A Ssd1 homolog impacts trehalose and chitin biosynthesis and contributes to virulence in Aspergillus fumigatus

Regulation of fungal cell wall biosynthesis is critical to maintain cell wall integrity in the face of dynamic fungal infection microenvironments. In this study, we observe that a yeast ssd1 homolog, ssdA, in the filamentous fungus Aspergillus fumigatus is involved in trehalose and cell wall homeostasis. An ssdA null mutant strain exhibited an increase in trehalose levels and a reduction in colony growth rate. Over-expression of ssdA in contrast perturbed trehalose biosynthesis and reduced conidia germination rates. The ssdA null mutant strain was more resistant to cell wall perturbing agents while over-expression of ssdA promoted increased sensitivity. Over-expression of ssdA significantly increased chitin levels and both loss and over-expression of ssdA altered sub-cellular localization of the class V chitin synthase CsmA. Strikingly, over-expression of ssdA abolished adherence to abiotic surfaces and severely attenuated the virulence of A. fumigatus in a murine model of invasive pulmonary aspergillosis. In contrast, despite the severe in vitro fitness defects observed upon loss of ssdA, neither surface adherence or murine survival was impacted. In conclusion, A. fumigatus SsdA plays a critical role in cell wall homeostasis that alters fungal-host interactions. Importance Life threatening infections caused by the filamentous fungus Aspergillus fumigatus are increasing along with a rise in fungal strains resistant to contemporary antifungal therapies. The fungal cell wall and the associated carbohydrates required for its synthesis and maintenance are attractive drug targets given that many genes encoding proteins involved in cell wall biosynthesis and integrity are absent in humans. Importantly, genes and associated cell wall biosynthesis and homeostasis regulatory pathways remain to be fully defined in A. fumigatus. In this study, we identify SsdA, a model yeast Ssd1p homolog, as an important component of trehalose and fungal cell wall biosynthesis in A. fumigatus that consequently impacts fungal virulence in animal models of infection.


Introduction
in trehalose biosynthesis and support a global role for SsdA in fungal fitness when glucose is 174 the sole carbon source. 175 176

SsdA is important for cell wall integrity 177
A. fumigatus trehalose null mutants including tslA have altered cell wall integrity and 178 previous research identified a link between yeast Ssd1p and Neurospora crassa GUL-1 179 (SSD1 homolog) function and the cell wall (39,40,50,54). We next utilized the cell wall 180 perturbing agents congo red (1 mg/mL), calcofluor white (CFW, 50 g/mL), and the 181 echinocandin caspofungin (2 g/mL) to test the hypothesis that A. fumigatus SsdA is 182 important for cell wall integrity. ssdA exhibited increased resistance to cell wall perturbing 183 agents while OE:ssdA exhibited increased susceptibility, particularly to congo red and 184 calcofluor white (Fig. 2). 185 We hypothesized that the altered growth and change in susceptibility to cell wall 186 perturbing agents observed in SsdA mutants comes from altered cell wall composition and/or 187 organization. To initially test this hypothesis, CFW and wheat germ agglutinin (WGA) were 188 used to interrogate total and exposed chitin respectively, while soluble human dectin-1-FC 189 was used to examine -1,3-glucan exposure. We observed a large decrease in the intensity of 190 CFW and WGA staining of ssdA germlings while in contrast germlings of OE:ssdA showed 191 increased intensity with these chitin binding molecules (P=0.0322, ssdA to the wild type, 192 P<0.0001, OE:ssdA to the wild type) (Fig. 3A). For -1,3-glucan, we observed a decrease in 193 soluble dectin1-FC staining on both the ssdA and the OE:ssdA germlings suggestive of a 194 decrease in -1,3-glucan exposure (P=0.0389, ssdA to the wild type, P<0.0001, OE:ssdA 195 to the wild type) (Fig. 3). While additional quantitative cell wall composition analyses are 196 needed, these data support the hypothesis that SsdA impacts A. fumigatus cell wall integrity.
Given the changes in the cell wall of the ssdA and the OE:ssdA strains, we next 198 tested their ability to adhere to an abiotic surface. Using the crystal violet adherence assay, 199 we observed no difference in adherence between the wild-type, ssdA, and reconstituted 200 strains. However, a striking loss of adherence was observed in the OE:ssdA strain (Fig. 4A). 201 To investigate this adherence difference further, spinning disk confocal microscopy, in 202 combination with the galactosaminogalactan binding FITC labeled soy bean agglutinin 203 (SBA), was utilized. Given the decreased adherence of the overexpression strain, we were 204 surprised that increased expression of ssdA resulted in much greater levels of SBA staining, 205 revealing striking differences compared to the wild-type and ssdA strains (Fig. 4B). As SBA 206 binds to oligosaccharides with alpha-or beta-linked N-acetylgalactosamine and, to a lesser 207 extent, galactose residues, we tested whether mRNA levels of the UDP-glucose 4-epimerase 208 involved in galactosaminogalactan biosynthesis were altered in the ssdA mutant strains (55). 209 No significant difference in uge3 mRNA levels were observed under the conditions 210 examined, suggesting a role for ssdA in post-transcriptional regulation of the 211 galactosaminogalactan polysaccharide (Fig. 4C). Taken together, these data suggest that ssdA 212 expression levels impact fungal adherence. 213 Given the responses of the ssdA mutant strains to agents and reagents that inhibit or 214 bind to chitin, a non-radioactive chitin synthase activity assay was next utilized to further 215 define the impact of SsdA levels on the A. fumigatus cell wall (56,57). Consistent with the 216 cell wall immunohistochemistry results, chitin synthase activity in ssdA was significantly 217 reduced while in contrast chitin synthase activity in OE:ssdA was significantly increased 218 (P=0.0029, csmA to the wild type; P=0.0208, ssdA to the wild type; P<0.0001, OE:ssdA 219 to the wild type) (Fig. 5A). We previously observed that activity and localization of the chitin 220 synthase CsmA was perturbed by loss of the trehalose regulatory protein TslA (41). As TslA was found to also physically interact with SsdA, we hypothesized that SsdA levels may also 222 impact CsmA sub-cellular localization. 223 To study CsmA sub-cellular localization when SsdA levels are altered, we introduced 224 a C-terminal GFP-tagged csmA allele into the respective ssdA mutant strains. Using spinning 225 disk confocal microscopy, we observed that alteration of ssdA mRNA levels (loss or 226 increase) led to an altered CsmA localization pattern compared to the wild-type and 227 reconstituted strains (Fig. 5B). CsmA:GFP puncta observed in ssdA are mainly focused at 228 the hyphal tip with a few puncta also localized along the lateral hyphal walls but no visible 229 localization at the conidial septum. In contrast, in OE:ssdA CsmA:GFP puncta were 230 dispersed throughout the hyphae with no visible puncta at the hyphal tip or conidial septum 231 ( Fig. 5B). Intriguingly, this latter result is similar to the diffuse sub-cellular localization of 232 CsmA:GFP in the absence of TslA (41). Taken together, these results suggest SsdA levels 233 affect sub-cellular localization of the chitin synthase CsmA. 234 235

SsdA levels are critical for Aspergillus fumigatus virulence 236
Given the trehalose, cell wall, and biofilm phenotypes associated with alterations in SsdA 237 levels, we hypothesized that SsdA plays an important role in A. fumigatus fungal-host 238 interactions. To understand the importance of SsdA in the A. fumigatus-host interaction, we 239 first utilized the triamcinolone (steroid) murine model of IPA (58). Strikingly, we observed 240 that overexpression of SsdA significantly decreased A. fumigatus virulence compared to the 241 wild type (P= 0.0033, OE:ssdA to the wild type) (Fig. 6A). This reduction in virulence was 242 associated with a large reduction in immune cell infiltrate in the bronchoalveolar lavage fluid 243 (BALs) (P=0.0159, OE:ssdA to the wild type, Mann-Whitney t-test) (Fig. 6B). Perhaps 244 correspondingly, we observed a significant reduction in fungal growth within the OE:ssdA 245 inoculated lungs compared to other strains (Fig. 6C).
In contrast, complete loss of SsdA did not alter median murine survival time between 247 the wild type and ssdA (median survival = 3 days). However, despite the in vitro growth 248 defect of ssdA, fungal burden observed by histopathology revealed modest increases in 249 ssdA fungal burden at day three post inoculation compared to wild-type (Fig. 6C). 250 Surprisingly, despite equivalent or increased fungal burden compared to the wild type, a 251 significant reduction in immune cell infiltrate in the bronchoalveolar lavage fluid (BALs) is 252 apparent in animals inoculated with ssdA (P=0.0159, ssdA to the wild type, Mann-253 Whitney t-test) (Fig. 6C). These results support the hypothesis that changes in ssdA levels 254 impact the fitness of A. fumigatus in vivo and alter host immune responses. 255 Given the striking ssdA in vitro growth defect observed but full virulence (as 256 measured by murine mortality) in the steroid IPA model, we hypothesized that SsdA would 257 be essential for virulence in a leukopenic IPA model with significant immune cell depletion 258 (41). However, surprisingly, and similar to the corticosteroid model, ssdA also had 259 persistent if not slightly increased virulence in the leukopenic model (P = 0.005) (Fig. 6D). 260 Also similar to the steroid model, OE:ssdA had significant virulence attenuation compared to 261 the wild type (P = 0.0049) (Fig. 6D). Median survival of the wild type, ssdA, and OE:ssdA-262 inoculated mice was 3.5, 2, and 9.5 days, respectively. Histopathology from this leukopenic 263 model revealed less fungal growth from lungs of OE:ssdA-inoculated mice while ssdA-264 inoculated mice, in contrast to the in vitro growth phenotype, had substantial invasive hyphal 265 growth compared to the wild type (Fig. 6F). In contrast to the steroid model, inflammatory 266 cell infiltrations were the same between ssdA mutants and the wild type in this leukopenic 267 model possibly reflecting the significant chemical mediated immune suppression (Fig. 6E). 100,000g for 40 min at 4°C as described before. After that, the nonradioactive chitin synthase 458 activity assay was performed in a 96-well plate as previously described (56,57). 459 460

Trehalose measurement 461
Trehalose content in conidia and mycelia was as previously described (40). Briefly, A. 462 fumigatus strains were grown on GMM plates at 37°C for 3 days. A total of 2 x 10 8 conidia 463 were used for the conidial stage of the trehalose assay, and 1 x 10 8 conidia in 10mL LGMM 464 were cultured overnight for the mycelial stage as described by d 'Enfert C and Fontaine 465 (1997)  Three mice in each group (CEA10, ΔssdA, ΔssdA+ssdA-GFP, and OE:ssdA) were humanely 485 euthanized at day 3 post-inoculation. Lungs were harvested from each group and fixed in 486 10% formalin before embedding in paraffin. 5μm-thick sections were taken and stained with 487 either H&E (Hematoxylin and Eosin) or GMS (Gomori-Methenamine Silver stain) as 488 previously described (85). The microscopic examination was performed on a Zeiss Axioplan 489 II microscope and engaged imaging system. Images were captured at 50x magnification as 490