Competition of Candida glabrata against Lactobacillus is Hog1 dependent

Abstract Candida glabrata is a common human fungal commensal and opportunistic pathogen. This fungus shows remarkable resilience as it can form recalcitrant biofilms on indwelling catheters, has intrinsic resistance against azole antifungals, and is causing vulvovaginal candidiasis. As a nosocomial pathogen, it can cause life‐threatening bloodstream infections in immune‐compromised patients. Here, we investigate the potential role of the high osmolarity glycerol response (HOG) MAP kinase pathway for C. glabrata virulence. The C. glabrata MAP kinase CgHog1 becomes activated by a variety of environmental stress conditions such as osmotic stress, low pH, and carboxylic acids and subsequently accumulates in the nucleus. We found that CgHog1 allows C. glabrata to persist within murine macrophages, but it is not required for systemic infection in a mouse model. C. glabrata and Lactobacilli co‐colonise mucosal surfaces. Lactic acid at a concentration produced by vaginal Lactobacillus spp. causes CgHog1 phosphorylation and accumulation in the nucleus. In addition, CgHog1 enables C. glabrata to tolerate different Lactobacillus spp. and their metabolites when grown in co‐culture. Using a phenotypic diverse set of clinical C. glabrata isolates, we find that the HOG pathway is likely the main quantitative determinant of lactic acid stress resistance. Taken together, our data indicate that CgHog1 has an important role in the confrontation of C. glabrata with the common vaginal flora.

causing significant discomfort and inconvenience for the afflicted (Sobel, 2007(Sobel, , 2016. In the northern hemisphere, Candida albicans and Candida glabrata are the two most common species isolated from VVC patients. C. glabrata belongs to the Nakaseomyces group, which includes other pathogenic yeasts and is phylogenetically related to Saccharomyces cerevisiae (Gabaldon et al., 2013). The intrinsic resistance of C. glabrata to fluconazole (and occasional resistance to echinocandins) advanced C. glabrata to the second most isolated Candida spp. and, due to the few available classes of antifungal drugs, renders effective treatment difficult (Rodrigues et al., 2014, Sardi et al., 2013, Silva et al., 2012. VVC often coincides with increased pH due to a dysbiosis of the vaginal microbiome, allowing Candida growth (Green et al., 2015). The native bacterial flora, which is often dominated by Lactobacillus spp., maintains a low pH and exerts a fungistatic effect against Candida spp. (Parolin et al., 2015, Chew et al., 2015, Hutt et al., 2016. In addition, certain Lactobacillus spp.
are also known to enhance recruitment of phagocytic cells and propagate efficient release of pro-inflammatory cytokines (Villena et al., 2011). Restoring the vaginal microbiome with Lactobacillus spp. is suggested as safe alternative treatment in case of antifungal-resistant Candida spp. (Van Kessel et al., 2003, Villena et al., 2011. C. glabrata has a highly malleable genome (Polakova et al., 2009) with an expanded family of EPA genes facilitating adherence, some of them not shared with S. cerevisiae , Desai et al., 2011. Within the host, C. glabrata survives by modulating the content of the phagolysosome and preventing phagosome maturation, enabling it to escape (reviewed in Kasper et al., 2015). C. glabrata is taken up by macrophages more efficiently than C. albicans but adapts to the situation, reproduces within the macrophage, and then gets released after the macrophage bursts (Seider et al., 2011). Adaptable resistance to abiotic stress is an important determinant for the competitiveness of any microorganism, especially if it encounters fluctuating environments. In fungi, the high osmolarity glycerol (HOG) pathway is activated in response to osmotic stress caused by high extracellular salt or sugar concentrations. This pathway has been explored in great detail in S. cerevisiae, where it mainly reacts to osmotic stress (for review, see Saito et al., 2012, Brewster et al., 2014, but also to other stress signals (Mollapour et al., 2006, Lawrence et al., 2004, Bilsland et al., 2004, Rodriguez-Pena et al., 2010. Hog1 is the MAPK of the HOG signalling pathway and upon phosphorylation relays information to many different targets both in the cytosol and the nucleus to increase the intracellular glycerol level to preserve an optimal turgor pressure. Deletion of HOG1 leads to attenuated virulence, for example, in C. albicans (Alonso-Monge et al., 1999), Cryptococcus neoformans (Bahn et al., 2005) and C. glabrata (Srivastava et al., 2015).
Interestingly, despite the high degree of conservation, regulation of the HOG pathway differs between species. In Schizosaccharomyces pombe, the orthologous Spc1 MAPK pathway is not only activated by osmotic stress but also by a wide range of environmental stresses such as UV radiation and heat stress (Toone et al., 1998, Chen et al., 2003. Furthermore, C. glabrata CgHog1 is activated by weak organic acid stress (Jandric et al., 2013, Gregori et al., 2007. In C. albicans, the transcriptional response to osmotic stress and heavy metal stress is partly Hog1 dependent (Enjalbert et al., 2006). C. albicans Cahog1Δ mutants are sensitive against oxidative stress (Brown et al., 2009, Correia et al., 2016, Alonso-Monge et al., 2003, but CaHog1 appears to be dispensable for induction of oxidative stress resistance. Therefore, the signals relayed by the HOG pathway differ even among closely related organisms.
The HOG pathway can influence a host-pathogen relationship in many ways as it is at the crossroad of many mechanisms involved in host-pathogen interactions. Here, we investigate the role of CgHog1 under different environmental conditions and to explore its involvement in virulence-associated traits. We found that C. glabrata CgHog1 protects against the secreted metabolites of Lactobacillus spp. and allows it to compete with co-colonising Lactobacillus spp.
2 | RESULTS 2.1 | CgHog1 protects C. glabrata from growth inhibition caused by weak organic acids and low pH Here, we explored the role of CgHog1 in response to alternative stress types. Unlike in S. cerevisiae, C. glabrata Hog1 confers resistance not only to osmotic stress and sorbic acid stress but also to other stress types (Jandric et al., 2013, Srivastava et al., 2015, Gregori et al., 2007. To cover monocarboxylic acids, we chose lactic acid and PAA in addition to sorbic acid as model substrates. Lactic acid commonly occurs as a by-product of Lactobacillus spp. metabolism. PAA and the derivatives 3-phenyl lactic acid, 3-indole acetic acid, and indole-3-carboxylic acid have fungicidal activity. Wild type, Cghog1Δ and a reconstituted Cghog1Δ mutant (Cghog1ΔHOG1) were spotted on agar containing the selected compounds as well as a number of other stress agents. Tests with acids and nitrite were performed at pH 4, whereas all other substances were tested at neutral pH. Low pH causes a slight growth inhibition of the Cghog1Δ strain and weak organic acids further enhance this effect ( Figure 1a). The mutant strain also shows a slight inhibition when challenged with H 2 O 2 or NO 2 − . Interestingly, osmotic stress caused by 2.4 M of glycerol had no effect on the Cghog1Δ mutant, despite the similar osmotic strength to 1.5 M of NaCl (3 osmol/L of NaCl vs. 2.4 osmol/L of glycerol).
To measure subtle phenotypes, which might have escaped the qualitative analysis of spotting assays, we quantified growth parameters, such as the peak growth rate μ max [h −1 ] during the exponential phase of growth, the lag phase [h] or the maximum growth level [AU]. Results of wild type and Cghog1Δ strains are shown in Figure 1b. Compared with the wild type, Cghog1Δ showed significantly reduced growth rates when exposed to osmotic stress, organic acids, and low pH. Some of the conditions tested caused other parameters of the medium to change as well. The effect of the low pH was subtracted by preparing a separate control sample adjusted to pH 4 using HCl when evaluating the effect of nitrite, sorbic acid, PAA, and lactic acid, which were all performed at pH 4. We calculated the final growth rate as μ final = μ measured * μ YPD /μ Ctrl , where μ measured is the initial noncorrected growth rate, μ YPD is the growth rate obtained in YPD, and μ Ctrl is the growth rate obtained in the separate control assay, with adjusted pH or osmolarity. With these corrections, we calculated the CgHog1-dependent resistance not only to sorbic acid but also to the other weak acids lactic acid and PAA as well as to pH 3.6 ( Figure 1b). For glycerol, no sensitivity phenotype was observed, despite its osmotic properties. High concentrations of NaCl inhibited growth in both the mutant and the wild type, whereas equimolar amounts of glycerol did not ( Figure S1A). We did not observe any hypersensitivity of the mutant to fluconazole, iron starvation, and high temperature and prolonged desiccation (Figure 1b, Figure S1B).
Interestingly, oxidative and nitrosative stress did not result in reduced growth rate (μ max ) in the mutant, as our previous results with   Growth experiments were run at least in triplicate with three biological replicates on the same plate. Maximal growth rate μ [h −1 ] was calculated with GROFIT. BG14 wild type, BG14 hog1Δ, and the BG14 hog1ΔHOG1 strains were cultivated in YPD or under indicated stress conditions in 96-well format for 24 hr at 37°C. (b) Different stress conditions reveal sensitivity phenotypes of the Cghog1Δ mutant. Significant sensitivity was observed for 1.5 M of NaCl (p < 0.0001), 2 mM of sorbic acid (p < 0.01), 7 mM of PAA (p < 0.0001), pH 3.6 (p < 0.01), and 100 mM of lactic acid (p < 0.0001). (c) A range of concentrations as indicated was tested for each stress condition. (d) Growth rates of BG14 wild type and hog1Δ when grown in YPD containing lactic acid adjusted to pH 7. Asterisks indicate a significant difference between wild type and Cghog1Δ (p value <0.001) drop tests on solid medium would suggest ( Figure 1a). It appears, however, that oxidative and nitrosative stress both lead to an increased lag phase ( Figure S1C). This suggests a reduced ability of the mutant to adapt to the nitrosative and oxidative stress. In addition, we tested a wide range of concentrations and pH values and observed that the Cghog1Δ strain was hypersensitive to any tested concentration of sorbic acid (0.5-2 mM), PAA (4-7 mM), and lactic acid (11-180 mM) and to low pH when compared with the wild type ( Figure 1c). Results obtained from weak acid experiments were corrected for the low pH effect. Cghog1Δ was also sensitive to lactic acid at high concentrations at neutral pH, (Figure 1d). Therefore, Cghog1Δ mutants are hypersensitive to physiological concentrations (Owen et al., 1999) of lactic acid and other weak organic acids.
2.2 | CgHog1 in vitro protects C. glabrata from cocultured Lactobacillus spp Lactobacillus spp. are a common part of the vaginal microbiome. They create a first line of defence against potentially pathogenic, microorganisms by production of stress factors, for example, organic acids.
We tested the role of CgHog1 in the C. glabrata-Lactobacillus spp.
competition. Indeed, we observed impaired growth of the Cghog1Δ mutant when directly challenged with six different clinical isolates of Lactobacillus spp. (L. rhamnosus, L. gasseri, L. paracasei, L. fermentum, L. casei, and L. crispatus) in a co-cultivation on MRS agar plates. In comparison, the wild type strain was less affected (Figure 2a). To exclude a pure pH effect, we challenged C. glabrata with pH-adjusted supernatants from three of the Lactobacillus spp. and still observed growth inhibition of the Cghog1Δ mutant ( Figure 2b). This effect also occurred in heat-or protease-treated supernatants (data not shown), thus excluding a polypeptide as a possible cause. We investigated the activity of CgHog1 upon exposure to culture supernatants obtained from L. rhamnosus. As shown in Figure 2c, the level of double phosphorylated Hog1 increased immediately (5 min) after the challenge, but remained lower than the osmotic stress control treatment.
Additionally, the response of Hog1 rapidly diminished after 15 min and decreased even below the basal level of untreated cells. Adjusting the supernatant to a neutral pH did not change the activation kinetics of Hog1, and thus, the culture pH does not contribute to the observed transient activation. The combination of low pH and lactic acid is therefore a major contributor to inhibition of C. glabrata by Lactobacillus spp., and the HOG pathway is contributing to the equilibrium between these species in co-culture.

| CgHog1 localises into the nucleus following treatment with low pH stress or lactic acid
Activation of the HOG pathway is leading to Hog1 phosphorylation and subsequent nuclear accumulation. A fully complementing GFPtagged CgHog1 (Jandric et al., 2013) was used for microscopy to determine the ratio of nuclear versus cytosolic fluorescence (N/Cratio) ( Figure 3a). As reference for nuclear accumulation of CgHog1, 0.6 M of NaCl was used, which showed an N/C ratio of 1.9 (p < 0.0001), whereas the untreated control had an N/C ratio of 1.4.
Increased localisation of CgHog1 was observed for treatment with 100 mM of lactic acid, 2.4 M of glycerol (p ≤0.0001) and low pH ( Figure 3b). Treatments that did not inhibit growth of Cghog1Δ failed to enhance nuclear localisation of CgHog1 (i.e., 250 μM of BPS, 32 μg/ml of fluconazole, 20 mM of H 2 O 2 or 42°C). However, glycerol treatment caused significant accumulation of CgHog1-GFP in the nucleus, although no growth phenotype was observed previously. Activated CgHog1 isoforms were detected by immunoblotting using phospho-specific antibodies recognising double phosphorylation at T180 and Y182 (CgHog1-PP). As positive control, cells were challenged with 0.6 M of NaCl, which led to an approximate fivefold increase of CgHog1-PP signal. Treatment with sorbic acid, PAA, lactic acid, low pH, glycerol, H 2 O 2 , and, unexpectedly, also fluconazole led to significantly increased levels of CgHog1-PP ( Figure 3c).  (Srivastava et al., 2015). We conclude from our data that the HOG pathway plays a role for defence against macrophages but no significant role in systemic infections, at least under the tested conditions in the CD1 mouse model.

| The HOG pathway is essential for lactic acid resistance in clinical isolates of C. glabrata
To define other factors contributing to weak acid resistance, we identified lactic acid stress sensitive and resistant strains in a collection of 240 C. glabrata clinical isolates. This was done by quantitative growth analysis of the isolates when exposed to a high dose of lactic acid  Western blot using α-Hog1-PP antibodies to detect the double phosphorylated form of CgHog1 in C. glabrata strain BG14 when challenged with supernatants from a L. rhamnosus strain. Pgk1 was used as loading control. Fold-changes of Hog1 phosphorylation signal relative to a nontreated control in YPD and osmotic stress (0.5 M of NaCl) are indicated. L. rhamnosus supernatant was supplemented with YPD and added directly (pH 5.3) or adjusted to neutral pH 6.9 HOG pathway is a central stress signalling pathway and has been explored in depth in S. cerevisiae (for reviews, see Saito et al., 2012, Brewster et al., 2014. In yeast, this pathway predominantly senses osmotic changes and is the major contributor of hyperosmotic stress resistance. Furthermore, ScHog1 is involved in sensing other stresses, that is, H 2 O 2 , citric acid, or acetic acid stress (Lee et al., 2017, Lawrence et al., 2004, Mollapour et al., 2006 and low pH (de Lucena et al., 2012, Claret et al., 2005. In C. albicans, CaHOG1 confers resistance to multiple stress types (Alonso-Monge et al., 2003, Smith et al., 2004, Enjalbert et al., 2006, Brown et al., 2009  for a variety of stress responses in C. glabrata (Gregori et al., 2007, Kaloriti et al., 2012, Jandric et al., 2013, Srivastava et al., 2015.
Compared with S. cerevisiae, C. glabrata CgHog1 is activated by different stress types apart from osmotic stress. The results of our phenotypic screen concur with previous reports obtained with a C. glabrata hog1Δ mutant, which showed an increased susceptibility towards oxidative stress and low pH and no effect of BPS (Srivastava et al., 2015). However, we applied a quantitative approach by analysing additional cultivation parameters such as growth rate and lag phase. Interestingly, we did not observe a growth defect of HOG FIGURE 3 CgHog1-GFP relocates to the nucleus in response to several stress conditions (a). Representative live microscopy pictures of BG14 hog1Δ expressing CgHog1-GFP without stress or under stress conditions. (b) Nuclear accumulation of Hog1-GFP upon stress induction results in a higher nucleus to cytosol signal ratio. Fluorescence intensity ratios (nucleus to cytosol) of cells grown exposed to indicated conditions are given (n ≥ 40). Compared with the nonstressed control, exposure to NaCl, low pH 4, lactic acid, and glycerol significantly increased nuclear accumulation of Hog1-GFP. FLZ = fluconazole. The Kruskal-Wallis test was used to test for significance (***p < 0.001; ****p < 0.0001). (c) Levels of dually phosphorylated CgHog1 detected in BG14 wild type when exposed to different stress agents. CgHog1 was detected using a polyclonal antiphospho-p38 MAPK antibody. All values were referenced to an untreated control sample. Treatments with NaCl, weak acids, low pH, glycerol, fluconazole, and H 2 O 2 led to increased phosphorylation levels, whereas iron starvation (BPS), heat stress (42°C), and exposure to Lactobacillus supernatant do not activate CgHog1. (d) Macrophage engulfment causes transient nuclear accumulation of CgHog1-GFP. Cells were incubated at 37°C with macrophages in a 4:1 ratio. For microscopy, cells were fixed at indicated time points and stained with Phalloidin Texas-Red. Fluorescence microscopy shows accumulation of CgHog1-GFP in the nucleus directly after phagocytosis and proliferation of Candida glabrata within the phagolysosome after engulfment after 12 hr and 24 hr pathway mutants at 42°C in contrast to other reports (Srivastava et al., 2015, Gregori et al., 2007. This discrepancy, however, remained isolated and is possibly due to growth methods or strain background differences. Patients defective in their macrophage or neutrophil response, leads to impaired intracellular survival in C. glabrata when challenged FIGURE 4 CgHog1 promotes survival in macrophages but not in a systemic mouse model (a) Cghog1Δ as well as the reconstituted mutant strain were added to macrophages in a 1:2 ratio and nonphagocytosed yeast cells were removed after 45 min. Macrophage cells were lysed after 12 hr and Candida glabrata cells were spread on YPD plates; colonies were counted after incubation at 37°C for 2 days. (b) CFU count of C. glabrata wild type and hog1Δ cells after intravenous infection of CD1 mice. Five mice were infected per time point via the lateral tail vein with C. glabrata ranging from 2.5 × 10 4 to 6.25 × 10 6 CFU/g body weight.
Samples of brain, kidneys, spleen, and liver were plated from mice after 12 hr, 72 hr, and 7 days of postinfection and CFU determined after 48 hr of incubation at 37°C. PBS mockinfected animals served as controls with human THP-1 macrophages (Srivastava et al., 2015). It was speculated that oxidative stress and high concentration of free iron within the macrophage cause the increased susceptibility of the mutant. We propose CgHOG1 to provide additional protection from macrophage killing. We show that, upon H 2 O 2 challenge, CgHOG1 is required to restart the cell cycle as it reduced lag phase. A function for reducing the lag phase has also been reported for S. cerevisiae ScHog1 where it is required to restart the cell cycle after growth arrest (Escote et al., 2011). CgHog1 becomes phosphorylated but seems not to translocate into the nucleus. In fact, ScHog1 and CaHog1 translocation to the nucleus is not required for the activation of downstream events (Westfall et al., 2008, Lee et al., 2017, Day et al., 2017. Even though CgHog1 upon H 2 O 2 challenge does not enrich in the nucleus, this does not exclude cycling between nucleus and cytosol. In any case, CgHog1 downstream targets are regulated, and it thus contributes to the rapid recovery promoting growth in the engulfed situation.
We speculate that the combined action of low pH and oxidative stress within mammalian macrophage phagosomes necessitate the recalcitrance of C. glabrata during macrophage engulfment.
To further elucidate whether CgHOG1 plays a role in the pathogenicity of C. glabrata, we conducted an infection experiment in mice. Intravenous infection of mice is one of the most widely used Candida spp. virulence assays to date (Maccallum, 2012). Immunocompetent mice were described as a suitable model for testing the in vivo fitness of C. glabrata strains (Jacobsen et al., 2010, Brunke et al., 2015. Although in mice C. glabrata infections elicit only mild symptoms and a low induction of pro-inflammatory cytokines, they can be recovered from host organs up to 28 days postinfection, even from immunocompetent mice (Brieland et al., 2001). Similar results were obtained when testing clinical strains of C. glabrata (Arendrup et al., 2002). We observed no altered fitness of the C. glabrata hog1Δ mutant in vivo. An earlier study in BALB/c mice (Srivastava et al., 2015) reported significant loss of recovered hog1Δ yeast cells 7 days postinfection. Our data collected from a time series show a rapid decline of fungal burden in the kidney, spleen, and liver within the first 3 days, except for the brain. We found no significant difference between wild type and hog1Δ mutant.  (Goncalves et al., 2016). Lactobacillus spp.
secrete large amounts of lactic acid and a variety of other weak carboxylic acids thereby acidifying their environment and rendering it less hospitable for other microbes (Parolin et al., 2015, Larsen et al., 2001, Tachedjian et al., 2017. Their strategies encompass not only direct inhibition of competitive microbiota but also indirect inhibition through depletion of colonisation sites or competition for nutrients (Larsen et al., 2001). Interestingly, C. albicans responds to lactate with masking of β-glucan, a key pathogen-associated molecular pattern to reduce its visibility to the immune system and also indicating a long history of adaptation (Wheeler et al., 2008, Ballou et al., 2016.
Resistance to weak acids and low pH would allow C. glabrata to resist the Lactobacillus-dominated vaginal microflora of a healthy host.
Indeed, we found that CgHOG1 confers resistance to direct challenge with Lactobacillus spp. as well as to their late-stationary phase supernatant. The growth-inhibitory effect of Lactobacillus spp. on C. glabrata is likely caused by weak acids. We found lactic acid concentrations as Deletion of HOG1 in these selected isolates resulted in a uniform hypersensitive phenotype irrespective of the original phenotype.
Therefore, the different phenotype is likely caused by differences downstream of CgHog1. Besides CgHog1, other components such as, for example, CgHaa1 might contribute to the variation of intrinsic resistance of isolates observed here. CgHaa1 regulates about 75% of all acetic acid induced genes (Bernardo et al., 2017) and deletion of Haa1 resulted in a growth phenotype on lactic acid and other weak organic acids. However, our results confirm CgHOG1 as a bottleneck in the complex regulative pattern of weak acid resistance.
In conclusion, the phenotypes conferred by CgHOG1 were associated with two important virulence factors: the ability to compete with vaginal Lactobacillus spp. and the ability to resist macrophage-assisted killing. CgHOG1 also confers resistance to individual stresses such as oxidative, nitrosative, and weak acid stress enabling C. glabrata to compete with vaginal Lactobacillus spp. and to overcome the host's innate immune system. Finally, we report that the HOG pathway appears to be a bottleneck in the regulation network governing intrinsic weak acid resistance in C. glabrata. As a next step, the relevance of the HOG pathway for virulence might be assessed further, either in an improved in vivo model, such as a murine VVC model (Nash et al., 2016), or cell culture-based models mimicking the vaginal microbiome. We propose that CgHog1 has a vital function in the host-commensal relation.
The Cghog1Δ mutant and the pHog1-GFP plasmid were created as described previously (Jandric et al., 2013 West Pico detection solution (Thermo Scientific). As a loading control, a rabbit antiserum raised against yeast phosphoglycerate kinase (Pgk1) was used (Kuchler et al., 1993). All signals were normalised to Pgk1 and to a reference protein extract of BG14 grown in YPD, which was run on each gel.

| Macrophage assays
Primary bone marrow-derived macrophages (BMDMs) were obtained from the femur bone marrow of 6-to 10-week-old C57Bl/6 mice housed under specific pathogen-free conditions. Cells were cultivated in Dulbecco's Modified Eagle's medium supplemented with 10% fetal calf serum (FCS) in the presence of L cell-derived CSF-1 as described (Baccarini et al., 1985). For infection assays, BMDMs were seeded at 5 × 10 5 cells per dish in 3.5 cm dishes containing medium without antibiotics. Log-phase C. glabrata cells were washed with phosphate buffered saline (PBS) supplemented with 0.1% glucose and added to macrophages in a 4:1 ratio and incubated at 37°C. For microscopy, cells were fixed with 2% formaldehyde for 5 min. After washing with PBS, cells were incubated in 1% Triton X-100 for 1 min. After another washing cycle, cells were dyed with Phalloidin Texas-Red for 30 min.
Coverslips were fixed to slides with Mowiol. For CFU assays, BMDMs were seeded at 2 × 10 5 cells per dish. Exponentially growing C. glabrata cells were washed with PBS supplemented with 0.1% glucose and added to macrophages in a 1:2 ratio and incubated at 37°C. After 45 min, cells were washed three times with PBS to remove nonphagocytosed yeast cells, and fresh medium was added. At the indicated times, deionised water was added to lyse macrophage cells.
C. glabrata cells were spread on YPD plates; colonies were counted after incubation at 37°C for 2 days.

| Murine infection model
The infection was performed as described previously (Jacobsen et al., 2014). Briefly, C. glabrata cells from liquid YPD overnight cultures were washed twice with ice-cold PBS and adjusted to the desired concentration of 5 × 10 8 cells/ml in PBS. On Day 0, five female, 8-10 weeks old, CD1 mice per time point were infected via the lateral tail vein with 2.5 × 10 7 CFU C. glabrata in 200 μl of PBS.
The infection dose was confirmed by plating. After infection, the health status of the mice was examined twice a day by a veterinarian. Mice were humanely sacrificed 12 hr, 72 hr, and 7 days of postinfection. Immediately after euthanasia, brain, kidneys, spleen, and liver were removed aseptically, rinsed with sterile PBS, weighed, and placed in sterile PBS. Following homogenisation using an UltraThurrax, serial dilutions were prepared in PBS and plated on YPD for determination of CFU.

| Ethics statement
All animal experiments were performed in accordance with the German animal protection law and were approved by the

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Beyer R, Jandric Z, Zutz C, et al.