Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Introduction 47 of repeated live-imaging of larvae over the course of multi-day infections [19][20][21][22][23][24]. Additionally,A. 73 fumigatus infections in zebrafish can be successfully treated with clinically relevant antifungals [25], 74 allowing us to screen for changes in drug susceptibility of ZfpA mutants in vivo. 75 Here, we sought to determine whether ZfpA-mediated changes to hyphae could impact tissue invasion, 76 resistance to host defenses, and antifungal susceptibility during infection. Using a combination of in vivo 77 zebrafish experiments and human neutrophil killing assays, we show that loss of ZfpA does not impede 78 tissue invasion during infection but limits virulence by increasing fungal susceptibility to neutrophil killing. 79 Further, ZfpA deletion increases susceptibility to caspofungin, but not voriconazole, during infection of 80 neutrophil-deficient hosts. Notably, ZfpA overexpression decreases susceptibility to both neutrophil killing 81 and antifungal treatment. We found that ZfpA confers protection against caspofungin via regulation of 82 chitin synthesis during hyphal development, offering mechanistic insight into the function of this 83 transcription factor during echinocandin exposure. Together, these findings establish a role for ZfpA in 84 tolerance to stress induced by the host immune response and antifungal drugs during infection. 85

Results 86
ZfpA regulates virulence and fungal burden but does not affect immune cell recruitment in wild-type 87 hosts 88 We hypothesized that the effects of ZfpA on hyphal development may impact tissue invasion and resistance 89 to host defenses during infection. Among other phagocyte defects, neutrophil-deficiency or neutropenia is 90 a major risk factor for IA development [26]. Therefore, to test whether ZfpA is important for pathogenesis 91 in a clinically relevant host background, we used a larval zebrafish model of a human leukocyte adhesion 92 deficiency in which neutrophils express a dominant-negative Rac2D57N mutation (mpx:rac2D57N) that 93 impairs recruitment to infection and host survival [19,27]. We found that virulence of ∆zfpA was attenuated 94 in wild-type control larvae (mpx:rac2wt) while virulence of OE::zfpA was similar to WT CEA10 (Fig 1). 95 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint measured the percent of germlings able to escape neutrophil contact by extending hyphae outside of 121 surrounding neutrophil clusters. We found that ∆zfpA germlings never escaped surrounding neutrophils, 122 while WT CEA10 and OE::zfpA escaped at similar frequencies, suggesting that the enhanced ability of 123 OE::zfpA to withstand neutrophil activity is not due to an increased ability to evade neutrophils via branch 124 production ( Fig 3C). These data also suggest that susceptibility to neutrophil killing underlies the virulence 125 defect of ∆zfpA in wild-type hosts and that ZfpA confers protection from host defenses. 126

ZfpA contributes to cell wall integrity but is not implicated in osmotic or oxidative stress 127
Alterations in stress resistance in the ZfpA mutants could underpin differences in virulence and 128 susceptibility to neutrophil-killing mechanisms such as reactive oxygen species. We therefore challenged 129 ZfpA mutants with cell wall, osmotic, and oxidative stressors using spot-dilution assays. ZfpA deletion was 130 previously shown to increase susceptibility to the common cell wall stressor calcofluor white (CFW) which 131 impairs cell wall integrity by disrupting assembly of chitin chains in the cell wall [11,29]. Accordingly, 132 ∆zfpA showed increased susceptibility to CFW while OE::zfpA was more resistant (Fig 4). There were no 133 clear differences between strains in susceptibility to the osmotic stressor sorbitol or the oxidative stressor 134 H2O2 (Fig 4), suggesting cell wall defects as the primary driver of differential stress resistance in these 135 mutants. 136

ZfpA overexpression decreases voriconazole susceptibility in vitro and during infection 137
We next wanted to test whether ZfpA affects antifungal susceptibility during infection. Tri-azoles are a 138 first-line therapy for invasive aspergillosis that suppress fungal growth by impairing ergosterol synthesis 139 and membrane integrity [30]. Further, zfpA was upregulated in a previous study of the transcriptional 140 response of A. fumigatus to voriconazole treatment [16]. To assess voriconazole susceptibility in the ZfpA 141 mutants, we measured colony diameter after 4 days of growth on solid GMM supplemented with 0.1 or 142 0.25 µg/mL voriconazole. To account for the effects of ZfpA manipulation on hyphal development and 143 colony size, we report changes in growth for each strain as colony diameter relative to growth on GMM. 144 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 At both concentrations tested, ZfpA overexpression reduced voriconazole susceptibility relative to WT 145 CEA10 ( Fig 5A); while the effect of ZfpA deletion was less pronounced with a slight decrease in ∆zfpA 146 relative colony diameter compared to WT CEA10 at 0.25 µg/mL ( Fig 5A). 147 Antifungals can work in concert with the host to clear fungal infection, and therefore drug efficacy and the 148 mechanisms driving fungal killing can vary between in vitro and in vivo scenarios [25]. Our lab has 149 successfully used voriconazole to treat A. fumigatus infection in larval zebrafish and previously reported 150 that voriconazole completely protects larvae from death at 1 µg/mL [25]. Therefore, we selected a sub-151 effective dose of 0.1 µg/mL to screen for differences in voriconazole susceptibility during infection. We 152 injected neutrophil-deficient Rac2D57N larvae with spores of WT CEA10, ∆zfpA, or OE::zfpA, and added 153 0.1 µg/mL voriconazole to the larval water. As expected, voriconazole treatment improved survival of all 154 larvae relative to the solvent-treated controls. However, voriconazole was least effective in animals infected 155 with OE::zfpA ( Fig 5B). Loss of ZfpA did not improve efficacy of voriconazole when compared to WT 156 CEA10, similar to observations in our in vitro analyses ( Fig 5A). 157

ZfpA is required for echinocandin tolerance 158
We have previously shown that ZfpA deletion increases susceptibility to cell wall perturbations and the 159 echinocandin caspofungin (Fig 4) [11]. Here, we wanted to expand our analysis to include multiple 160 echinocandins and test the effect of ZfpA overexpression on tolerance to this class of antifungals. To assess 161 caspofungin susceptibility in the ZfpA mutants, we measured relative colony diameter after 4 days of 162 growth on solid GMM supplemented with 0.25, 0.5, 1, and 8 µg/mL caspofungin or micafungin. As 163 expected, ∆zfpA was most susceptible to caspofungin up to 1 µg/mL. ZfpA overexpression significantly 164 improved caspofungin tolerance at these same concentrations (Fig 6A-B). We selected 8 µg/mL to test 165 whether ZfpA mutants were capable of paradoxical growth, a phenomenon in which drug efficacy decreases 166 with increased drug concentrations [31]. At 8 µg/mL, colony diameter expanded for all strains, indicating 167 that ZfpA is not essential for paradoxical growth. Micafungin exhibited greater inhibition of colony growth 168 than caspofungin, with all strains having severely restricted growth at all concentrations tested 169 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint S1). Similar to the effects of caspofungin, ∆zfpA was the most susceptible while OE::zfpA was most tolerant. 170 There was no evidence of paradoxical growth at these concentrations of micafungin. 171 ZfpA-mediated changes in basal chitin content underpin differences in caspofungin tolerance 172 Caspofungin exposure stimulates a compensatory increase in chitin synthesis that is associated with 173 decreased drug susceptibility [32]. We have previously reported that ZfpA deletion decreases chitin, while 174 overexpression drastically increases chitin in the cell wall [11]. To test whether ZfpA is involved in 175 compensatory chitin synthesis in response to caspofungin, we grew WT CEA10, ∆zfpA, and OE::zfpA in 176 liquid GMM supplemented with 1 µg/mL caspofungin and visualized chitin content with calcofluor white 177 (CFW) staining and fluorescence microscopy. As seen previously [11], ∆zfpA had reduced chitin and 178 OE::zfpA had increased chitin relative to WT CEA10 ( Fig 7A). Notably, all strains increased chitin in 179 response to caspofungin, suggesting that ZfpA is not required for compensatory chitin production during 180 caspofungin exposure (Fig 7A). 181 Despite the ability of ∆zfpA to upregulate chitin during drug exposure, it still displayed increased 182 susceptibility to caspofungin compared to WT and OE::zfpA (Fig 6A-B). We thus hypothesized that 183 temporal control of chitin synthesis is important for caspofungin tolerance. To test this hypothesis, we 184 increased cell wall chitin prior to caspofungin exposure by pretreating spores with a combination of CaCl2 185 and CFW to activate the Ca 2+ -calcineurin and PKC (protein kinase C) stress response pathways responsible 186 for maintenance of cell wall integrity [32]. Spores were grown in GMM or GMM supplemented with 187 CaCl2/CFW for 8 hours before exchanging media for GMM with or without caspofungin (Fig 7B). Using a 188 resazurin-based viability reagent, we measured fungal viability after 12 hours of caspofungin exposure. 189 CaCl2 and CFW pretreatment improved viability of WT CEA10 by 16% and by 21% in ∆zfpA compared to 190 untreated controls ( Fig 7C). However, ∆zfpA viability was still lower than WT CEA10. There was no effect 191 on OE::zfpA, which maintained high tolerance to caspofungin with and without pretreatment (Fig 7C). 192 These data suggest that ZfpA-mediated changes in basal chitin levels during hyphal development are largely 193 responsible for the differences in caspofungin tolerance among ZfpA mutants. 194 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint

Loss of ZfpA enhances caspofungin susceptibility during infection 195
As ZfpA is a determinant of caspofungin tolerance in vitro, we wanted to test the importance of ZfpA for 196 caspofungin tolerance in a live host. We injected Rac2D57N larvae with WT CEA10, ∆zfpA, or OE::zfpA 197 spores and added 1 µg/mL caspofungin to the larval water. Caspofungin had only a slight protective effect 198 in larvae infected with WT CEA10 relative to the solvent-treated controls (Fig 8), in agreement with the 199 reported fungistatic nature of caspofungin. No protective effect was seen in larvae infected with OE::zfpA 200 (Fig 8), contrary to our observations during voriconazole treatment ( Fig 5B). Survival of ∆zfpA-infected 201 animals was significantly improved relative to controls (Fig 8), consistent with our in vitro analyses ( ZfpA activity may be especially relevant in hosts with some preserved neutrophil function. 216 ZfpA deletion increased, while overexpression decreased, susceptibility to neutrophils but not to reactive 217 oxygen species, suggesting that ZfpA is important for resistance against non-oxidative killing mechanisms. 218 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10. 1101/2023 Alterations in cell wall composition have been previously shown to impact virulence and neutrophil killing 219 of A. fumigatus. For example, the exopolysaccharide galactosaminogalactan (GAG) is a virulence factor 220 that specifically mediates resistance to neutrophil extracellular traps [33]. Although we have not assessed 221 GAG levels of the ZfpA mutants, we know ZfpA deletion decreases, while overexpression increases, chitin 222 deposition. Genetic depletion of A. fumigatus chitin synthases or pharmacologic inhibition of chitin 223 synthesis has been previously shown to increase susceptibility to neutrophil killing in vitro and attenuate 224 virulence in corneal infection of mice [34]. It is unclear whether chitin protects against neutrophils by 225 serving as a physical barrier to antimicrobial effectors or if it impacts phagocyte recognition of other 226 immunologically relevant cell wall polysaccharides like β-1,3-glucan. Although ZfpA deletion and 227 overexpression resulted in no detectable changes to neutrophil recruitment, increased β-1,3-glucan 228 exposure could increase neutrophil killing capacity via activated dectin-1 signaling. More comprehensive 229 studies comparing cell wall composition of the ZfpA mutants will be needed to fully appreciate how ZfpA 230 mediates hyphal-phagocyte interactions. 231 How does hyphal branching impact virulence and interactions with neutrophils? Previous in vitro analyses 232 of neutrophil-hyphae interactions suggest that branching is a double-edged sword. It serves as an evasive 233 maneuver to escape neutrophils but may also increase opportunities for neutrophils to exert their 234 microbicidal functions [14]. Septum formation is closely associated with branching and creates physical 235 barriers within hyphae to protect hyphal compartments from damage. During infection of mice, septation 236 deficiency severely limits tissue invasion and virulence, however, it is unclear whether these phenotypes 237 result from decreased hyphal strength or ability to withstand host immunity [35]. While the pleiotropic 238 effects of ZfpA manipulation make it challenging to determine the precise contributions of septation and 239 branching to virulence of these strains, the live-imaging techniques used in this study provide some insights 240 on how ZfpA protects against the host immune response. Repeated live-imaging of infected larvae revealed 241 successful colonization of host tissue by the ZfpA deletion mutant. This experiment suggests the branching 242 and septation defects of this strain do not limit tissue invasion but may contribute to the decreased fungal 243 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint burden observed later in infection. We speculate this is due to increased susceptibility to phagocytes, as we 244 saw this strain was completely unable to escape neutrophil killing in vitro. The hyperbranching ZfpA 245 overexpression strain did not escape surrounding neutrophils more frequently than wild-type A. fumigatus 246 yet survived longer, suggesting that increased branching was not advantageous in this in vitro scenario. We 247 suspect that any potential detrimental effects of excessive branch production in this strain are offset by 248 enhanced cell wall integrity and stress resistance. 249 The ZfpA-mediated changes to A. fumigatus hyphae that impact resistance to host defenses are likely also 250 responsible for our observations of altered antifungal drug susceptibility.

Fish lines and maintenance 288
Adult zebrafish and larvae were maintained as described previously [19]. Larvae were anesthetized in E3 289 water (E3) + 0.2 mg/mL Tricaine (ethyl 3-aminobenzoate, Sigma) prior to all experiments. To prevent 290 pigment formation, larvae used in live-imaging experiments were treated with E3 + 0.2 mM N-291 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint phenylthiourea (PTU, Sigma) beginning at 1 day post fertilization (dpf). All zebrafish lines used in this 292 study are listed in Table 1. 293

Aspergillus strains and growth conditions 294
Aspergillus fumigatus conidial stocks were maintained at -80°C in glycerol suspension until being streaked 295 on solid Glucose minimal media (GMM), supplemented with the appropriate amounts of uridine (0.5 296 mg/mL), uracil (0.5 mg/mL), or arginine (1 mg/mL) when necessary. Liquid GMM with 0.5% yeast extract 297 was used to extract genomic DNA. Conidia were harvested from solid GMM culture grown in darkness at 298 37°C for 3-4 days for a short-term (1 month at 4°C) working stock by scraping with an L-shaped cell 299 spreader in 0.01% Tween-water. The conidial suspension was then passed through sterile miracloth to 300 remove hyphal fragments. To prepare conidia for microinjection, 10 6 conidia were plated on solid GMM 301 and grown in darkness at 37°C for 3-4 days. Conidia were harvested as described above and the conidial 302 suspension was centrifuged at 900 x g for 10 minutes at room temperature. The resulting pellet was 303 resuspended in 1X PBS and spun again. Conidia were resuspended in 1X PBS, passed through sterile 304 miracloth, and counted using a hemacytometer. The conidial concentration was adjusted to 1.5 x 10 8 305 conidia/mL for the injection stock (1 month at 4°C). All Aspergillus strains used in this study are listed in 306 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint strains, two 1 kb fragments immediately upstream and downstream of zfpA translational start site were 316 amplified by PCR from Af293 genomic DNA. A. parasiticus pyrG::A. nidulans gpdA(p) were used as the 317 selectable marker and overexpression promoter, respectively, and were amplified from the plasmid pJMP9 318 [42]. The three fragments were fused by double joint PCR and transformed into TCDN6.7 to create strain 319 TJW216.1. Single integration of the transformation construct was confirmed by PCR and Southern blotting 320 using PciI restriction enzyme digests and both the P-32 labeled 5′ and 3′ flanks (S3 Fig). To create the 321 prototrophic wildtype control strain TDGC1.2 from TCDN6.7, 2 kb A. fumigatus pyrG was amplified to 322 complement pyrG auxotrophy. All of primers for this study is listed in Table 3. DNA extraction, restriction 323 enzyme digestion, gel electrophoresis, blotting, hybridization, and probe preparation were performed by 324 standard methods [43]. 325

In vitro chemical perturbation assays 326
To assess radial growth, GMM plates supplemented with 0.25, 0.5, 1, and 8 µg/mL caspofungin or 327 micafungin, or 0.1 or 0.25 µg/mL voriconazole were point inoculated with 10 4 spores for each strain and 328 grown at 37˚C for four days before measuring colony diameter. To assess cell wall, osmotic, and ROS stress 329 tolerance, square plates were inoculated with 10 5 , 10 4 , 10 3 , and 10 2 spores in a volume of 2 µL of each strain 330 and grown at 37˚C for 48 hours. All plates were solid GMM supplemented with 30 µg/mL CFW, 1.2 M 331 sorbitol, or 3 mM H2O2. Both radial growth and dilution plating experiments were completed in triplicate 332 or quadruplicate. 333

Spore microinjections 334
Larvae (2 dpf) were anesthetized and 3 nL of A. fumigatus conidial suspension was microinjected into the 335 hindbrain ventricle via the otic vesicle as previously described [19,39]. The conidial stock was mixed 2:1 336 with 1% Phenol Red prior to injection to visualize the inoculum in the hindbrain. After injection larvae 337 were rinsed 3X with E3 without methylene blue (E3-MB) to rinse off Tricaine and remained in E3-MB 338 throughout all experiments. Larvae were transferred to individual wells of a 96-well plate for survival 339 experiments or individual wells of 24-or 48-well plates for imaging experiments. For survival experiments, 340 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10.1101/2023.01.25.525624 doi: bioRxiv preprint larvae were checked daily for 7 days and considered dead if there was no visible heartbeat. To determine 341 the number of conidia injected for each experiment, 8 larvae/condition were collected after injection and 342 individually added to microcentrifuge tubes in 90 µL 1X PBS with 500 µg/mL kanamycin and 500 µg/mL 343 gentamycin. Larvae were homogenized for 15 sec using a mini-bead beater and then plated on solid GMM 344 plates. Colony forming units (CFUs) were counted and averaged after 2-3 days incubation at 37°C. The 345 CFU averages for each condition and experiment are reported in figure legends. 346

Zebrafish drug treatments 347
Caspofungin (Cat# 501012729, Fisher) and voriconazole (PZ0005, Sigma) were reconstituted in DMSO at 348 1 mg/mL and stocks were stored in small aliquots at -20°C to avoid repeated freeze-thaw cycles. Larvae 349 were treated with caspofungin diluted 1:1,000 (f.c. 1 µg/mL) in E3-MB and the media was exchanged daily 350 for fresh drug solution. Larvae were treated with voriconazole diluted 1:10,000 (f.c. 0.1 µg/mL) in E3-MB 351 and the media was exchanged daily for fresh drug solution. 352

Calcofluor white (CFW) staining and caspofungin treatment 353
To visualize chitin content, 2,500 spores were grown in 1 mL GMM with 0.1% DMSO or 1 µg/mL 354 caspofungin for 14 h on a glass coverslip in individual wells of a 12-well plate. Coverslips were then rinsed 355 once with PBS and inverted on a 200 µL drop of 0.1 mg/mL CFW for 10 min. Coverslips were then washed 356 with water for 10 min on a rocker and mounted on slides immediately before imaging. CFW was kept at 357 room temperature in darkness at a stock concentration of 1 mg/mL in water. 358

CaCl2/CFW treatment and PrestoBlue viability assay 359
For CaCl2 and CFW treatment, 2.5 x 10 5 spores in 100 µL liquid GMM or GMM supplemented with 0.2 M 360 CaCl2 and 0.1 mg/mL CFW were plated in a 96-well plate and incubated at 37°C for 8 h. Media was then 361 removed from the germlings and replaced with GMM + 0.1% DMSO or 1 µg/mL caspofungin and 362 incubated for 11 h at 37°C. After 11 h, media was replaced with GMM + PrestoBlue viability reagent (f.c. 363 1:10, ThermoFisher Cat# P50200) with 0.1% DMSO or 1 µg/mL caspofungin. Plates were then incubated 364 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 at 37°C for an additional hour before fluorescence was measured at 555/590 nm using a PerkinElmer 365 Victor3V plate reader. 366

Human neutrophil isolation and co-incubation with A. fumigatus 367
All blood samples were obtained from healthy donors and were drawn according to our institutional review 368 board-approved protocols per the Declaration of Helsinki. Neutrophils were isolated immediately after 369 blood collection using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltyeni Biotec #130-104-370 434) and manufacturer instructions. Neutrophils were centrifuged for 5 min at 200 x g and the pellet was 371 resuspended in 1 mL PBS for counting. Neutrophils were centrifuged again and resuspended to a final 372 concentration of 4 x 10 5 cells/mL in RPMI + 2% fetal bovine serum and used immediately. For live-imaging 373 of neutrophil-fungal interactions, 2 x 10 3 spores/well were grown until the germling stage (8 h at 37°C) in 374 500 µL liquid GMM in a 24-well plate. GMM was then removed and replaced with 500 µL of the neutrophil 375 suspension (200,000 neutrophils, neutrophil:spore 100:1). The 24-well plate was then immediately brought 376 to the microscope for imaging. 377

Image acquisition 378
Transgenic larvae were pre-screened for fluorescence using a zoomscope (EMS3/SyCoP3; Zeiss; Plan-379 NeoFluor Z objective). For multi-day imaging experiments, larvae were anesthetized and mounted in a Z-380 wedgi device [39,44] where they were oriented such that the hindbrain was fully visible. Z-series images 381 (5 µm slices) of the hindbrain were acquired on a spinning disk confocal microscope (CSU-X; Yokogawa) 382 with a confocal scanhead on a Zeiss Observer Z.1 inverted microscope, Plan-Apochromat NA 0.8/20x 383 objective, and a Photometrics Evolve EMCCD camera. Between imaging sessions larvae were kept in E3-384 MB with PTU in individual wells of 24-or 48-well plates. Neutrophil-fungal interactions were imaged 385 using an inverted epifluorescence microscope (Nikon Eclipse TE3000) with a Nikon Plan Fluor 20x/0.50 386 objective, motorized stage (Ludl Electronic Products) and Prime BSI Express camera (Teledyne 387 Photometrics). Environmental controls were set to 37°C with 5% CO2. Images were acquired every 3 min 388 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 for 12 h. Imaging of A. fumigatus stained with CFW was performed using an upright Zeiss Imager.Z2 LSM 389 800 laser scanning confocal microscope with Airyscan detection and a Plan-Apochromat 20x /0.8 objective. 390 A single z plane image was acquired for each hypha. Images were captured using identical laser and 391 exposure settings for each condition.

Statistical analyses 404
The number of independent replicates (N) and larvae or plates (n) used for each experiment are reported in 405 the figure legends. Survival analyses of larvae and fungal germlings were performed with RStudio using 406 Cox proportional hazard regression analysis with experimental condition included as a group variable, as 407 previously described [19]. Pair-wise P values and hazard ratios are included in the main figure or figure  408 legend for all survival experiments. Analysis of germination rate and percent of germlings to escape 409 neutrophils were performed with Student's t-tests (GraphPad Prism version 9). 2D area of fungal growth, 410 neutrophils, and macrophages represent least-squared adjusted means±standard error of the mean 411 (LSmeans±s.e.m.) and were compared using ANOVA with Tukey's multiple comparisons (RStudio). 412 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10. 1101/2023 Relative colony diameters in Figs 5 and 6 were compared using ANOVA with Tukey's multiple 413 comparisons (GraphPad Prism version 9). Comparison of fungal viability in Fig 7C was  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Institute of Allergy and Infectious Diseases (NIAID) of the NIH to N.P.K. T.J.S. was supported by the 430 National Institute on Aging of the National Institutes of Health under Award Number T32AG000213. The 431 content is solely the responsibility of the authors and does not necessarily represent the official views of 432 the NIH. The funders had no role in study design, data collection and analysis, decision to publish, or 433 preparation of the manuscript. 434 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Figure captions
The copyright holder for this preprint this version posted January 26, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 26, 2023. ; https://doi.org/10. 1101/2023 Spot-dilution assays to test susceptibility of ZfpA mutants to the cell wall stressor calcofluor white (CFW), 581 osmotic stressor sorbitol, or the oxidative stressor H2O2. Spores were point-inoculated on solid glucose 582 minimal medium (GMM) ± stressors at concentrations of 10 5 -10 2 and incubated for 48 hours at 37℃. 583 Images are representative of growth from 3 plates per condition. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Example of primary human neutrophils swarming around A. fumigatus germling. Germling is indicated by 658 black arrow. The first neutrophil contact is indicated by a blue asterisk. Note the morphology change of 659 surrounding neutrophils after this first cell makes contact and the subsequent rapid accumulation of 660 neutrophils around the germling. Scale bar = 20 µm. 2 frames/s. 661

Fig 1 663
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Fig 2 664
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