless otherwise indicated. Rich media was YPD (1% yeast extract, 2% peptone, 2% glucose) supplemented with 80 μg/mL uridine. Synthetic media was made with 0.67% w/v yeast nitrogen base without amino acids or carbohydrate and with ammonium sulfate (US Biological Y2025) with 80 μg/mL uridine and the appropriate amino acids. Repressive conditions for the ERMES mutants mmm1↓, mdm10↓ and mdm12↓ were achieved by addition of 2.5 mM methionine and 0.5 mM cysteine to the media.

For analysis of sensitivities to various drugs and chemicals, ten-fold serial dilutions of cultures starting form an OD₆₀₀ = 0.5 were dropped on control plates, or plates containing the compounds indicated in the Figures. Plates were incubated at 30°C for 2 days and then photographed.

For testing filamentation on plates, C. albicans strains were plated on Spider plates (1% nutrient broth, 1% D-mannitol, 2 g K₂HPO₄) or RPMI-1640 plates. Plates were incubated for up to 5 days at 37°C or 30°C, and colonies examined and photographed with a stereo dissecting microscope (Olympus SZX 16 and Olympus MVX10).

For assaying filamentous growth in liquid media, cells were inoculated to an OD₆₀₀ = 0.3 into pre-warmed YPD+10% FBS, Spider media or RPMI and incubated at 37°C for the times indicated in the figure legends.

MICs were determined using the broth microdilution method according to CLSI guidelines M27-A3. Drug concentrations ranged from 0.004 to 2 μg/ml for caspofungin; One-hundred-microliter portions of 2-fold serial dilutions of the drugs prepared in YPD were added into wells of 96-well plates. Overnight cultures were diluted to a density of 1 × 10³ to 5 × 10³ CFU/ml, and 100 μl was added to each well of a 96-well plate. Plates were incubated for 48 h at 30°C (the longer incubation time was additionally used to compensate for slow growth of the gem1Δ/Δ mutant strain). The MIC was defined as the concentration resulting in an inhibition of at least 50% of fungal growth.

Growth conditions and experimental set up are described in the figure legends. Total RNA was obtained by extraction with hot acid phenol. DNase treatment of 10 μg of RNA using TURBO DNase (Ambion) was performed according to the manufacturer's instructions. One μg of DNase-treated RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Quantitative PCR was done on LightCycler 480 (Roche) using the FastStart Universal SYBR Green Master Rox (Roche) reagent mix. The data was analyzed using the LinReg software (Ramakers et al., 2003; Ruijter et al., 2009). 18S RNA or SCR1 served as control genes for normalization. Statistical analysis of qPCR data was performed using 2-way-ANOVA analysis with multiple comparison, and Tukey's multiple comparison test.

For assaying cell wall stress pathway activation, overnight cultures of yeast strains were diluted to an OD₆₀₀ of 0.2 into fresh YPD and grown to early log phase, followed by treatment with 125 ng/ml caspofungin for the times indicated in the Figure. For ERMES shutdown strains mmm1↓, mdm10↓ and mdm12↓ a single colony was streaked in a patch on SD plates with all amino acids plus Met/Cys to achieve ERMES gene repression. The patch was taken up in PBS-buffer. Liquid SD medium containing all amino acids and Met/Cys was then inocculated to and OD₆₀₀ of 0.2, and from there on treated as described above for the cultures grown in YPD. Whole-cell protein extracts were prepared by trichloroacetic acid (TCA) precipitation, and Western blotting was performed. Phospho-Mkc1 and phospho-Cek1 were detected using the rabbit monoclonal anti-phospho-p44/42 mitogen-activated protein (MAP) kinase antibody (Cell Signaling). Actin was detected with an anti-actin monoclonal antibody (Millipore). Cek1-HA (C-terminally tagged) was detected with the anti-HA antibody (Biolegend). Quantification of Western blot data for the phospho-Cek1 and actin bands in Figure 3B (from data shown in Figure 3A and Figure S3) was performed with 16-bit grayscale pictures of scanned films using the Toolbox module of ImageQuant 1D version 7.0. Background signals were subtracted using the local median method performed by the software. Chemiluminescent signals for protein levels of Cek1-3xHA (Figures 3D,E) were captured with the Biorad ChemiDoc^TM Touch Imaging System, and quantification of Cek1-3xHA and actin bands was performed using the Biorad Image Lab^TM software Version 6.0.0 build 26.

All cell imaging was performed using an Olympus BX60 microscope equipped with Olympus with Spot Advanced Software, with bright field or fluorescent settings. Mitochondrial staining was performed with 1 μM MitoTracker Red CMXRos (Life Technologies, M7512), with the stock diluted into the growth media to give a final concentration of 0.1 μM, and then used as per the manufacturer's instructions. Images were taken with a 100 × objective, 72 DPI resolution (1,600 × 1,200 pixels) using an Olympus BX60 fluorescence microscope equipped with Spot Advanced Software.

Calcofluor white staining (250 μg/ml) was performed at room temperature in the dark for 10 min, followed by three washes in distilled water.

Immunofluorescence was performed after induction of hyphal formation in Spider media at 37°C for 3h. Cells were fixed with 3.7% formaldehyde (final concentration). Blocking with BSA was carried out for 1 h at room temperature. Primary anti-1, 3 beta glucan antibody (Biosupplies) incubation was carried out for 1 h (1:500 dilution), and secondary antibody anti-mouse Alexa Fluor488 (Invitrogen) incubation was carried out for 45min at room temperature (1:800 dilution). Antibodies were diluted in 3% BSA in PBS.

Cultures were grown in YPD until mid-log phase and 20 OD units of each strain were harvested and washed three times with H₂O. Cells were then resuspended in 400 μl of methanol and disrupted by vortexing with 100 μl of glass beads. Then 800 μl of chloroform was added and samples were vortexed and centrifuged to remove insoluble material. The organic supernatant was removed and washed once with 0.9% NaCl. The lipid containing phase was collected and dried under the stream of nitrogen. Lipids were separated by thin layer chromatography (TLC) on Slica 60 plates (Millipore) in chloroform/ethanol/water/trimethylamine (30:35:7:35). Dried TLC plates were stained with 470 mM CuSO₄ in 8% o-phosphoric acid and incubated at 180°C for 15 min. Lipids standards (Avanti Polar Lipids) were used to identify cardiolipin (CL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Phospholipids were quantified using FIJI software (version 2.0.0-rc43/1,5k).

The worm-C. albicans infection assay was performed as described previously (Uwamahoro et al., 2012). Briefly, young adult nematodes were allowed to feed for 4 h on lawns of C. albicans wild type, gem1Δ/Δ or gem1Δ/Δ+GEM1 grown on solid BHI media (DIFCO) + kanamycin (50 μg/ml). Worms were washed with M9 media and transferred into wells of a six-well microtiter dish (Corning) containing 2 ml of liquid media (80% M9 and 20% BHI) at 60 to 80 worms per well. The plates were incubated at 25°C, and worms were assessed at 24 h intervals for C. albicans filamentation using a DIC microscope and photographed using an Olympus IX81 microscope with the Olympus cellSense software. Worms were scored as follows: alive (no penetrating hyphae visible), dead with hyphae penetrating, and dead without penetrative hyphae (i.e., death caused by other reason unrelated to hyphal formation). Only 13 out of 1,925 worms (i.e., ≈0.67%) died without filamentation across the three experiments. In order to focus on the filamentation-induced host death, these 13 animals were excluded from the analyses. The percentage of dead worms with penetrative filamentation was determined from three independent experiments after 72 h of infection. Means and the standard deviation were calculated.

The C. albicans Gem1 is encoded by C1_00890W (orf19.6016), with characteristic domain structure consisting of GTPase domains I and II, Ca²⁺ binding domains I and II and a transmembrane domain (Figure 1A). Interestingly, a protein sequence alignment uncovered a stretch of 56 amino acids located in the GTPase Domain I that is unique to C. albicans Gem1, and not found in either S. cerevisiae Gem1 nor human Miro1 or Miro2 (Figure 1B and Figure S1). A BLAST search of this stretch of amino acids revealed that, in addition to C. albicans, it exists in only one other organism, the closely related species Candida dubliniensis. It is not clear what impact this stretch has on the structure or activity of GTPase Domain I in C. albicans, but we do anticipate the GTPase Domain I to function normally, as the predicted GTP-binding site, the G1 motif, is highly conserved in the C. albicans Gem1 protein. The C. albicans homozygous deletion mutant gem1Δ/Δ showed reduced growth on rich glucose media at 30°C, but this growth defect was not exacerbated at 37°C or in media containing the non-fermentable carbon sources glycerol or lactate (Figure 1C). Two independent gem1Δ/Δ deletion clones showed comparable phenotypes, and we could restore fitness to the mutant by re-introduction of the wild type GEM1 gene (Figure 1C). In the gem1Δ/Δ mutant the tubular mitochondrial network structure was lost, and collapsed tubules and some mitochondrial globular spheres were evident (Figure 1D). Occasionally, we noticed some cells with no detectable mitochondrial staining, however this was comparable between the wild type and gem1Δ/Δ mutant strains and occurred in 1–5% of the cell population. Unlike the drastic difference in mitochondrial morphology between the wild type and the gem1Δ/Δ mutant, no differences in steady state phospholipid levels were observed in the mutant compared to the wild type strain (Figure 1E).

Mitochondria are centrally important to cellular physiology under normal conditions, and also in response to various stresses. We therefore tested the susceptibility of the gem1Δ/Δ mutant to a range of stresses that are relevant for C. albicans in a disease context. The growth defect of the C. albicans gem1Δ/Δ was not exacerbated by osmotic or oxidative stress (Figure 2A), indicating that the Gem1 does not participate in the response to these stressors. In contrast, the gem1Δ/Δ mutant was susceptible to cell wall stress caused by calcofluor white and the 1,3 ß-glucan synthase inhibitor caspofungin, and displayed susceptibility to the solvent DMSO that compromises membrane integrity (Figures 2A,B). Despite the susceptibility to cell wall stress, the gem1Δ/Δ mutant displayed normal distribution of chitin (as judged by calcofluor white staining), and also normal distribution of 1, 3 ß-glucan (as judged using immunofluorescence with the anti-1, 3 ß-glucan antibody) (Figures 2C,D). Therefore, we next assessed whether the mutant was able to activate the cell wall stress pathways in response to caspofungin-inflicted cell wall damage. For this, we used Western blots to monitor the appearance of the phosphorylated form of the kinase Mkc1 (which reports on the activation of the Cell Wall Integrity (CWI) pathway), and the appearance of the phosphorylated form of the kinase Cek1 (which reports on the activation of the Cek1-dependent pathway). Mutant strains of MKC1 and CEK1 were used to ascertain the identities of the phospho-Cek1 and phospho-Mkc1 bands (Figure 3A). The gem1Δ/Δ mutant was able to trigger activation of the CWI pathway normally (Figure 3A). However, unlike in the wild type strain, where the phospho-Mkc1 signal declined over time as cells repaired the damage to their cell wall and therefore turned the pathway off, Mkc1 remained phosphorylated for a prolonged time in the gem1Δ/Δ mutant, consistent with reduced ability of the mutant to repair cell wall damage (Figure 3A). In contrast to prompt activation of the CWI pathway, activation of the Cek1 pathway was delayed in the gem1Δ/Δ mutant (Figure 3A). Although Western blots are semi-quantitative, to gage the extent of the defect in the appearance of the phospho-Cek1 band in the gem1Δ/Δ mutant, we quantified the phospho-Cek1 signal at 120 min after caspofungin treatment relative to the actin loading control (the primary data used in these quantifications is shown in Figure 3A and Figure S3. Note that five out of six experiments were used for quantification due to fact that in experiment 6 the phospho-Cek1 signal in the mutant displayed no detectable intensity levels during quantification). This analysis showed a consistent reduction of phospho-Cek1 in the gem1Δ/Δ mutant compared to the wild type (Figure 3B). The gem1Δ/Δ mutant was able to up-regulate transcription of the CEK1 gene following caspofungin treatment (Figure 3C), and the total protein levels of Cek1 in the gem1Δ/Δ mutant were comparable to the wild type strain in the presence or absence of caspofungin treatment (Figures 3D,E; HA-tagged Cek1 was used in these experiments to detect total Cek1 protein levels). Therefore, low levels of Cek1 pathway activation in the gem1Δ/Δ mutant are likely due to a signaling defect, rather than low expression levels of Cek1. Cek1 is known to be phosphorylated in response to re-entry of cells into active growth (Román et al., 2009a). Given that the gem1Δ/Δ mutant is hyper susceptible to caspofungin, we wondered whether the delayed phosphorylation of Cek1 was caused by slower resumption of growth after cell wall stress. However, growth assays did not support this hypothesis: in both wild type and gem1Δ/Δ mutant cultures, caspofungin treatment caused a growth arrest that lasted for several hours (Figure 3F). Yet, the wild type strain was triggering Cek1 phosphorylation during the prolonged growth arrest (i.e., despite lack of growth in caspofungin-containing media), while the gem1Δ/Δ mutant was not able to activate Cek1 for a prolonged period of time (Figure 3A). Mutants in the ERMES complex also displayed caspofungin susceptibility, which was even more pronounced at 37°C compared to 30°C (Figure 4A). We note that ERMES genes are near essential in C. albicans, and therefore we used conditional mutants in which the expression of the ERMES genes is driven by the MET3 promoter and is therefore repressible by methionine and cysteine (Tucey et al., 2016). ERMES mutants were generally able to promptly trigger activation of the CWI pathway in response to cell wall stress (see phospho-Mkc1 Western blots in Figure 4B), although somewhat reduced levels of phospho-Mkc1 were seen in the mmm1 and mdm10 mutants at the early time points (Figure 4B). Cek1 pathway activation in response to caspofungin was clearly retarded in the mmm1 mutant, while the mdm10 and mmm12 mutants did not display a major defect (Figure 4B). We next assessed how inhibition of mitochondrial respiratory chain activity would affect cell wall stress pathway activation. To this end, we used a mutant in the mitochondrial respiratory complex I (nuo1Δ/Δ), as complex I activity has been shown to be required for cell wall integrity in C. albicans (She et al., 2013, 2015, 2016). The nuo1Δ/Δ was susceptible to caspofungin (Figure 4C), but activated Cek1 normally in response to caspofungin treatment (Figure 4D).

The capacity for invasive hyphal growth is important for pathogenicity of C. albicans, due to roles such as tissue invasion and evasion of innate immune responses (Sudbery, 2011; Jacobsen and Hube, 2017). Hyphal growth of the gem1Δ/Δ mutant was drastically reduced on mannitol-containing solid Spider media (Figure 5A). Hyphal outgrowth from the colonies was only sparsely observed, and the hyphae were much shorter than those emanating from wild type colonies, even after prolonged growth (Figure 5A). A similar hyphal defect was seen on RPMI plates (Figure 5A). Unlike on solid plates, the gem1Δ/Δ mutant was able to produce hyphae in liquid Spider, YPD + serum and RPMI media, although fewer cells underwent the transition from yeast to hyphae and in some cases shorter hyphae were observed in the mutant possibly due to slower growth (Figure 5B). Mitochondrial morphology in the hyphal cells of the gem1Δ/Δ mutant was consistent with what we observed in yeast cells, in that large collapsed mitochondria were seen, and they were distributed along the length of the filaments showing they can traffic between hyphal cells (segments) (Figure 5C). We have previously reported that the ERMES mutant mmm1 is able to filament in liquid RPMI media (Tucey et al., 2016). Given the specific defect of the gem1Δ/Δ mutant in filamentation on a solid substrate (i.e., plates), and a very similar mitochondrial morphology defect in hyphal cells of the gem1Δ/Δ mutant (Figure 5C) and the mmm1 mutant (Tucey et al., 2016), we wondered whether, similarly to Gem1, ERMES is also needed for filamentation on solid substrates. Due to a growth defect of ERMES strains at 37°C (Tucey et al., 2016), we performed the experiment at 30°C on Spider plates to separate general growth defects from reduced hyphal morphogenesis. We also confirmed that Spider media was repressive for the MET3 promoter driving ERMES gene expression in the conditional mutants (Figure 5D). As shown in Figure 5E (top panel), same as the gem1Δ/Δ mutant, the ERMES mutants mmm1, mdm10 and mdm12 were unable to produce the penetrative filaments surrounding the colonies on Spider plates. The colonies of the ERMES mutants grew well under these conditions: the mdm12 mutant grew as well as the wild type, and the mmm1 and mdm10 mutants displayed reduced growth, but were very clearly growing substantially. This shows that ERMES function in hyphal invasion on agar plates can be uncoupled from growth defects (Figure 5E, middle and bottom panel).

Unlike on plates, where hyphae invade into the agar, there is no resistance to hyphal growth in liquid media. Therefore, we interpret our hyphal morphogenesis results to mean that Gem1 and ERMES have roles in invasive hyphal growth, rather than hyphal morphogenesis per se. To further corroborate this proposition we used the worm infection model. The worm model relies on hyphal formation by C. albicans to pierce through the cuticle of the worm to kill it. This model has been used successfully to define C. albicans genes required for virulence, and it recapitulates virulence processes in mammalian infection models (Pukkila-Worley et al., 2009, 2011; Jain et al., 2013; Issi et al., 2017). As shown in Figure 6A, the gem1Δ/Δ mutant was drastically compromised in its ability to cause death of the worm host by hyphal penetration, with only 10–20% of the worms killed by the mutant 72 h following infection. Most worms infected by the gem1Δ/Δ mutant did not display any penetrative filamentation and survived the infection. We considered that the slow growth phenotype of the gem1Δ/Δ mutant might mean that a longer time is needed for penetrative filamentation to be observed. However, even at day 7 post infection, the worms infected with the mutant were mostly devoid of fungal filaments (data not shown). Accounting for the 10–20% of host killing in infection with the gem1Δ/Δ mutant (Figure 6A), occasionally hyphae were seen emanating from the worm (Figure 6B). However, these hyphae never achieved the length and numbers seen in infection with the control strains (Figure 6B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.