bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 available under aCC-BY-NC-ND 4.0 International license.

1 Host-pathogen transcriptomics of macrophages, Mucorales and their endosymbionts: a 2 polymicrobial pas de trois 3 4 Authors 5 Poppy Sephton-Clark1, José F Muñoz2, Herbert Itabangi1, Kerstin Voelz1, Christina A Cuomo2, 6 Elizabeth R Ballou1* 7 8 1Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, 9 Edgbaston, Birmingham, B15 2TT, UK

10 2Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, 11 Cambridge, Massachusetts, USA

12 * To whom correspondence should be addressed 13 ERB: [email protected] 14 15 Abstract 16 17 Mucorales spores, the causative agents of mucormycosis, interact with the innate immune 18 system to cause acute, chronic, or resolving infection. Understanding the factors that 19 influence disease initiation and progression is key to understanding mucormycosis and 20 developing new treatments. Complicating this, mucormycosis can be caused by a number of 21 species that span the Mucorales genus and may be host to bacterial endosymbionts. This 22 study sets out to examine the differences between two species in the Mucorales order by 23 characterising their differential interactions with the innate immune system, and their 24 interactions with environmental bacterial endosymbionts. Through a holistic approach, this 25 study examines the transcriptional responses of Rhizopus delemar and Rhizopus 26 microsporus, two of the most commonly diagnosed species, to innate immune cells. This 27 study also examines the immune cell response and assesses the variation in these 28 responses, given the presence or absence of bacterial endosymbionts within the fungi. We 29 see that the fungal response is driven by interaction with innate immune cells. The effect of 30 the bacterial endosymbiont on the fungus is species specific, with a minimal in the absence 31 of stress, but strongly influencing fungal transcripts during interaction with innate immune 32 cells. In contrast, we observe that the macrophage response varies depending on the 33 infecting fungal species, but also depending on endosymbiont status. The most successful 34 macrophages elicit a pro-inflammatory response, and we see that through germination 35 inhibition macrophage survival is enhanced. This work reveals species-specific host 36 responses to related Mucorales spores and shows that bacterial endosymbionts impact the 37 innate immune cell response. 38 39 Introduction 40 41 Mucormycosis is a devastating, environmentally acquired fungal infection caused by a 42 variety of Mucorales species. Understanding the interaction between Mucorales spores and 43 innate immune cells is key to understanding mucormycosis. Studies frequently focus upon 44 the interaction of a single species of the Mucorales order with innate immune cells (Warris bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

45 et al. 2005; Chamilos et al. 2008; Schmidt et al. 2013; Kraibooj et al. 2014; Inglesfield et al. 46 2018). However, between Mucorales species, and across infecting isolates, numerous 47 phenotypic and genomic differences can be observed (Hoffmann et al. 2013; Mendoza et al. 48 2014; Schwartze et al. 2014). Whether each species within this family employs the same 49 mechanism to cause infection remains unclear, however the range of virulence profiles 50 displayed across the family indicates there may be diversified mechanisms at work (Petraitis 51 et al. 2013). For example, it has been shown that pathogenicity of these species is linked to 52 the variable copy number of the gene encoding CotH, a family of cell surface proteins 53 important for the spores’ interactions with and adherence to endothelial cells. Iron- 54 scavenging and melanisation pathways have also been shown to play a role in pathogenicity 55 (Chibucos et al. 2016, Andrianaki et al. 2018). In addition to fungal genome plasticity and 56 transcriptional responses to host stimuli, fungal-host interactions can be further modulated 57 by bacterial endosymbionts (Itabangi et al., 2019;(Partida-Martinez & Hertweck 2005). The 58 majority of Mucorales species have been shown to harbour bacterial endosymbionts whose 59 species can vary between Mucorales isolates (Ibrahim et al. 2008; Kobayashi & Crouch 2009; 60 Mondo et al. 2017; Itabangi et al. 2019). We have recently shown that a bacterial 61 endosymbiont influences the outcome of Rhizopus microsporus infections in both zebrafish 62 and murine models through modulation of both fungal and phagocyte phenotypes (Itabangi 63 et al. 2019). However, the impact of endosymbiont on fungal and host transcriptional 64 response remains unclear. 65 66 While disease can be caused by several species in the Mucorales order, Rhizopus delemar 67 and Rhizopus microsporus are responsible for the majority of infections (Liu et al. 2018). Risk 68 factors for developing the disease include, but are not limited to: innate immune cell 69 deficiencies, uncontrolled diabetes, immune suppression, skin barrier breaches and iron 70 overload (Baldin & Ibrahim 2017). With limited antifungal treatments effective against these 71 invasive infections, there is often a need for surgical debridement or amputation (Spellberg 72 et al. 2016). In disseminated cases, mucormycosis may present with a mortality rate of over 73 90% (Mendoza et al. 2014). Reports of chronic mucormycosis have also emerged, affecting 74 both immunocompromised and immunocompetent patients (Alan et al. 2019). The innate 75 immune response to Mucorales spores is key to infection control: macrophages are required 76 to induce a differential immune response on contact with Rhizopus spp. and may inhibit 77 germination or kill hyphal forms upon contact in healthy individuals (Ghuman & Voelz 2017; 78 Andrianaki et al. 2018). Single and multi-species studies have shown that phagosome 79 maturation is arrested by melanin within the cell walls of Aspergillus spp. and Rhizopus spp., 80 however iron limitation allows macrophages to more effectively kill Rhizopus spp.(Liu et al. 81 2015; Ibrahim et al. 2010; Andrianaki et al. 2018). Several works comparing Aspergillus spp. 82 to Rhizopus spp. have revealed similar immunostimulatory capacities, but differences in 83 their responses to host stress (Warris et al. 2005; Chamilos et al. 2008; Schmidt et al. 2013; 84 Kraibooj et al. 2014). Exploring and understanding fungal responses to the host is essential 85 to improving our understanding of mucormycosis, yet it remains unclear how Mucorales 86 species respond to, and interact with, the innate immune system, and to what extent this 87 varies by species. 88 89 Our work explores the interplay between the innate immune system, R. delemar, and R. 90 microsporus using isolates of both species found to harbour bacterial endosymbionts 91 (Itabangi et al. 2019). We investigate the differences between these two fungal species, how bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

92 they respond transcriptionally to innate immune cells, and how their respective bacterial 93 endosymbionts affect this interaction. We also investigate the transcriptional response of 94 innate immune cells to these infectious spores and determine how this interaction is 95 influenced by the presence of an endosymbiont. We reveal a large difference between the 96 fungal transcriptional profiles of R. delemar and R. microsporus during in vitro monoculture. 97 There is a small conserved response to exposure to innate immune cells, including key 98 changes in cell wall genes, consistent with germination. Conversely, we see that the host 99 innate immune response differs significantly between fungal species and is also influenced 100 by the presence or lack of an endosymbiont. The innate immune response to R. delemar and 101 R. microsporus mirrors the relative aggressiveness of infection between these two species. 102 We also observe that through the activation of innate immune cells, or upon inhibition of 103 chitin synthase, we can improve the ability of innate immune cells to control the fungal 104 spores. Our work represents a broad analysis of the transcriptional interplay between innate 105 immune responders and infectious Mucorales spores, revealing species-species differences 106 which question the current model of ‘one species represents all’, when it comes to 107 mucormycosis. 108 109 Results 110 111 Experimental Design 112 113 We set out to investigate paired transcriptional responses of host and fungal cells, whilst 114 also exploring the influence of the endosymbiont on this interaction (Figure 1a). Fungal 115 spores from Rhizopus delemar and Rhizopus microsporus were either cured via ciprofloxacin 116 treatment to remove the bacterial endosymbiont (cured) or maintained in media permissive 117 to bacterial endosymbiosis (wt). Cured spores were passaged twice in the absence of 118 ciprofloxacin to limit the impact of the drug on transcriptional responses. The cured and wt 119 spores of both R. delemar and R. microsporus were allowed to swell in sabouraud broth 120 until 95% of the population had reached mid isotropic phase. Due to the differences in 121 germination rates between the species (Figure 1b), this occurred at 2 hours for R. delemar 122 and 4 hours for R. microsporus. Swollen spores were then used to infect the J774.1 murine 123 macrophage-like cell line. Fungal spores were co-cultured with macrophages for one hour, 124 after which unengulfed spores were removed, and phagocytosed spores were incubated 125 within the macrophages for a further two hours. The cells from the resulting infection were 126 processed to explore their transcriptional response to this infection scenario (Figure 1a). 127 Macrophages that had phagocytosed fungal spores were isolated and sequenced via the 128 10X Genomics Chromium Single Cell Sequencing platform. Macrophages left unexposed to 129 the fungi were used as a negative control. RNA was also isolated from fungal spores (cured 130 and wt) which had been engulfed by macrophages, and this was sequenced with a bulk 131 RNA-Seq approach. Unexposed fungi (cured and wt) were incubated in macrophage media 132 for a matched time and used as a negative control. The data shows the fungal response to 133 phagocytosis by macrophages, as well as the fungal response to the presence of its 134 endosymbiont. The macrophage response to the two species is also revealed, a response 135 which appears to differ when the endosymbiont is present for both fungal species. 136 bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

137 Comparative genomics predicts alternative transcriptional responses 138 139 In order to better understand differential disease progression, we chose to compare R. 140 delemar and R. microsporus, as they cause a large proportion of mucormycosis infections 141 but appear morphologically dissimilar. R. delemar germinates more quickly than R. 142 microsporus, with 50% of spores germinating by 3 hours, and a spore body size which 143 reaches 12.8 μm over the course of germination (Figure 1b, c). R. microsporus germinates at 144 a significantly slower rate, taking 6 hours for 50% of spores to germinate, with a final spore 145 body size of 13.9 μm (Figure 1b, c). To better understand their relationship to one another, 146 we compared the gene content to explore the similarity and differences between the two 147 species. Previous work has established that the R. delemar genome (45.3 Mb, 17,513 genes) 148 is larger, and contains an increased number of genes, compared to R. microsporus genome 149 (26 Mb, 10,959 genes) (Ma et al. 2009; Mondo et al. 2017). Our results show that, compared 150 to R. microsporus, the genome of R. delemar is enriched for genes with protein domains 151 (PFAM) associated with ion binding, carbohydrate derivative binding, nucleic acid binding, 152 cytoskeletal protein binding, poly(A) binding, NAD+ ADP-ribosyltransferase activity, protein 153 kinase C activity, translation initiation factor binding and inorganic phosphate 154 transmembrane transporter activity (Supplemental Figure S1, Figure 1d). R. microsporus is 155 enriched for genes with protein domains corresponding to nucleoside phosphate binding, 156 early endosome activity and DNA repair complex activity (Supplemental Figure S1, Figure 157 1d). The clear differences illustrated by genome size and gene content indicate the 158 likelihood of alternative transcriptional responses. 159 160 Alternative transcriptional profiles are presented by R. delemar and R. microsporus in 161 response to innate immune cells 162 163 First we examined overall trends in fungal responses to phagocytosis, obtained through our 164 bulk RNA-Seq approach. We analysed the signal obtained from R. delemar and R. 165 microsporus samples with principle component analysis (PCA) (Figure 2). We observed large 166 differences between the transcriptomes of both fungal species, when exposed or 167 unexposed to macrophages, while the presence or absence of their respective 168 endosymbionts had a weak but differential effect on PCA. The presence or absence of the 169 endosymbiont appears to have very little bearing on the transcriptional patterns displayed 170 by R. delemar, as samples fell into two distinct clusters, most strongly influenced by 171 macrophage status (Figure 2a). R. microsporus exhibits a similar trend upon exposure to 172 macrophages, however the presence of the endosymbiont also influenced clustering (Figure 173 2b). We investigate the phenotypic consequences of this interaction in the companion 174 paper by Itabangi et al., showing that the presence of the endosymbiont Ralstonia pickettii 175 impacts fungal cell wall organization, resistance to host-relevant stress, spore germination 176 efficiency, and pathogenesis (Itabangi et al., 2019). Therefore, we focus here on the 177 transcriptional analysis of the host-pathogen-endosymbiont interaction across the two 178 species. 179 180 There are 2,493 genes that are significantly differentially expressed (Log fold change > 2; 181 false discovery rate < 0.05) in R. delemar across all conditions (Figure 3a), while R. 182 microsporus only exhibits 40 genes significantly differentially expressed across all conditions 183 (Log fold change > 2; false discovery rate < 0.05) (Figure 3b). The theme of a muted bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

184 transcriptional response from R. microsporus is also seen within pairwise comparisons of 185 conditions. Pairwise comparisons of differential expression across each experimental 186 condition show similar trends in responses between R. delemar and R. microsporus, 187 however R. microsporus responds with a reduced gene set (Figure 4). Pairwise comparisons 188 showed the biggest shift in transcriptional response when comparing phagocytosed fungal 189 spores to those unexposed to macrophages, regardless of endosymbiont status. When 190 phagocytosed (Supplemental Figure S2), R. microsporus upregulates genes enriched in GO 191 categories corresponding to thiamine metabolism, sulfur metabolism, glycerol metabolism, 192 alcohol dehydrogenase activity and transmembrane transporter activity (hypergeometric 193 test, corrected P value < 0.05). This is consistent with the fungal response seen to 194 macrophage stress (Parente-Rocha et al. 2015), and the micronutrient scavenging response 195 to nutritional immunity (Ballou and Wilson 2016; Shen et al., 2018; Andrianaki et al., 2018). 196 Phagocytosed R. microsporus downregulated genes enriched in GO categories 197 corresponding to rRNA processing, ribosome biogenesis and ribosome localization 198 (hypergeometric test, corrected P value < 0.05) (Supplemental Figure S2), consistent with 199 growth arrest within the phagolysosome (Inglesfield et al. 2018; Andrianaki et al. 2018). 200 201 Comparisons of R. delemar conditions reveal that, upon phagocytosis, spores upregulate 202 genes enriched in KEGG classifications corresponding to MAPK signalling, phenylalanine 203 metabolism, tyrosine metabolism, glutathione metabolism and fatty acid synthesis 204 (hypergeometric test, corrected P value < 0.05). Upregulation of these processes is 205 consistent with melanin biosynthesis (Eisenman et al. 2011; Andrianaki et al. 2018) and 206 intra-phagosomal survival (Yadav et al., 2011; Lorenz and Fink, 2005). Unexposed R. delemar 207 spores upregulate genes enriched in KEGG classifications corresponding to ketone body 208 synthesis, protein processing via the endoplasmic reticulum, amino sugar and nucleotide 209 sugar metabolism (hypergeometric test, corrected P value < 0.05). This is consistent with 210 metabolic activation and cell wall biogenesis (Figure 5). 211 212 R. delemar and R. microsporus harbour distinct bacterial endosymbionts (Itabangi et al., 213 2019). When comparing transcriptional profiles of wt and cured spores incubated in serum- 214 free DMEM (sfDMEM) for 3 hours (time matched to phagocytosis assay), we observed very 215 few transcriptional changes in either species in response to curing (Supplemental Table 1). 216 In R. delemar, a single gene, predicted to be a putative protein phosphatase, was repressed. 217 In R. microsporus, three genes were induced: an autophagy-related protein, a C2H2 zinc 218 finger transcription factor, and ribosomal protein L2. 219 220 Despite these small changes, loss of the endosymbiont significantly impacted the 221 transcriptional responses of both fungal species to macrophages (Figure 4). Specifically, we 222 observed an overall increase in the number of fungal genes differentially regulated upon 223 phagocytosis for both species. Exposure of wt R. microsporus to macrophages induced the 224 expression of one gene with no known function and repressed 6 genes. The repressed genes 225 include an autophagy-related protein and a zinc finger transcription factor, as well as 4 un- 226 annotated genes (Figure 4, Supplemental Table S1). In contrast, when cured R. microsporus 227 spores were exposed to macrophages, 277 genes were significantly induced and 82 228 repressed, compared to unexposed cured spores (Figure 4). Induced genes were enriched 229 (hypergeometric test, corrected P value < 0.05) for the following GO categories: organelle 230 organisation, pre-ribosome and ribosome activity, ATPase activity, hydrolase activity, bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

231 pyrophosphatase activity, helicase activity, nucleic acid binding, RNA metabolism, nitrogen 232 metabolism, chromatin silencing. Repressed genes were enriched (hypergeometric test, 233 corrected P value < 0.05) for the following GO categories: oxidoreductase activity, hydrogen 234 sulphide metabolism, glycolysis, sulphur metabolism, hexose catabolism, siderophore activity, 235 iron assimilation, nitrogen metabolism, carboxylic acid metabolism. This suggests an overall 236 failure to properly respond to host stresses such as iron starvation in the absence of the 237 bacterial endosymbiont. 238 239 A similar impact of the endosymbiont was observed for R. delemar. While the overall fungal 240 response to phagocytosis is characterized by a robust transcriptional response, induced 241 genes in wt samples (1137 genes, Figure 4) were enriched for KEGG classifications 242 corresponding to: alanine metabolism, PPAR signalling, aromatic compound biosynthesis 243 and degradation, lysine metabolism, lipid metabolism, MAPK signalling, sugar metabolism, 244 tyrosine metabolism, secondary metabolite biosynthesis (Figure 5). Repressed genes in wt 245 samples (472 genes, Figure 4) were enriched for KEGG classifications corresponding to: 246 carbohydrate metabolism, secondary metabolite biosynthesis, ketone body processing, 247 protein processes, MAPK signalling (Figure 5). In contrast, induced genes in cured samples 248 (1285 genes, Figure 4) were enriched for KEGG classifications corresponding to: Fatty acid 249 metabolism, DNA replication, amino acid metabolism, glycan metabolism, pyruvate 250 metabolism, secondary metabolite processing (Figure 5). Repressed genes in cured samples 251 (878 genes, Figure 4) were enriched for KEGG classifications corresponding to: sugar 252 metabolism, amino acid metabolism, lipid metabolism, MAPK signalling, NOD-like receptor 253 signalling. Again, this suggests that the endosymbiont has an overall suppressive impact on 254 fungal transcription in response to macrophage challenge. 255 256 Next, we directly compared the transcription patterns of genes shared across the two fungal 257 species. When comparing the transcriptional responses of orthologous genes shared by R. 258 delemar and R. microsporus, we saw only a small proportion behave similarly (213 genes). 259 When phagocytosed, wt spores from both species upregulate orthologues genes involved in 260 fatty acid catabolism, transcription, regulation via polymerase II, and organelle organization. 261 Phagocytosed cured spores from both species upregulate orthologues genes involved in 262 RNA processing, chromosome organization and condensed chromosome pathways. When 263 unexposed, we see wt spores upregulate orthologous genes involved in translocation, 264 protein binding, siderophore activity, cobalmin processing, and post-translational protein 265 targeting. Cured unexposed spores upregulate orthologous genes with roles in siderophore 266 activity and transferase activity. Overall, R. delemar and R. microsporus both respond 267 transcriptionally to the presence of macrophages, however the size and composition of this 268 response differs between species. 269 270 Finally, we examined the regulation of genes predicted to be involved in ferrous iron 271 transport, as previous work has linked iron scavenging to survival within the phagolysosome 272 (Andrianaki et al., 2018). There are 12 genes in the R. delemar genome with predicted 273 ferrous iron roles. While 8 showed no significant change over the tested conditions, 3 274 (R0G3_006623, R0G3_007727, R0G3_011864) appeared highly expressed in wt and cured 275 phagocytosed spores, compared to unexposed spores. The last gene, ROG3_009943, is 276 highly expressed in wt spores unexposed to macrophages. Together, this suggests there bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

277 may be condition dependent specialization in the expression of ferrous iron transport in R. 278 delemar. 279 280 Innate immune cell transcription varies with fungal species and endosymbiont 281 presence 282 283 To investigate the innate immune response when challenged with R. delemar and R. 284 microsporus, we carried out single cell RNA-Seq of J774.A1 murine macrophages, unexposed 285 and exposed for 3 hours to the four types of pre-swollen spores (Figure 1a). Transcription of 286 both challenged and unchallenged macrophages displayed underlying population 287 heterogeneity (Supplemental Figure S3). To identify the transcriptional patterns of genes 288 responding to the spores, we focused on the expression of a subset of genes previously 289 identified as immune response genes (Muñoz et al. 2018). Principle component analysis of 290 the aggregated transcriptional data shows there is a clear difference in transcription 291 between macrophages that have and have not been exposed to the fungi (Figure 6). Across 292 all exposed conditions, relative to unexposed macrophages, there was a profile consistent 293 with cytokine activation, response to stimulus, and activation of the NF-Kb pathway. This 294 was accompanied by repression of CCL5, which is involved in T-cell recruitment (Figure 7). 295 However, different macrophage profiles can be seen in response to the two fungal species, 296 and these are further influenced by the presence of the endosymbiont. While the response 297 to wt R. delemar shows the most deviation from the macrophage-only control, exposure to 298 cured R. delemar also elicited a strong and distinct macrophage response (Figures 6,7). 299 Exposure to wt R. delemar elicits increased expression of general markers of activation, 300 including GTPase activity and MHC class II protein binding (LAG3 repressor of T-cell 301 activation, H2-M2, IFN-gamma induced IIGP1, MX1, KCTD14, PNP2), growth factor binding, 302 IL1 receptor agonist activity and endocytosis (SERPINE1, ENG, FGFBP3, GM8898, GCNT2, 303 IL1F6). Specifically, we observe modest increases in the expression of IFN- γ responsive 304 CXCL10 (3.1 fold) and IRG1/IRG11 (5.9 fold), pro-inflammatory SAA3 (3.5 fold), and ENPP4 305 (2.8 fold), but also induction of the M2 polarizing PSTPIP2/21 (6.7 fold), the IL-4 responsive 306 signaling modulator CISH (5.4 fold), and the vascular damage responsive F3/F31 (5.9 fold) 307 (Martinez et al. 2013). These latter genes are not as strongly induced during exposure to R. 308 microsporus, which may reflect the aggressive nature of infection by R. delemar relative to 309 R. microsporus. In contrast, infection with cured R. delemar showed a decrease in the 310 induction of these M2-polarisation markers (PSTPIP2, 3.5 fold relative to uninduced). The 311 transcriptional profile is instead shifted to include increased transcription of genes involved 312 in G protein signaling and phosphoinositide binding (PDE7B, CCL1, SCARF1, RGS16, 313 PLEKHA4) (Figure 7). 314 315 A similar change in macrophage polarization was observed during exposure to wt and cured 316 R. microsporus. The phenotypic analysis of this is discussed more fully in Itabangi et al. 2019. 317 For both wt and cured R. microsporus, expression of IFN- γ responsive CXCL10, SAA3, and 318 ENPP4 was comparable to unexposed macrophages. Compared to R. delemar, exposure to 319 wt R. microsporus induced a more limited expression of genes with roles in cytokine 320 activation, ERK1 and ERK2 regulation, and regulation of NF-kB cascade (Figure 7). There was 321 also a weaker induction of the vascular damage responsive F3/F31 genes, and a relatively 322 stronger upregulation of the PLA2G16 phospholipase, TRIM30D, SLC1A2 and the M2 323 polarizing IL-6. However, other key polarizing genes, particularly PSTPIP2/21, SAA3, and bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

324 ENPP4 were only weakly induced (Supplemental Table S2). Overall, this profile suggests a 325 weak M1-like activation consistent with poor phagocytosis and reduced overall antifungal 326 activity that we observe in macrophages interacting with endosymbiont-harbouring spores 327 (Itabangi et al., 2019). 328 329 Finally, cured R. microsporus induced a strong pro-inflammatory response, which included 330 upregulation of CXCL3, the neutrophil chemoattractant, consistent with our observations of 331 differences in phagocyte recruitment in zebrafish upon infection with wt vs. cured spores 332 (Itabangi et al., 2019). Markers of NF-kB activation were also strongly induced in this 333 population. Cured R. microsporus also strongly induced the expression of TNFRSF8 (CD30), a 334 marker of lymphocyte activation occasionally associated with subcutaneous fungal 335 infections. Overall, this is suggestive of a shift to a more pro-inflammatory profile. In our 336 companion paper, we observed that cured R. microsporus is more sensitive to phagocyte- 337 mediated killing and phagocyte recruitment compared to wt (Itabangi et al., 2019). We 338 therefore went on to test whether a more successful response to the spores could be 339 mounted via the induction of a pro-inflammatory response. 340 341 Chitin synthase inhibition and pro-inflammatory priming regulate infection outcome 342 343 R. delemar exhibits rapid germination followed by hyphal extension (Sephton-Clark et al., 344 2018). The transcriptional profile we observe here in macrophages exposed to R. delemar is 345 consistent with a strong damage response, likely prompted by the germination of R. 346 delemar spores. We therefore hypothesised that macrophages may be better able to 347 control the infection if the fungi were slowed in their developmental progress. The necessity 348 of genes involved in chitin synthesis and regulation appeared important for both R. delemar 349 and R. microsporus in response to phagocytosis (Supplemental Figure S4). In previous work 350 we also highlighted the importance of chitin synthase for germination (Sephton-Clark et al. 351 2018). When fungal spores were pre-treated with the chitin synthesis inhibitor Nikkomycin Z 352 (24 μg/ml), spores failed to germinate, displayed less chitin/chitosan in their outer cell wall 353 (Supplemental Figure S5) and macrophage survival was increased at 7.5 hours post infection 354 (Figure 8). At Nikkomycin Z concentrations lower than those used to pre-treat spores for 355 phagocytosis, we see the spores are able to swell, however development appears halted 356 after swelling (Supplemental Figure S5). As the macrophages are better able to control 357 these spores, this suggests that spores undergoing the initial stages of germination may 358 offer less of a challenge for the macrophages. 359 360 Our transcriptional data show a strong M2 alternative activation signal during R. delemar- 361 macrophage interaction, but a weaker M2 polarisation during R. microsporus-macrophage 362 interaction that was further shifted towards NF-kB-mediated M1 upon endosymbiont cure. 363 We therefore hypothesized that shifting the macrophage polarization towards M1 classical 364 activation might have a protective effect upon Mucorales infection. Consistent with this, the 365 pre-treatment of macrophages with NF-kB activating lipopolysaccharide (LPS) significantly 366 improved the ability of macrophages to control R. microsporus. At 7.5 hours post infection, 367 59.7% of macrophages survived when pre-treated with LPS, compared to 24.6% without 368 (Figure 8). 369 bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

370 Discussion 371 372 In this work we show the fungal response to innate immune cells differs by species in the 373 Rhizopodaceae family. Although R. delemar and R. microsporus share a small conserved 374 response to exposure to macrophages, the majority of their response differs. We show that 375 the ability to germinate prior to phagocyte control appears to be key to virulence, as 376 blocking spore germination with the chitin synthase inhibitor Nikkomycin Z improves 377 macrophage survival. A range of germination and virulence phenotypes can be seen 378 throughout the Mucorales, and this highlights a need for further investigation into these 379 differences, to better understand the infections they cause. 380 381 We have also shown that the fungal transcriptional response appears largely unperturbed 382 by the presence, or lack, of an endosymbiont, in the absence of stress. However, the 383 presence of an endosymbiont greatly effects the response of and to the host. It has been 384 shown that endosymbionts influence asexual development and sporulation through the 385 regulation of Ras2 (Mondo et al. 2017). While we observed limited changes in fungal 386 transcription when comparing wt and cured spores, we did observe changes in the 387 expression of fungal genes upon exposure to macrophages. In our companion paper, 388 Itabangi et al show that the presence of an endosymbiont is important for virulence of R. 389 microsporus. The endosymbiont enhances virulence through the secretion of an anti- 390 phagocytic factor. The endosymbiont also impacts organisation of the fungal plasma 391 membrane, as well as environmental stress resistance and resistance to macrophage- 392 mediated killing (Itabangi et al., 2019). We show here that this is mirrored by the 393 transcriptional response of the WT and cured spores of both Rhizopus species to 394 macrophages, and in the macrophage response to infection. 395 396 As anticipated, activation of pro-inflammatory pathways increased macrophage survival in 397 response to the spores. This consolidates several studies which demonstrate improved 398 innate immune cell response to fungal pathogens when primed (Rogers et al. 2013; Municio 399 et al. 2013; Blasi et al. 1995). It has been shown that the early immune response to a Mucor 400 circinelloides infection is dependent on the formation of a pro-inflammatory TNF-α- 401 expressing granuloma-like structure that controls but does not kill spores in the zebrafish 402 model (Inglesfield et al. 2018). Ungerminated Rhizopus spores are highly resistant to 403 ROS/RNS which may mediate survival within granulomata. Consistent with this, the 404 induction of a strong proinflammatory response by cured R. microsporus, hypersensitive to 405 ROS/RNS stress, allows macrophages to better control the spores. 406 407 Interestingly, we see the chitin synthase inhibitor, Nikkomycin Z, is able to inhibit 408 germination in both R. delemar and R. microsporus. This demonstrates the requirement of 409 chitin synthase for spore development in these species. Treated spores swell, but do not 410 polarise. The ability to germinate also has consequences for virulence. The ability of 411 macrophages to control swollen spores, but not subsequently polarised ones, highlights that 412 the developmental stage of the spore is key to innate immune success and control. Whilst 413 many fungal pathogens are virulent in an ungerminated form, germination appears a key 414 virulence factor for these filamentous fungi. This is confirmed by work which shows that the 415 developmental stage of both R. microsporus and Rhizopus oryzae spores impacts 416 pathogenesis in zebrafish and murine models (Itabangi et al., 2019; Andrianaki et al., 2018). bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

417 Specifically, infection with pre-swollen spores leads to evasion of macrophage-mediated 418 immunity and increased pathogenesis (Itabangi et al., 2019; Andrianaki et al., 2018). In both 419 models, spore clearance is dependent on phagocyte recruitment, and we show that 420 phagocyte recruitment (in Itabangi et al.) and activation (Figure 7) is influenced by 421 endosymbiont status (Itabangi et al., 2019; Andrianaki et al., 2018). In addition, we 422 extended this analysis by revealing profound differences in the host response to two closely 423 related Rhizopus species. In particular, we observe a M2/damage-associated response 424 during infection with wt R. delemar spores that is shifted towards an M1 protective 425 response upon infection with cured R. microsporus spores. We reinforce this finding through 426 experimental modulation of macrophage polarization, showing that exposure to strongly 427 M1-polarizing LPS is sufficient to reduce macrophage killing by wt R. delemar spores. 428 Therefore, our data provide a framework for beginning to understand differences in the 429 relative virulence of pathogenic Mucorales species and underpin our finding of cross- 430 kingdom fungal-bacterial symbiosis influencing mammalian disease. 431 432 Methods 433 434 Fungal Culture 435 R. delemar and R. microsporus were cultured with Sabouraud dextrose agar (SDA) or broth 436 (10 g/liter mycological peptone, 20 g/liter dextrose), sourced from Sigma-Aldrich, at room 437 temperature. Spores were harvested, 10 days after plating, with phosphate-buffered saline 438 (PBS), centrifuged for 3 min at 3,000 rpm, and washed. Appropriate concentrations of 439 spores were used for further experiments as indicated. To cure spores of their respective 440 bacterial endosymbionts, spores were cultured with ciprofloxacin, as described in Itabangi 441 et al. 2019. Once cured, spores were subcultured at least twice in ciprofloxacin-free media 442 before use.

443 Macrophage Culture 444 Macrophages from the J774.A1 cell line were cultured in Dulbecco's Modified Eagle 445 Medium, (complemented with 10% foetal bovine serum, 1% penicillin, 1% streptomycin and

446 1% L-glutamine). Macrophages were grown at 37°C, in 5% CO2. 447 448 Phagocytosis Assay 449 Macrophages were incubated for one hour in serum-free DMEM prior to infection. Spores 450 were pre-swollen in SAB (2 hr for R. delemar, 4 hr for R. microsporus). Washed spores were 451 incubated at 5:1 MOI with 1 x 105 macrophages as described in Itabangi et al, to ensure that 452 >95% of macrophages contained one spore or more. After a 1 hour of incubation, excess 453 spores were washed off the surface and the macrophages were incubated for a further 2 454 hours, before processing for RNA-Seq experiments. For live cell imaging experiments, 455 images were taken starting immediately after the excess spores were removed. 456 457 Live Cell Imaging 458 Time course images were taken to determine how LPS pre-treatment (100ng/ml) (Myers et 459 al. 2010) of macrophages, and Nikkomycin Z pre-treatment (120ug/ml over the course of 460 swelling in SAB) of spores effected phagocytic outcome. Images were taken at 20x on a Zeiss 461 Axio Observer, with images taken every 5 minutes. Bright-field and fluorescent images were bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

462 then analysed using ImageJ V1.

463 Comparative Genomics and Enrichment Analysis 464 Fishers exact test was used to detect enrichment of Pfam terms between R. delemar and R. 465 microsporus, terms with a corrected P value of < 0.01 were considered significant. 466 Orthologue genes of R. delemar and R. microsporus were identified using blast+. R (version 467 3.3.3) was used to carry out hypergeometric testing of KEGG and GO terms to determine 468 enrichment. 469 470 Bulk RNA-Seq 471 RNA was extracted from spores which had either been incubated with the macrophages, or 472 incubated in DMEM for the equivalent time period. To remove the macrophages, triton at 473 1% was used to lyse the macrophages, the resulting solution was then centrifuged for 3 min 474 at 3,000 rpm, and washed, leaving only spores. The DMEM control also received the same 475 treatment. To extract total RNA, the washed samples were immediately immersed in TRIzol 476 and lysed via bead beating at 6,500rpm for 60s. Samples were then either immediately 477 frozen at -20°C and stored for RNA extraction or placed on ice for RNA extraction. After lysis, 478 0.2 ml of chloroform was added for every 1 ml of TRIzol used in the sample preparation. 479 Samples were incubated for 3 min and then spun at 12,000 g at 4°C for 15 min. To the 480 aqueous phase, an equal volume of 100% ethanol (EtOH) was added, before the samples 481 were loaded onto RNeasy RNA extraction columns (Qiagen). The manufacturer’s 482 instructions were followed from this point onwards. RNA quality was checked by Agilent, 483 with all RNA integrity number (RIN) scores above 7 (Schroeder et al. 2006). One microgram 484 of total RNA was used for cDNA library preparation. Library preparation was done in 485 accordance with the NEBNext pipeline, with library quality checked by Agilent. Samples 486 were sequenced using the Illumina HiSeq platform; 100-bp paired-end sequencing was 487 employed (2 x 100 bp) (>10 million reads per sample).

488 Single Cell RNA-Seq 489 For single cell sequencing experiments, macrophages were infected with fungal spores, as 490 outlined above. Uninfected macrophages, used as a negative control, were treated in the 491 same manner and underwent mock washes and media changes at identical time points to 492 infected macrophages. Macrophages were isolated and released from the bottom of their 493 wells with accutase, as per manufacturer’s instructions (Technologies n.d.). Once in solution, 494 the macrophages were loaded onto the 10X genomics single cell RNA sequencing pipeline 495 for single cell isolation and library preparation. In total, 1082 single cells were sequenced. 496 The libraries were sequenced on the Illumina Sequencing Platform. 497 498 Data Analysis 499 For the bulk RNA-Seq data, FastQC (version 0.11.5) was employed to ensure the quality of 500 all samples. Hisat2 (version 2.0.5) was used to align reads to the indexed genome of 501 Rhizopus delemar RA 88-880 (PRJNA13066, Ma et al. 2009) and the indexed genome of 502 Rhizopus microsporus (Mondo et al. 2017). HTSeq (version 0.8.0) was used to quantify the 503 output (Anders et al. 2015). Trinity and edgeR (Robinson et al. 2009)(version 3.16.5) were 504 then used to analyse differential expression (Grabherr et al. 2013). For the single cell RNA- 505 Seq data, the 10X genomics analysis pipeline (Loupe Cell Browser V 2.0.0, Cell Ranger 506 Version V 2.0.0) was used to align reads to the mus musculus genome (version MM10), and bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

507 quantify the output. For single cell analysis, the samples were then aggregated using this 508 pipeline, to allow comparisons between samples.

509 Data Availability 510 Data will be available upon request. 511 512 Acknowledgments 513 We are grateful to the University of Birmingham’s Genomics Services Facility and to 514 Deborah Croom-Carter for her technical support. 515 516 Author contributions 517 PSC conceived and designed the experiments, collected the data, performed the analysis 518 and interpretation, and wrote the manuscript. JFM, KV and CAC contributed to 519 interpretation of the data, and contributed to the manuscript. ERB conceived and designed 520 the experiments, contributed to interpretation of the data, and wrote the manuscript. 521 522 Funding 523 PSC was supported by a BBSRC MIBTP PhD Studentship (BB/M01116X/1). This work was 524 supported by a Wellcome Trust Seed award to KV (108387/Z/15/Z). HI was supported by the 525 Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377). 526 CAC and JFM were funded by the National Institute of Allergy and Infectious Diseases, 527 National Institutes of Health, under award U19AI110818 to the Broad Institute. ERB was 528 supported by the UK Biotechnology and Biological Research Council (BB/M014525/1) and a 529 Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society 530 (211241/Z/18/Z). 531 532 533 References 534 535 Alan, E.C. et al., 2019. An Emergent Entity: Indolent Mucormycosis of the Paranasal Sinuses 536 . A Multicenter Study. , (2193), pp.92–100.

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649

650 Figure legends 651 652 Figure 1 Phenotypic and genomic comparisons. A) Experimental setup: Pre-swollen (Sab) 653 WT or cured fungal spores from Rhizopus delemar (2hr) and Rhizopus microsporus (4hr) in 654 mid-isotropic phase were co-cultured with J774.1 murine macrophages (sfDMEM) at 5:1 655 MOI for 1hr, washed to remove unengulfed spores, then co-incubated for a further 2 hr and 656 RNA for fungi and host cells harvested. Pre-swollen spores (Sab) incubated in sfDMEM for 657 3hr served as the negative control. B) Germination percentage over time for R. delemar and 658 R. microsporus grown in Sab. C) Spore sizes for R. delemar and R. microsporus grown in SAB. 659 D) Pfam terms enriched (corrected P < 0.01) in R. delemar vs R. microsporus genomes, with bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

660 colour indicating the extent of enrichment (Log10 Count).

661 Figure 2 Principal component analysis of fungal genes differentially expressed across all 662 samples. A) R. delemar wt and cured, macrophage engulfed or sfDMEM control B) R. 663 microsporus wt and cured, macrophage engulfed or sfDMEM control. Replicates (n=3) are 664 shown for each sample.

665 Figure 3 Clustering of fungal transcriptional changes. A) Heatmap displaying differentially 666 expressed genes in R. delemar. Expression levels are plotted in Log2, space and mean- 667 centered (FDR < 0.001) B) Heatmap displaying differentially expressed genes in R. 668 microsporus. Expression levels are plotted in Log2, space and mean-centered (FDR < 0.001)

669 Figure 4 Differential expression of fungal genes in compared conditions. A) The number of 670 genes significantly differentially expressed (multiple corrected P value < 0.05) between 671 samples. Blue bars indicate genes with an increase in expression (LogFC > 2), whilst orange 672 bars indicate genes with a decrease in expression (LogFC < -2).

673 Figure 5 Gene functions of genes differentially expressed in R. delemar. Enriched KEGG 674 categories for the up/down regulated genes over sample comparisons. The enrichment of 675 the category is indicated by the colour bar. White corresponds to no enrichment, and yellow 676 to red corresponds to the given P value of the enrichment.

677 Figure 6 Principal component analysis of macrophage genes differentially expressed 678 across all samples. Single cell sequencing was performed on uninfected and infected 679 macrophages. Transcriptional data from the experiment was analysed with the 10X 680 genomics analysis pipeline, and aggregated prior to principle component analysis.

681 Figure 7 Clustering of macrophage transcription. Heatmap displaying immune response 682 genes significantly differentially expressed between macrophage populations. Expression 683 levels are plotted in Log2 (FDR < 0.001)

684 Figure 8 Macrophage survival following exposure to R. delemar and R. microsporus spores. 685 Macrophages +/- LPS pre-treatment were infected (MOI 5:1) with fungal spores, pre-swollen 686 in SAB consistent with single cell experiments. Macrophages were infected with fungal 687 spores that were pre-treated with +/- Nikkomycin Z (24 μg/ml; n=3 for each sample). 688 Macrophage survival was determined 7 hr post infection. Significant differences between 689 samples is indicated by (*= p < 0.01).

690 Supplementary Figure 1 Table of Pfam terms found to be enriched (corrected P < 5 x 10-8) in 691 R. delemar and R. microsporus upon genome comparison.

692 Supplementary Figure 2 Clustering of fungal transcription with GO annotation. Heatmap 693 displaying all differentially expressed genes, across all conditions, in R. microsporus. 694 Expression levels are plotted in Log2, space and mean-centered (FDR < 0.001).

695 Supplementary Figure 3 Clustering and heterogeneity in single cell analysis of J774.1 696 macrophages. Single cell plot generated by loupe cell browser. Colors indicate experimental 697 conditions as indicated. Spacial distribution indicates relative similarity across all detected 698 transcripts. 1082 single cells represented. bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

699 Supplementary Figure 4 Expression of chitin synthase genes. A) Heatmap showing the 700 expression of chitin synthase genes in R. delemar B) Heatmap showing the expression of 701 chitin synthase genes in R. microsporus

702 Supplementary Figure 5 Chitin synthase inhibition of R. delemar and R. microsporus 703 germination. R. delemar and R. microsporus treated with Nikkomycin Z in SAB at indicated 704 concentrations for the indicated times. Fluorescence indicates calcofluor white staining, and 705 thus the availability of chitin/chitosan in the cell wall. Labels indicate time and 706 concentration of inhibitor.

707 Supplemental Table 1 Selected genes differentially regulated in pairwise comparisons, with 708 Pfam annotations provided where available.

709 Supplementary Table 2 Table displaying the LogFC values for a subset of genes displayed in 710 Figure 7.

711 bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

A

B C

D bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

A Macrophages + R. delemar WT B Macrophages + R. microsporus WT R. delemar WT R. microsporus WT Macrophages + R. delemar cured Macrophages + R. microsporus cured R. delemar cured R. microsporus cured 0.2 0.0 0.2 PC 2 (9.67%) PC 2 (5.98%) 0 10 20 30 0.4

30 20 10 0 10 20 0.35 0.30 0.25 0.20 0.15

PC 1 (72.54%) PC 1 (91.72%) bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

Color Key Color Key samples vs. features samples vs. features Color Key diffExpr.P0.001_C2.matrix.log2.centered diffExpr.P0.001_C2.matrix.log2.centered

−6 −2 2 6 −4 0 2 4 Value Value LogFC LogFC

−6 −2 2 6 R. delemar curedcond_3 R. microsporuscond_3 cured MacrophagesValue +R. delemar curecond_2d Macrophages + R. microsporuscond_2 cured R. delemarcond_1 WT R. microsporuscond_1 WT Macrophages +R. delemarcond_0WT Macrophages + R. microsporuscond_0WT

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RO3G_003867RO3G_001808RO3G_005447RO3G_014874RO3G_015798RO3G_009128RO3G_006189RO3G_008227RO3G_015889RO3G_006100 RO3G_011448RO3G_009889RO3G_004813RO3G_009414RO3G_017006RO3G_004010RO3G_007225RO3G_009541RO3G_017073RO3G_000704 RO3G_004174RO3G_003605RO3G_010132RO3G_002307RO3G_001063RO3G_003293RO3G_003618RO3G_008995RO3G_002456RO3G_012572 RO3G_003859RO3G_000984RO3G_013624RO3G_004086RO3G_009343RO3G_003912RO3G_013623RO3G_002325RO3G_002533 RO3G_008929RO3G_005089RO3G_011966RO3G_008351RO3G_012076RO3G_000988RO3G_013888RO3G_005037RO3G_011806RO3G_003166 RO3G_000266RO3G_000017RO3G_004981RO3G_000249RO3G_013469RO3G_004160RO3G_013571RO3G_016889RO3G_012981RO3G_006622 RO3G_005708RO3G_010002RO3G_013746RO3G_015170RO3G_006145RO3G_007825RO3G_000386RO3G_014298RO3G_012662RO3G_011989 RO3G_003867RO3G_001808RO3G_005447RO3G_014874RO3G_015798RO3G_009128RO3G_006189RO3G_008227RO3G_015889RO3G_006100 RO3G_000431RO3G_001255RO3G_013281RO3G_011928RO3G_011987RO3G_016325RO3G_013995RO3G_010187RO3G_011399RO3G_011754 RO3G_004174RO3G_003605RO3G_010132RO3G_002307RO3G_001063RO3G_003293RO3G_003618RO3G_008995RO3G_002456RO3G_012572 RO3G_014971RO3G_007723RO3G_000974RO3G_015579RO3G_013542RO3G_014650RO3G_010433RO3G_005008RO3G_006318RO3G_005899 RO3G_008929RO3G_005089RO3G_011966RO3G_008351RO3G_012076RO3G_000988RO3G_013888RO3G_005037RO3G_011806RO3G_003166 RHIMIDRAFT_96114 RO3G_007400RO3G_005904RO3G_006119RO3G_011533RO3G_008558RO3G_016939RO3G_016704RO3G_015490RO3G_013446RO3G_001677 RO3G_005708RO3G_010002RO3G_013746RO3G_015170RO3G_006145RO3G_007825RO3G_000386RO3G_014298RO3G_012662RO3G_011989 RO3G_006615RO3G_015449RO3G_012452RO3G_009658RO3G_007835RO3G_015881RO3G_003009RO3G_017703RO3G_009187RO3G_016195 RO3G_000431RO3G_001255RO3G_013281RO3G_011928RO3G_011987RO3G_016325RO3G_013995RO3G_010187RO3G_011399RO3G_011754 RO3G_008500RO3G_007795RO3G_014057RO3G_001097RO3G_014140RO3G_008818RO3G_001707RO3G_016207RO3G_015442RO3G_016268 RO3G_014971RO3G_007723RO3G_000974RO3G_015579RO3G_013542RO3G_014650RO3G_010433RO3G_005008RO3G_006318RO3G_005899 RO3G_017376RO3G_014162RO3G_001432RO3G_001476RO3G_005274RO3G_006645RO3G_011183RO3G_005137RO3G_013060RO3G_016089 RO3G_007400RO3G_005904RO3G_006119RO3G_011533RO3G_008558RO3G_016939RO3G_016704RO3G_015490RO3G_013446RO3G_001677 RO3G_002010RO3G_004555RO3G_010673RO3G_013999RO3G_001769RO3G_014851RO3G_005588RO3G_005148RO3G_016300RO3G_012201 RO3G_015449RO3G_012452RO3G_009658RO3G_007835RO3G_015881RO3G_003009RO3G_017703RO3G_009187RO3G_016195 RO3G_014481RO3G_001557RO3G_002536RO3G_009159RO3G_008028RO3G_008578RO3G_011184RO3G_004001RO3G_006206RO3G_008727 RO3G_007795RO3G_014057RO3G_001097RO3G_014140RO3G_008818RO3G_001707RO3G_016207RO3G_015442RO3G_016268RO3G_006615 RO3G_002901RO3G_011817RO3G_003862RO3G_016107RO3G_004721RO3G_006891RO3G_008545RO3G_006894RO3G_015812RO3G_016843 RO3G_014162RO3G_001432RO3G_001476RO3G_005274RO3G_006645RO3G_011183RO3G_005137RO3G_013060RO3G_016089RO3G_008500 RHIMIDRAFT_242387 RO3G_014327RO3G_013872RO3G_014340RO3G_011832RO3G_001923RO3G_003091RO3G_014401RO3G_011172RO3G_010903RO3G_005470 RO3G_004555RO3G_010673RO3G_013999RO3G_001769RO3G_014851RO3G_005588RO3G_005148RO3G_016300RO3G_012201RO3G_017376 RO3G_010990RO3G_006780RO3G_003602RO3G_015213RO3G_004678RO3G_005437RO3G_014646RO3G_005277RO3G_017052RO3G_005059 RO3G_001557RO3G_002536RO3G_009159RO3G_008028RO3G_008578RO3G_011184RO3G_004001RO3G_006206RO3G_008727RO3G_002010 RO3G_009506RO3G_000908RO3G_008048RO3G_008370RO3G_000468RO3G_012203RO3G_015628RO3G_004544RO3G_003695RO3G_001423 RO3G_011817RO3G_003862RO3G_016107RO3G_004721RO3G_006891RO3G_008545RO3G_006894RO3G_015812RO3G_016843RO3G_014481 RO3G_000748RO3G_001824RO3G_016254RO3G_003106RO3G_013722RO3G_017160RO3G_009881RO3G_013380RO3G_010796RO3G_002214 RO3G_013872RO3G_014340RO3G_011832RO3G_001923RO3G_003091RO3G_014401RO3G_011172RO3G_010903RO3G_005470RO3G_002901 RO3G_011162RO3G_002323RO3G_010829RO3G_000954RO3G_002500RO3G_004683RO3G_008249RO3G_006968RO3G_017547RO3G_003102 RO3G_003602RO3G_015213RO3G_004678RO3G_005437RO3G_014646RO3G_005277RO3G_017052RO3G_005059RO3G_014327 RO3G_012320RO3G_017274RO3G_006358RO3G_012780RO3G_016571RO3G_003922RO3G_009949RO3G_011212RO3G_010607RO3G_009742 RO3G_008048RO3G_008370RO3G_000468RO3G_012203RO3G_015628RO3G_004544RO3G_003695RO3G_001423RO3G_010990RO3G_006780 RHIMIDRAFT_245669 RO3G_009836RO3G_007558RO3G_009004RO3G_001510RO3G_006479RO3G_008489RO3G_001310RO3G_015620RO3G_002493RO3G_013882 RO3G_016254RO3G_003106RO3G_013722RO3G_017160RO3G_009881RO3G_013380RO3G_010796RO3G_002214RO3G_009506RO3G_000908 RO3G_007433RO3G_016212RO3G_008411RO3G_014396RO3G_015861RO3G_011864RO3G_001309RO3G_009644RO3G_013422RO3G_004367 RO3G_010829RO3G_000954RO3G_002500RO3G_004683RO3G_008249RO3G_006968RO3G_017547RO3G_003102RO3G_000748RO3G_001824 RO3G_008711RO3G_000900RO3G_000949RO3G_014501RO3G_003598RO3G_004193RO3G_014305RO3G_016120RO3G_010417RO3G_005315 RO3G_006358RO3G_012780RO3G_016571RO3G_003922RO3G_009949RO3G_011212RO3G_010607RO3G_009742RO3G_011162RO3G_002323 RO3G_014451RO3G_001190RO3G_011698RO3G_007377RO3G_014467RO3G_007769RO3G_014471RO3G_011512RO3G_010037RO3G_016857 RO3G_009004RO3G_001510RO3G_006479RO3G_008489RO3G_001310RO3G_015620RO3G_002493RO3G_013882RO3G_012320RO3G_017274 RO3G_005635RO3G_017389RO3G_005396RO3G_015241RO3G_006584RO3G_016752RO3G_014734RO3G_010189RO3G_014933RO3G_006623 RO3G_008411RO3G_014396RO3G_015861RO3G_011864RO3G_001309RO3G_009644RO3G_013422RO3G_004367RO3G_009836RO3G_007558 RO3G_014380RO3G_006165RO3G_006326RO3G_011802RO3G_001062RO3G_008322RO3G_010934RO3G_007683RO3G_012567RO3G_009678 RO3G_000949RO3G_014501RO3G_003598RO3G_004193RO3G_014305RO3G_016120RO3G_010417RO3G_005315RO3G_007433RO3G_016212 RHIMIDRAFT_283813 RO3G_016742RO3G_017422RO3G_009519RO3G_013940RO3G_014198RO3G_012571RO3G_013538RO3G_005645RO3G_000866RO3G_007382 RO3G_007377RO3G_014467RO3G_007769RO3G_014471RO3G_011512RO3G_010037RO3G_016857RO3G_008711RO3G_000900 RO3G_015694RO3G_009424RO3G_012739RO3G_016465RO3G_015312RO3G_009281RO3G_000465RO3G_003906RO3G_016415RO3G_001012 RO3G_015241RO3G_006584RO3G_016752RO3G_014734RO3G_010189RO3G_014933RO3G_006623RO3G_014451RO3G_001190RO3G_011698 RO3G_005875RO3G_002036RO3G_009513RO3G_007365RO3G_006644RO3G_016651RO3G_001550 RO3G_011802RO3G_001062RO3G_008322RO3G_010934RO3G_007683RO3G_012567RO3G_009678RO3G_005635RO3G_017389RO3G_005396 RO3G_013940RO3G_014198RO3G_012571RO3G_013538RO3G_005645RO3G_000866RO3G_007382RO3G_014380RO3G_006165RO3G_006326 RO3G_016465RO3G_015312RO3G_009281RO3G_000465RO3G_003906RO3G_016415RO3G_001012RO3G_016742RO3G_017422RO3G_009519 RO3G_005875RO3G_002036RO3G_009513RO3G_007365RO3G_006644RO3G_016651RO3G_001550RO3G_015694RO3G_009424RO3G_012739 Without With Macrophages With Without Macrophages Macrophages Macrophages cond_3_rep2 cond_3_rep1 cond_1_rep3 cond_1_rep1 cond_1_rep2 cond_3_rep3 cond_0_rep2 cond_2_rep1 cond_0_rep3 cond_0_rep1 cond_2_rep3 cond_2_rep2 A B cond_2_rep1 cond_0_rep2 cond_0_rep1 cond_2_rep2 cond_2_rep3 cond_3_rep1 cond_3_rep2 cond_3_rep3 cond_1_rep1 cond_0_rep3 cond_1_rep2 cond_1_rep3 cond_3_rep2 cond_3_rep1 cond_1_rep3 cond_1_rep1 cond_1_rep2 cond_3_rep3 cond_0_rep2 cond_2_rep1 cond_0_rep3 cond_0_rep1 cond_2_rep3 cond_2_rep2 − No. Differentially− Expressed Genes (Log2FC > 2 or < −2, FDR < 0.05) − 1500 1000 1000 1500 500 500 0 1137 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples DEM v DE − 472 DEM v DE

MEM v ME 1 − − − − − No. Differentially Expressed Genes6 (Log2FC > 2 or < −2, FDR < 0.05) − 1500 1000 1000 1500 − 1500 1000 MEM v ME 1000 1500 500 500 500 500 0 0 DEM v DM 0 − − No. Differentially Expressed Genes0 (Log2FC > 2 or < −2, FDR < 0.05) − 1500 1000 DEM v DM 1000 1500 500 500 1137 1137 0 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemar Samples Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples MEMDEM vv MMDE 0 − − 472 − − 472

No. Differentially Expressed Genes0 (Log2FC > 2 or < −2, FDR < 0.05) − 1500 1000 1000 1500 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder

DEM v DE bioRxiv preprint − − 500 MEMDEM vv MMDE No. Differentially Expressed500 Genes (Log2FC > 2 or < −2, FDR < 0.05) 1137 − 1500 1000 1000 1500 1072 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples 500 500 0 1 DEM v DE 1 MEMDEM v MEv D 0 − − 472 − − − − No. Differentially590 Expressed Genes (Log2FC > 2 or < −2, FDR < 0.05) 6 − 1500 1000 1000 1500 doi: MEMDEM vv MEDE 6 500 MEMDEM v MEv D 500 1137 https://doi.org/10.1101/580746 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples 0 336 1 1137 0 DEM v DE 0 MEMDEMMEM v vDMME M Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples DEM v DE − − 472 − − 209 0 6 0

DEM v DE 472 MEMDEMMEM v vDMME M DEM v DE 1137 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples 778 1 0 0 MEMDEMDEM vv DMMEDE 0 WT+ MacrophageMEMDE vv MMDM vs 1 −

MEM v ME − 472 − 1325

WT 0 a 6 0 0 CC-BY-NC-ND 4.0Internationallicense

DEM v DE ; MEMDEM v DMME − MEM v MM this versionpostedMarch20,2019. 6 MEMDE vv MMDM − − − No. Differentially− Expressed Genes (Log2FC > 2 or < −2, FDR < 0.05)

MEM v ME − 1500 1000 1000 1500 − 1072 1500 1000 1000 1500 1072 1 0 500 500 500 500 0 MEMMEMDEM vv MMDMME 8 0 0 WT+ MacrophageMEDEM v MMv Dvs 0 DEM v DM − − − 0 590 0

Cured + Macrophage 6 − MEM v ME 590 MEMDEM vv MMDM 2 0 MEDEM v MMv D − No. Differentially− Expressed Genes (Log2FC > 2 or < −2, FDR < 0.05) − 1500 1000 1000 1500 DEM v DM 1072 336 500 500 0 0 336 1137 1137 MEMDEM vv MMDM 0 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples 0

DEM v D Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples MEM v M 0 R. delemarR. WT+ MacrophageMEMDE vv M Dvs MEM v MM − DEM v DE − − 590 0 0 209 − 209 − DEM vCured DM −

MEM v MM 1 472 472

DEM v D − − MEM v M 0 MEMDE vv MD No. Differentially Expressed Genes (Log2FC > 2 or < −2, FDR < 0.05) The copyrightholderforthispreprint(whichwasnot − 1072 1500 1000 1000 1500 MEMDEM vv MMDE .

DEM v DE 336 500 500 778 0 778 1137 1072 3

MEM v MM Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples MEMDEM vv MD 0 1 DEME v vDM M 1 − − WTDEM vs Cured v DE + − MEMDEM v MEv D − 590 1325 209 0 1325 − − 0 MacrophageMEM v MM 472

DEM v D 590 − − − MEMDEME v vDM M No. Differentially Expressed Genes− (Log2FC > 2 or < −2, FDR < 0.05) − 6 1500 1000 1000 1500 6 DEM v DE 1072 DEM v D 336

MEM v ME 500 500

MEM v ME 778 1285 1137 8 336 8 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples MEMDEM vv MD 0 DE v DM 1 0 MEDM v MMv D 0 − − Cured +MacrophageMEMDEMDEMMEM vv vDMMEDE M −

DEM v DM 1325 590 − 209 − − − − 878 472

vs Cured − DEM v D 2 209 MEMDE v vDM M 2 0 0 DEMMEDM v v MM vDE D 6

MEM v ME 336 DEMMEM v vDM M 778 1137 277 8 0 Differentially ExpressedGenesfrom Pairwise ComparisonsofR.microsporus andR.delemarSamples 778 MEM v M 0 DE v DM 1 ME v MM 0 0 DE v D 0 MMDE vv MD − MEMDEMDEM vv DMMEDE − MEMDE vv MMDM 1325 209 − − − WT vs Cured − − − 472 2 − 1325 82

MEM v M 1

DE v DM 1 ME v MM 0 6 0 MEMDEMMMDE vv vvMEDE MD 0 MEMDEMDE vv MMDM 778 8 0 1072 1072 3 3 DE v DM 1 ME v MM 0 DE v D 0 8

ME v M − MEMMEMDEMME vv MMvDMME M WT+ MacrophageMEDEM v MMv Dvs 1325 − − 2 − − − 0 1 DE v DM 0 0 ME v MM − 590 0 590 MEDE vvWT MD 6

ME v M 2 MEMMEMDEMME vv MMDMME DEM v D 1285 1285 8 0 1072 3 336 336 ME v MM 0 MEDE vv MD 0 DM v D 0 MEMDEMDEMDE vv MMDMv D

MEM v M − WT+ MacrophageMEM v M vs − − − − 878 0 2 1 878 − − 0 ME v MM 590 0 DE v D − 209 Cured + MacrophageMEDM vv MD 209 MEMDEMDM vv MMDMv D 1 MEMDEMDE vv MD 1285 277 0 1072 3 277 336 778 778 DE v D 0 R. microsporusR. MEDM vv MD MEMDEMMM v MMvv MD 3 MEMDEME v vDM M −

DE v DM −

WT+ Macrophage vs − 878 − − 0 − − − 1 82 590 82 1325 0 1325 MEDMDE vv MD 209 MEMMM vCured MMv M 0 MEMDEDEMME v vDMv MD 1285

DE v DM 277 1072 3 336 1285 MMMEDM vv MD 778 8 DEM v D 8 MEMEMDEDM vv MMvDMv MD − ME v MM − 878 − − 0 WT vs Cured + − 82 − 590 1325 ME v M 209

DM v D 878 − MacrophageMM v M − 2

DEM v D 2 MEMDE v vDM M 1285 MEDM v MMv D 277 336 778 MMDM vv MD 277 8 0 0

MEMEMDE vv MMvDM M − MMDE vv MD − 878 − −

Cured +Macrophage 82 1325 209 − MMDM vv MD − − − 82 vs Cured 2 1 MEMDE v vDM M 1 MEMMDE v MMvv MD 277 MM v M 778 8 0 3 MEDE vv MMDM 3

DE v D −

ME v M − 82 1325 −

WT MMvs Cured v M − 2 0 0 DE v DM 1 MEMEDE v MMvv MD 1285 1285 8 0 ME v MM 3 MEDMDE vv MD − − − − 878 0 878 2 ME v MM 1 MEDMDE vv MD 1285 277 277 0 DE v D 3 MMMEDM vv MD − − − − 878 0 1 82 DE v D 82 MMMEDM vv MD 1285 277 3 MMMEDM vv MD − − 878 0

MEDM vv MD 82 MM v M

1285

277 MMDM vv MD − − 878 MMDM vv MD 82 277 MM v M − MM v M 82 0.08 0.06 0.04 0.02 0 Fatty acid degradation acid Fatty metabolism sucrose and Starch biosynthesis neomycin and Butirosin biosynthesis Folate metabolism Galactose metabolism Tyrosine acid metabolism Arachidonic and absorption digestion Carbohydrate Endocytosis backbone biosynthesis Terpenoid DNA replication metabolism Pyruvate biosynthesis − ganglio seriesGlycosphingolipid dibasic acid metabolism C5−Branched Glycosaminoglycan degradation metabolism Tryptophan degradation Chloroalkane and chloroalkene Histidine metabolism and glucuronate interconversions Pentose sugar metabolism Amino sugar and nucleotide Cytosolic DNA−sensing pathway Glycerophospholipid metabolism Plant−pathogen interaction bodies of ketone Synthesis and degradation reticulum Protein processing in endoplasmic biosynthesis Streptomycin Peroxisome bicarbonate reclamation tubule Proximal Methane metabolism Fructose and mannose metabolism Arginine and proline metabolism phosphate pathway Pentose alpha−Linolenic acid metabolism MAPK signalling pathway receptor signalling pathway NOD−like Ether lipid metabolism acids fatty Biosynthesis of unsaturated degradation Lysine metabolism Ascorbate and aldarate Limonene and pinene degradation Isoquinoline alkaloid biosynthesis metabolism Phenylalanine alkaloid biosynthesis piperidine and pyridine Tropane, Butanoate metabolism acid metabolism Cyanoamino beta−Alanine metabolism Glutathione metabolism pathway signalling PPAR

Cured + Macrophage vs Cured (up)

Cured + Macrophage vs Cured (up)

WT vs Cured + Macrophage (down) . The copyright holder for this preprint (which was not The copyright holder for WT vs Cured + Macrophage (up)

WT + Macrophage vs Cured (down) this version posted March 20, 2019. this version posted March ; CC-BY-NC-ND 4.0 International license CC-BY-NC-ND 4.0 International a WT + Macrophage vs Cured (up)

WT + Macrophage vs WT (down) https://doi.org/10.1101/580746 doi:

WT + Macrophage vs WT (up) bioRxiv preprint certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a license to display the preprint in perpetuity. is the author/funder, who has granted bioRxiv certified by peer review) bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

S1 S2 S3 S4 S5 10 5 0 PC2 (18.71%) 5 −

R. delemar Cured R. delemar WT R. microsporus Cured R. microsporus WT Macrophage Control 10 − −10 −5 0 5 10 15 20

PC1 (49.17%) gamma production, production, gamma - cascade , Endopeptidase inhibitor , Endopeptidase kappaB - activity, MHC classbinding protein II 1 2 3 4 3 2 1 0 − − − GTPase inflammatory ofproduction, Regulation cytokine transferaseresponse, glutathione Glycoprotein, Receptor, G Protein Signaling,G Receptor, Glycoprotein, binding Phosphoinositide NK cell activation, Interferon NK activation, cell regulation cell of binding, T activation, leukocyte activation TNFRSF8, CDC247, CCL22 TNFRSF8, & response, CytokineERK1 activation, Immune stimulus, regulation, Response to ERK2 Regulation of NF Antigen presenting Antigen Extracellular region, Collagen, Cell Activation, Cell Proliferation, & Leukocyte Lymphocyte binding, receptor Endothelial activation, response, Inflammatory production, Interleukin Sugar binding CCL5 IRG F3, PSTPIP CXCL10, SAA3, ENPP4 Growth Factor Binding, IL1 receptor agonist,receptor IL1 Binding, Factor Growth binding protein endocytosis, M2 Q6 T24 AB1 − − − − IL20RB TNFRSF8 CD247 CCL22 AK4 HVCN1 IL1B IL23A AGRN ATP13A4 CX3CL1 HTRA1 GM8773 TGM2 AI504432 FLT4 NOD1 RSAD2 PLA2G16 TRIM30D SLC1A2 IL6 ADAMTS4 TNFSF10 AKR1C12 MACC1 BC147527 CSF3 SLFN5 IL12B BCL11A HTRA4 ADORA2A TRIM5 KLRK1 GBP4 PYHIN1 FABP3 PDE7B CCL1 SCARF1 RGS16 PLEKHA4 LAG3 H2 IIGP1 MX1 KCTD14 PNP2 GSTT1 GPR141 MEFV CD83 CD831 GM6377 TICAM2 CCL5 CCL51 F3 F31 IRG1 IRG11 PSTPIP2 PSTPIP21 CXCL10 SAA3 ENPP4 CISH SERPINE1 ENG FGFBP3 GM8898 GCNT2 IL1F6 ZFP558 CST7 H2 VCAN H2 LY75 IL27 AKAP2 SLAMF7 H2 AW112010 GM5431 CP FNDC7 CCND2 IL4I1 AA467197 COL4A2 COL27A1 EDN1 IFI205 R. R. cured

S4 Macrophage + Macrophage microsporus R. R. cured S2 delemar Macrophage + Macrophage T R. R. W

S3 microsporus Macrophage + Macrophage R. R. T W

. S1 The copyright holder for this preprint (which was not The copyright holder for delemar Macrophage + Macrophage

S5 control Macrophage Macrophage this version posted March 20, 2019. this version posted March ; CC-BY-NC-ND 4.0 International license CC-BY-NC-ND 4.0 International a 1 2 3 0 − − − 4 3 2 1 M2 Q6 T24 AB1 − − − − FABP3 PDE7B CCL1 SCARF1 RGS16 PLEKHA4 LAG3 H2 IIGP1 MX1 KCTD14 PNP2 GSTT1 GPR141 MEFV CD83 CD831 GM6377 TICAM2 IL1B IL23A AGRN ATP13A4 CX3CL1 HTRA1 GM8773 TGM2 AI504432 FLT4 NOD1 RSAD2 PLA2G16 TRIM30D SLC1A2 IL6 ADAMTS4 TNFSF10 AKR1C12 MACC1 BC147527 CSF3 SLFN5 IL12B BCL11A HTRA4 ADORA2A TRIM5 KLRK1 GBP4 PYHIN1 FGFBP3 GM8898 GCNT2 IL1F6 ZFP558 CST7 H2 VCAN H2 LY75 IL27 AKAP2 SLAMF7 H2 AW112010 GM5431 CP FNDC7 CCND2 IL4I1 AA467197 COL4A2 COL27A1 EDN1 IFI205 IL20RB TNFRSF8 CD247 CCL22 AK4 HVCN1 CCL5 CCL51 F3 F31 IRG1 IRG11 PSTPIP2 PSTPIP21 CXCL10 SAA3 ENPP4 CISH SERPINE1 ENG https://doi.org/10.1101/580746 doi:

S4 bioRxiv preprint certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a license to display the preprint in perpetuity. is the author/funder, who has granted bioRxiv certified by peer review)

S2

S3

S1

S5 bioRxiv preprint doi: https://doi.org/10.1101/580746; this version posted March 20, 2019. The copyright holder for this preprint (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 available under aCC-BY-NC-ND 4.0 International license.

* 100 * * 80 * * 60

40

20 Macrophage Survival (%) 0

R. delemarR. delemarR. delemar Macrophage R. microsporusR. microsporusR. microsporus Macrophage + LPS

Macrophage +

Macrophage + Macrophage + LPS +

Macrophage + LPS + Macrophage + Nikkomycin +

Macrophage + Nikkomycin +