Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans

Candida albicans cells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to α-ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen. C. albicans strains lacking GDH2 (gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression and mitochondrial function are derepressed as glucose levels are lowered from 2% (~110 mM) to 0.2% (~11 mM), or when glycerol is used as primary carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. In intact hosts, such as in fly and murine models of systemic candidiasis, gdh2-/- mutants are as virulent as wildtype. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence. Consistently, using the pH-sensitive dye (pHrodo), we observed no significant difference between wildtype and gdh2-/- mutants in phagosomal pH modulation. Following engulfment of fungal cells, the phagosomal compartment is rapidly acidified and hyphal growth initiates and sustained under consistently acidic conditions within phagosomes. Together, our results demonstrate that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate understanding of the microenvironment within macrophage phagosomes and the metabolic events underlying the survival of phagocytized C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome.

source. Using time-lapse microscopy, we document that gdh2-/-cells survive, 48 filament and escape from primary murine macrophages at rates indistinguishable 49 from wildtype. Consistently, gdh2-/-strains are as virulent as wildtype in fly and 50 murine models of systemic candidiasis. Thus, although Gdh2 has a critical role in 51 central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is 52 surprisingly dispensable for escape from macrophages and virulence, 53 demonstrating that amino acid-dependent alkalization is not essential for hyphal oxidative stressors and low pH (8-10). Acidification is important to optimize the 101 activity of the hydrolytic enzymes that target to the phagosome. 102 C. albicans can survive and even escape macrophage engulfment. This is thought 103 to depend on the ability of fungal cells to raise the phagosomal pH via ammonia 104 extrusion. It has been suggested that urea amidolyase (Dur1,2), localized to the 105 cytoplasm, catalyzes the reactions generating the ammonia extruded from cells by 106 the plasma membrane-localized Ato proteins (11,12). In addition to impairing the 107 activity of pH-sensitive proteolytic enzymes, phagosomal alkalization is thought to 108 initiate and promote hyphal growth (11,13). Consistent with this notion, C. albicans 109 lacking STP2, encoding one of the SPS (Ssy1-Ptr3-Ssy5) sensor controlled effector 110 factors governing amino acid permease gene transcription (14), fail to form hyphae 111 and escape macrophages (13). These observations led to a model that the reduced 112 capacity of stp2Δ strains to take up amino acids limits the supply of substrates of 113 Dur1,2 catalyzed deamination reactions, which would result in the reduced capacity 114 to alkalinize the phagosome (12, 13). 115 We have recently shown that the mitochondrial proline catabolism is required for 116 hyphal growth and macrophage evasion. The proline catabolic pathway is the 117 primary route of arginine utilization (15) and operates independently of the cytosolic 118 Dur1,2-catalyzed urea-CO2 pathway (15,16). In contrast to the proposed model (12), 119 we observed that dur1,2-/-cells retain the capacity to alkalinize a basal medium 120 containing arginine as sole nitrogen and carbon source (15). Furthermore, strains 121 carrying put1-/-or put2-/-mutations exhibit strong growth defects and 122 consequently, are incapable of alkalinizing the same medium, suggesting that 123 alkalization is linked to proline catabolism.
A potential source of ammonia responsible for alkalization is the deamination of 125 glutamate, a metabolic step downstream of Put2. In Saccharomyces cerevisiae, the 126 primary source of free ammonia is generated by the mitochondrial-localized NAD + -127 dependent glutamate dehydrogenase (Gdh2) catalyzed deamination of glutamate to 128 α-ketoglutarate, a reaction that generates NADH and NH3 (17). Importantly, the 129 reaction is anaplerotic and replenishes the tricarboxylic acid (TCA) cycle with α-130 ketoglutarate, a key TCA cycle intermediate between isocitrate and succinyl CoA, 131 and an important precursor for amino acid biosynthesis. 132 Here, we have examined the role of Gdh2 in morphological switching under in 133 vitro conditions in filament-inducing media, in situ in the phagosome of primary 134 murine macrophages, and in virulence in two model host systems. We show that 135 when C. albicans utilize amino acids as sole nitrogen-and carbon-sources they 136 extrude ammonia, which originates from Gdh2-catalyzed deamination of glutamate. 137 In contrast to current understanding regarding the importance of phagosomal 138 alkalization, we report that C. albicans strains lacking GDH2 filament and escape the 139 phagosome of engulfing macrophages at rates indistinguishable to wildtype. 140 Furthermore, we also report that the Gdh2-catalyzed reaction is dispensable for 141 virulence in both fly and murine models of systemic candidiasis. 142 143

C. albicans GDH2 is responsible for amino acid-dependent alkalization in vitro 145
Arginine is rapidly converted to proline and then catabolized to glutamate in the 146 mitochondria through the concerted action of two enzymes, proline oxidase (Put1; proline to Δ 1 -pyrroline-5-carboxylate or P5C) and P5C dehydrogenase (Put2; P5C to 148 glutamate) (Fig. 1A). C. albicans strains lacking PUT1 (put1-/-) and/or PUT2 (put2-/-) 149 are unable to grow efficiently in minimal medium containing 10 mM of arginine as 150 sole nitrogen and carbon source (YNB+Arg, pH = 4.0), and fail to alkalinize the 151 medium (15). In contrast, cells carrying null alleles of DUR1,2 (dur1,2-/-) grow 152 robustly and alkalinize the media (15). To test if the catabolism of amino acids other 153 than arginine and proline can be used as sole carbon source we examined the 154 growth characteristics of the strains in YNB containing 1% casamino acids, a 155 medium containing high levels of all proteinogenic amino acids (Fig. 1B). In this 156 media, dur1,2-/-cells grew as wildtype and readily alkalinized the media (compare 157 tube 5 with 6). In contrast, put1-/-cells exhibited poor growth and weakly alkalinized 158 the medium (tube 3). Cells lacking Put2 activity (put2-/-) did not grow and the culture 159 media remained acidic (tube 2). Interestingly, a put1-/-put2-/-double mutant strain 160 grew better than the single put2-/-mutant (compare tube 4 with 2). The severe 161 growth impairment associated with the loss of Put2 is likely due to the accumulation 162 of P5C, which is known to cause mitochondrial disfunction (18). These results 163 indicate that the amino acids metabolized via the proline catabolic pathway are 164 preferentially used as carbon sources when mixtures of amino acids are present, 165 e.g., in casamino acid preparations. The catabolism of these non-prefered amino 166 acids contribute only modestly to alkalization, consistent with reports that not all 167 amino acids can serve as carbon sources and contribute to environmental 168 alkalization (12). 169 The requirement of proline catabolism for growth suggested that the downstream 170 deamination of glutamate to α-ketoglutarate, catalyzed by glutamate media (Fig. 1C, compare columns 3 and 4 with 1). These observations confirm that 195 Gdh2 is responsible for alkalization of the external growth environment. 196 197 GDH2 is required for ammonia extrusion 198 Next, we analyzed whether the alkalization defect of the gdh2-/-mutant was due to 199 the lack of ammonia extrusion. The levels of volatile ammonia produced was 200 measured by colonies growing on solid YNB+CAA with 0.2% glucose medium 201 buffered with MOPS (pH = 7.4); the standard acidic growth medium (pH = 4.0) traps 202 ammonia (NH3) as ammonium (NH4 + ), decreasing the level of volatile ammonia and 203 thereby interfering with the assay. As shown in Fig. 1D, the gdh2-/-strain did not 204 release measurable ammonia. Consistent with their ability to alkalinize the growth 205 media (Fig. 1C), wildtype, dur1,2-/-and CRISPR control strains released substantial 206 and indistinguishable levels of ammonia. Together, these results indicate that the 207 reaction catalyzed by Gdh2 generates the ammonia that alkalinizes the growth 208 environment when C. albicans uses amino acids as the primary energy source. 209 210

Environmental alkalization originates in the mitochondria 211
We recently confirmed that mitochondrial activity in C. albicans can be repressed by 212 glucose (15), a finding that is consistent with existing transcriptional profiling data 213 (19). Consequently, the glucose repressible nature of extracellular alkalization in the 214 presence of amino acids could be linked to glucose repressed mitochondrial 215 function. To examine this notion, we first sought to confirm that Gdh2 localizes to 216 mitochondria. Cells (CFG273) expressing the functional GDH2-GFP reporter were grown in synthetic glutamate media with 0.2% glucose (SED0.2%) and YNB+CAA. 218 The GFP fluorescence in cells grown under both conditions clearly localized to the 219 mitochondria as determined by the precise overlapping pattern of fluorescence with 220 the mitochondrial marker MitoTracker Deep Red (MTR) (Fig. 2A). 221 To independently assess the role of mitochondrial activity in the alkalization 222 process, we grew the wildtype cells in standard YNB+CAA medium (without 223 glucose), in the presence of Antimycin A, a potent inhibitor of respiratory complex III. 224 No alkalization was observed in the medium even after 24 h of growth (Fig. 2B, 225 upper left panel). Antimycin A clearly impeded the growth of wildtype cells, which 226 phenocopies the gdh2-/-growth in YNB+CAA. To ascertain whether the failure to 227 alkalinize the medium was due to inhibiting mitochondrial respiration and not due to 228 cell death, we harvested the cells from antimycin-treated cultures and suspended 229 them in fresh medium; the cells regained their capacity to alkalinize the medium 230 and a low starting cell density, a delay in alkalization was observed initially (Fig. S2A), but then alkalization was virtually indistinguishable after 48 h, or when a high 242 starting cell density was used (data not shown). These results, together with our 243 observation that glucose availability influences Gdh2-dependent growth and 244 alkalization (Fig. 1C), support our conclusion that alkalization originates from 245 metabolism localized to mitochondria. 246

Gdh2 expression is repressed by glucose 247
To follow up on the observations that glucose negatively affects Gdh2 activity and 248 Gdh2 is a component of mitochondria ( Fig. 2A), we sought to visualize Gdh2 249 expression in living cells when shifted from repressing YPD (2% glucose) to non-250 repressing YNB+CAA. To do this, we used the same Gdh2-GFP reporter strain 251 described earlier ( Fig. 2A). This enabled us to observe Gdh2 expression in single 252 cells growing on a thin YNB+CAA agar slab over a period of 6 h. The Gdh2-GFP 253 signal was initially weak (t = 0 h), becoming more intense as time progressed and as 254 cells underwent several rounds of cell division, some leading to filamentous 255 pseudohyphal growth (Fig. 3A). 256 To relate this observation with the actual alkalization process, we analyzed the 257 levels of Gdh2-GFP in cells grown in liquid culture taken at similar time points. Cells, 258 pre-grown in YPD (2% glucose), were shifted to YNB+CAA and the levels of Gdh2-259 GFP were assessed by immunoblot analysis. To enable the recovery of adequate 260 amounts of cells for subsequent extract preparation, we increased the starting cell 261 density of the culture (i.e., OD600 ≈ 2.0). As shown in Fig. 3B (left panel) the Gdh2-262 GFP level in YPD-grown cells was initially low (t = 0 h) but within 2 h the level was 263 greatly enhanced and remained so during the entire 6 hr incubation. During the 264 course of growth, the media became successively alkaline, increasing from the starting pH of 4 to 7 (Fig. 3B, right panel). The finding that Gdh2 expression is 266 induced in cells growing in media rich in amino acids (i.e., YNB+CAA or YPG) 267 indicates that GDH2 expression in C. albicans, in contrast to S. cerevisiae (21), is not 268 subject to NCR. 269 Next, we examined the expression and stability of Gdh2-GFP in cells shifted from 270 YPD to YPG (Fig. 3C). Again, the level of Gdh2-GFP rapidly increased (lanes 1-3) 271 and remained high following the addition of glucose (2% final concentration) (

Inactivation of Gdh2 does not impair morphogenesis 281
Based on current understanding that the ability of C. albicans to alkalinize their 282 growth environments contributes to the induction of hyphal growth, and the 283 observation that gdh2-/-cells formed smooth macrocolonies on YNB+CAA (pH = 284 4.0) with non-repressing 0.2% glucose and 1% glycerol (Fig. 1C, column 2), we 285 examined whether the inactivation of Gdh2 would negatively affect morphogenesis. 286 To test this notion, we examined growth on Spider and Lee's media, two standard 287 media used to assess filamentation. These media contain amino acids, have a neutral pH, and are known to promote filamentous growth of wildtype cells. Similar 289 to wildtype and CRISPR control strains, the macrocolonies formed by the gdh2-/-290 strain were wrinkled and surrounded by an extensive outgrowth of hyphal cells (Fig.  291   4A). This indicates that Gdh2 function is dispensable for filamentation. This is 292 supported by the recent paper showing the capacity of gdh2Δ/Δ to switch in amino 293

acid-based medium (24). 294
This unexpected result led us to evaluate the capacity of gdh2-/-cells to filament 295 within phagosomes of engulfing macrophages. FITC-stained WT (SC5314) and 296 gdh2-/-(CFG279) cells were individually co-cultured with RAW264.7 macrophages. 297 Non-phagocytosed fungal cells were removed and then co-cultures were imaged 298 after 1.5 h of incubation. We readily observed macrophages containing gdh2-/-cells 299 that had formed hyphal extensions (Fig. 4B), clearly suggesting that amino acid-300 dependent alkalization of the phagosome is not a requisite for the induction of 301 filamentous growth. 302 303

Gdh2-GFP expression is rapidly induced upon phagocytosis by macrophages. 304
To assess the time-course of Gdh2-GFP expression in phagocytized C. albicans 305 cells, we co-cultured the Gdh2-GFP reporter strain CFG273 with RAW264.7 (RAW) 306 macrophages and followed the interaction by time-lapse microscopy. The Gdh2-307 GFP signal significantly increased after phagocytosis (Fig. 5A, see Video V1, 308 Supporting Information). We repeated the experiment using primary murine bone 309 marrow-derived macrophage (BMDM) and obtained a similar result. However, due to 310 the inherent green autofluorescence of BMDM, the GFP fluorescence appeared less pronounced (Fig. 5B, see Video V2, Supporting Information). These results 312 demonstrate that although Gdh2 expression is induced rapidly following 313 phagocytosis, presumably the reflection of limiting glucose availability and the 314 subsequent release from glucose repression. 315 316

Gdh2 activity is not required to escape macrophages 317
We directly compared the ability of wildtype and the gdh2-/-mutant cells to survive 318 and escape after being phagocytized by primary BMDM using a competition assay 319 ( Fig. 6). To carry out the experiment we created a wildtype strain constitutively 320 expressing GFP (ADH1/PADH1-GFP) and a gdh2-/-mutant strain constitutively 321 expressing RFP (gdh2-/-ADH1/PADH1-RFP). Both strains exhibited unaltered growth 322 characteristics (see Fig. S2B). Equal numbers of WT and gdh2-/-cells were mixed 323 (green:red; 1:1), and the fungal cell suspension was incubated with BMDM at a MOI 324 of 3:1 in HBSS for 30 min before washing non-phagocytosed fungal cells away. The 325 co-cultures were monitored by time-lapse microscopy. 326 Again, contrary to what we expected, the gdh2-/-mutant remained fully 327 competent to initiate hyphal growth in the phagosome of BMDM (Fig. 6). 328 Furthermore, given the perceived importance of environmental alkalization in the 329 onset of phagosomal escape by C. albicans, we anticipated that the gdh2-/-mutant 330 would be killed more efficiently than the wildtype. To test this notion, we performed 331 a colony forming unit (CFU) assay to quantify the survival of phagocytized cells. The 332 results show that Gdh2-mediated alkalization is not essential for survival in BMDM 333 Bomanin genes on chromosome 2 that encode for secreted peptides with 343 antimicrobial property (26). As shown in the survival curve, the gdh2-/-mutant 344 remained competent to infect Bom Δ55C flies similar to wildtype control (Fig. 7A). The 345 data indicate that Gdh2-dependent alkalization is not required for virulence in a 346

Drosophila infection model. 347
To further assess the importance of Gdh2 in virulence in a more complex host, we 348 used a tail vein infection model using C57BL/6 mice. Two groups of mice (n = 10) 349 were challenged with 3 x 10 5 wildtype or gdh2-/-cells and survival was monitored 350 for a period of up to 8 days. Similar to the fly model, we observed that the loss of 351 Gdh2 activity did not attenuate virulence (Fig. 7B); the gdh2-/-mutant exhibited 352 survival indistinguishable from wildtype. Consistently, the fungal burden of gdh2-/ 353 cells in the brain, kidney and spleen of infected mice 3 days post-infection did not 354 significantly differ to mice infected with wildtype (Fig. 7C). Next we performed a 355 competition assay; equal numbers of wildtype and gdh2-/-cells were intravenously 356 injected in mice and the ratio (R) of wildtype to gdh2-/-cells recovered from kidneys 3 days post-infection was determined. Consistent to our findings in mice individually 358 infected with each strain, the ratio of recovered cells did not significantly differ to 359 that of the inoculum ratio (I) (Fig. 7C). Together, our results indicate that Gdh2 is not 360 required for virulence, and that the loss of Gdh2 activity does not create a selective 361 disadvantage or significantly impair growth in infected model host systems. 362

364
In this work, we identified a major metabolic step that endows C. albicans with the 365 capacity to increase the extracellular pH by ammonia extrusion in the presence of 366 amino acids. We have shown that under in vitro growth conditions, environmental 367 alkalization is dependent upon GDH2, a gene that encodes the mitochondrial-368 localized glutamate dehydrogenase. Strikingly, the data clearly show that despite its 369 unambiguous role in alkalization in vitro, GDH2 remained dispensable for the 370 induction of hyphal growth and escape of C. albicans from macrophages, and also 371 dispensable for virulence in intact hosts. Our results, consistent with a recent report 372 (25), suggest that phagosomal alkalization is unlikely to be a defining event required 373 for hyphal initiation of C. albicans in phagosomes of engulfing macrophages. 374 Despite its role as a key enzyme of central nitrogen metabolism, the gdh2-/-375 mutant exhibited only a modest growth defect on synthetic glucose or glycerol 376 media containing, glutamate as sole nitrogen source (Fig. S1C). Consistent with 377 proline being catabolized to glutamate in a linear pathway mediated by Put1 and 378 Put2, a similar modest growth defect of gdh2-/-was observed when proline was the 379 sole source of nitrogen (Fig. S1C). However, when glucose or glycerol is removed from the media (i.e., YNB+CAA), and amino acids serve as both carbon and nitrogen 381 sources, the gdh2-/-mutant demonstrated a striking growth defect (Fig. 1B, 1C, 382 S2A). Under these conditions, the impaired growth of cells lacking Gdh2 is likely the 383 consequence of diminished levels of α-ketoglutarate. In the absence of Gdh2, cells 384 must rely entirely on the TCA cycle to generate α-ketoglutarate required to support 385 de novo biosynthetic needs, e.g., amino acid biosynthesis. In the absence of 386 glucose or glycerol, the supply of acetyl-CoA derived from pyruvate becomes 387 limiting, stalling the TCA cycle, and consequently, the growth of gdh2-/-cells. 388 Similar to S. cerevisiae, the Gdh2-catalyzed reaction is the primary source of 389 extruded ammonia in C. albicans. This conclusion is based on the following key 390 observations: 1) a mutant strain lacking GDH2 (gdh2-/-) is unable to alkalinize media 391 with amino acids as carbon and nitrogen source (i.e., YNB+CAA) and under non-392 repressing glucose conditions (Fig. 1C); 2) ammonia extrusion is impaired in gdh2-/-393 mutant (Fig. 1D); and 3) the Gdh2-catalyzed reaction responsible for alkalization 394 occurs in the mitochondria, and is subject to glucose repression and inhibited by 395 inhibitors of mitochondrial respiration (Fig. 2). 396 The capacity of glucose to repress mitochondrial activity (15) and Gdh2 as the external environment is acidic. Hence, the ability of the ammonia generated 420 by Gdh2 will likely be the consequence of Pma1 activity, the major proton pumping 421 ATPase in the PM. Alternatively, and according to several reports, putative ammonia 422 transport proteins, the Ato family of plasma membrane proteins, are thought to 423 facilitate ammonia export in C. albicans (11). Supporting this notion, the deletion of 424 ATO5 significantly delays alkalization. Interestingly, the requirement for the Ato 425 proteins suggests that the species traversing the plasma membrane from within the 426 cell is either charged or polar, thus, it is likely that the transported species is 427 ammonium (NH4 + ) and coupled to H + import as previously suggested in yeast (30, 31). Since cytoplasmic pH is tightly regulated, the conundrum persists as to how 429 extruding ammonium can facilitate steady-state alkalization. The underlying 430 mechanism of how Ato proteins facilitate alkalization needs to be precisely defined 431 and placed in context to the fact that ammonia can readily diffuse through 432 membranes, and in so doing, is expected to move directionally towards acidic 433 environments. trigger that causes filamentation (25), aligns well with our observations. Accordingly, 450 Westman et al. proposed that the step-wise alkalization of the phagosome could be 451 attributed to proton leakage out of the compartment due to the transient physical stress imposed by hyphal expansion. Also, hyphal formation was found to start prior 453 to a measurable change in pH (pH ~ 5-6), suggesting that alkalization is not the 454 primary stimulus triggering hyphal formation in the phagosome. Beyond the realm of 455 the phagosome, our work also suggests that environmental alkalization via ammonia 456 extrusion, a mechanism that is thought to facilitate virulence of fungal pathogens 457 (32), is dispensable for pathogenesis of C. albicans (Fig. 7) requiring us to rethink 458 the specific role of alkalization in fungal virulence. 459 We confirmed that DUR1,2 does not significantly contribute to alkalization.  Table S1 were 500 routinely cultivated in YPD agar medium (1% yeast extract, 2% peptone, 2% 501 glucose, 2% Bacto agar) at 30 °C after recovery from -80 °C glycerol stock. Where 502 needed, YPD medium was supplemented with 25, 100 or 200 µg/ml nourseothricin 503 were grown in liquid YPD for overnight at 30°C and then washed 3X with ddH2O. 568 Cells were diluted in the indicated alkalization media at OD600 ≈ 2 and then 569 incubated continuously in a rotating drum for 6 h at 37°C with sampling performed 570 every 2 h. In each sampling point, cells were harvested, washed once with ice-cold ddH2O, and then adjusted to OD600 ≈ 2. Whole cell lysates were prepared using 572 sodium hydroxide/ trichloroacetic acid (NaOH/TCA) method as described previously 573 with minor modifications (41). Briefly, 500 µl of adjusted cell suspension were added 574 to tube containing 280 µl of ice-cold 2 M NaOH with 7% ß-Mercaptoethanol (ß-Me) 575 for 15 min. Proteins were then precipitated overnight at 4°C by adding the same 576 volume of cold 50% TCA. Protein pellets were collected by high-speed 577 centrifugation at 13,000 rpm for 10 min (4°C) and then the NaOH/TCA solution 578 completely removed. The pellets were resuspended in 50 µl of 2X SDS sample 579 buffer with additional 5 µl of 1 M Tris Base (pH = 11) to neutralize the excess TCA. 580 Samples were denatured at 95-100°C for 5 min before resolving the proteins in 581 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4-582 12% pre-cast gels (Invitrogen). Proteins were analyzed by immunoblotting on 583 nitrocellulose membrane according to standard procedure. After transfer, 584 membranes were blocked using 10% skimmed milk in TBST (TBS + 0.1% Tween) 585 for 1 h at room temperature. For Gdh2-GFP detection, membranes were first 586 incubated with mouse anti-GFP primary antibody at 1:2,000 dilution (JL8, Takara) for 587 overnight at 4°C. For the detection of the primary antibody, an HRP-conjugated goat 588 anti-mouse secondary antibody (Pierce) was used. For loading control, α-tubulin 589 was detected with rat monoclonal antibody conjugated to HRP [YOL1/34] (Abcam). 590 For both secondary antibody and loading control, antibodies were used at 1:10,000 591 dilution in 5% skimmed milk in TBST incubated for 1 h at room temperature. 592 Immunoreactive bands were visualized by enhanced chemiluminescent detection 593 system (SuperSignal Dura West Extended Duration Substrate; Pierce) using Filamentation assay. Filamentation in solid Spider (42) or Lee's (43) media was 596 performed as described (44). Cells from overnight YPD liquid cultures were 597 harvested, washed 3X with sterile PBS, adjusted to OD600 ≈ 1 and then 5 µl of cell 598 suspensions were spotted onto the indicated media. Plates were allowed to dry at 599 room temperature before incubating at 37 ºC as indicated. 600 Macrophage culture. RAW264.7 murine macrophage cells (ATCC TIB-71) and 601 primary bone marrow-derived macrophages (BMDM) were cultured and passaged in 602 complete RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 603 U/ml penicillin and 100 mg/ml streptomycin (referred to as R10 medium in the text) 604 in a humidified chamber set at 37°C with 5% CO2. For BMDM differentiation, bone 605 marrows collected from mouse femurs of C57BL/6 wildtype mice (7-to 9-week old) 606 were mechanically homogenized and resuspended in R10 medium supplemented 607 with 20% L929 conditioned media (LCM). Differentiation was carried out initially for 608 3 days before boosting the cells with another dose of 20% LCM until harvested. 609 BMDM were used 7-10 days after differentiation. 610 C. albicans killing assay. To assess candidacidal activity by BMDM, we co-611 cultured C. albicans wildtype and gdh2-/-mutant with BMDM and then assessed 612 colony forming units (CFU) following co-incubation. About 16-24 h prior to co-613 culture, differentiated BMDM were collected by scraping, counted, and then seeded 614 at 1 x 10 6 cells/well into a 24-well microplate. C. albicans cells from overnight YPD 615 cultures were collected by centrifugation, washed 3X with sterile PBS, and then 616 added to macrophages at MOI 3:1 (C:M). The plates were briefly centrifuged at 500 617 x g for 5 min to collect the fungal cells at the bottom of each well and then co-618 cultured for 2 h in a humidified chamber. After co-culture, each well was treated with 0.1% Triton X-100 for 2 min followed by vigorous pipetting to lyse the macrophage 620 and release the fungal cells. Each well was rinsed seven times (7X) with ice-cold 621 ddH2O and collected in a 15-ml conical tube. Lysates were serially diluted and then 622 plated onto YPD. Plates containing colonies between 30-300 were counted. The     wildtype or gdh2-/-cells (upper panels) and survival (left) and weight loss (right) was 954 monitored at the timepoints indicated. Survival curves from two independent 955 experiments were statistically analyzed by the Kaplan-Meier method (a log-rank test, 956 GraphPad Prism), no significant difference. The fungal burden (lower panels) in brain 957 (left), kidney (middle), and spleen (right) extracted from mice 3 days post infection. 958 Each symbol represents a sample from an individual mouse and results were 959 compared by Student t-test, no significant difference. (C) Competition assay; mice 960 were infected via the tail vein with an inoculum (I) comprised of an equal number of 961 wildtype (SC5314) and gdh2-/-(CFG279), 1:1. At 3 days post infection, the 962 abundance and genotype of fungal cells recovered from kidneys was quantitated 963 and the ratio of wildtype:gdh2-/-recovered (R) was determined. The significance of 964 the log2(R/I) values was assessed using an unpaired t-test, no significant difference 965 (ns). V1. Gdh2-GFP is induced upon phagocytosis by murine RAW264.7 macrophages 974 (Fig. 5A). 975 V2. Gdh2-GFP is induced upon phagocytosis by primary murine BMDM (Fig. 5B). 976 V3. Competition assay to compare wildtype and gdh2-/-filamentation and 977

Supporting Information
survival upon phagocytosis by BMDM (Fig. 6). 978 979 Tables: 980   Table S1. Strains used in this study 981 Table S2. Primers used in this study 982 Tables   1054   1055   Table S1. Strains used in this study.