AfRip3, a RIP3-like kinase, is identified as a key modulator of necroptotic death in Aspergillus fumigatus

Aspergillus fumigatus exhibits autophagic and necroptotic process when its GPI anchor synthesis is suppressed. A putative kinase (AFUA_6G02590) is found to be overexpressed in response to GPI anchor suppression and identified as a RIP3-like protein, namely AfRip3. To elucidate its function, in this study a Afrip3-overexpressing strain OE-Afrip3 was constructed. Although OE-Afrip3 strain exhibited an increased cell death, neither apoptotic nor autophagic process was activated. Our evidences demonstrated that overexpression of Afrip3 gene in A. fumigatus only led to necroptosis, while the Afrip3-knockout mutant was unable to activate necroptotic process. Further analysis revealed that both JNK and SMase pathways were activated in OE-Afrip3 strain, by which an increase of reactive oxygen species (ROS) was induced. We also showed that expression of Afrip3 gene was induced by Ca2+. In addition, eEF1Bγ and adenylylsulfate kinase (ASK) were identified as potential candidates to interact with AfRip3. These results indicate that AfRip3 is a key modulator that activates necroptotic process in A. fumigatus, which can be induced by Ca2+ and in turn activate JNK (c-Jun NH2-terminal kinase) and SMase (sphingomyelinase) pathway. Our findings suggest that necroptotic pathway in A. fumigatus is distinct from that in mammalian cell and may provide a new strategy for development of anti-fungal drug. Author summary Aspergillus fumigatus is a human fungal pathogen and causes invasive aspergillosis (IA) in immunocompromised patients with high mortality (30-95%). Development of novel therapies is urgently needed. In this study, we confirm AfRip3 (AFUA_6G02590), a RIP3-like protein, is a key modulator that activates necroptotic process in A. fumigatus. We also find that cytosolic Ca2+ can induce the expression of Afrip3 and activated AfRip3 in turn activate JNK (c-Jun NH2-terminal kinase) and SMase (sphingomyelinase) pathway. Our findings suggest that necroptotic pathway in A. fumigatus is distinct from that in mammalian cell and may provide a new strategy for development of anti-fungal drug.

4 78 mechanism of RIP1-independent necroptosis remains unclear. 79 Aspergillus fumigatus is a human fungal pathogen capable of causing infections 80 ranging from allergic to invasive disease [17], and the major cause of invasive 81 aspergillosis (IA) in immunocompromised patients [18]. In these patients, the crude 82 mortality is 30-95%. Despite some effective drug treatments, mortality from fungal 83 infections remains about 50%, and new drugs are urgently needed due to the 84 inefficacy, side effects and resistance that have emerged as important factors limiting 85 successful clinical outcome [19][20][21][22][23]. A major barrier for the development of novel 86 therapies is the general lack of capacity in fungal pathogen research [24]. 87 Although PCD has already been discovered from bacteria to animals [25][26][27][28], in 88 contrast to that in mammalian cells, little is known about death process in filamentous 89 fungi. In filamentous fungi autolysis is a highly regulated and natural process that 90 occurs later in older, stationary phase cultures and leads to the progressive 91 disintegration of the mycelium. In A. fumigatus, it has been revealed that autolysis 92 during the stationary phase is an apoptotic process and caspase-dependent [29]. 93 However, autophagic and necroptotic process in A. fumigatus remain unclear. 94 Previously, we have shown that suppression of GPI anchor synthesis leads to both revealed that the expression of Afrip3 in OE-Afrip3 strain was 3.8 times of that in the 125 WT, indicating that OE-Afrip3 strain was successfully constructed (Fig 1B). 126 After 24 h of incubation, both WT and OE-Afrip3 strain reached their log-phase and 127 mycelia were stained with propidium iodide (PI), a dye that labels the nucleus in an elevated activity of CasA was induced ( Fig 3A) and exposure of PtdSer was 142 detected (Fig 3B), indicating an occurrence of dexamethasone-induced apoptosis in A. 143 fumigatus. However, as compared with that in WT, the CasA activity in OE-Afrip3 144 strain was declined by 39.3% ( Fig 3A)   no cell death was induced by TSZ (Fig 7). Taken together, our results confirm that    191 During autophagic process, the Vps34-Atg6/beclin1 class Ⅲ phosphoinositide  [15,30]. In this study, we also tested the expression 207 of these genes in OE-Afrip3 strain. As summarized in Table 1, Nfr1/AIF, 208 cyclophilins, JNK1, Hsp70 family proteins, glyoxalase family proteins and Rab7 were 209 induced (Table 1), which is consistent with that in the afpig-a conditional mutant [30]. 210 On the other hand, although Ca 2+ was able to induce an elevated expression of the 211 genes encoding PYGL, GLUL, GLUD1, JNK1 and SMase in the wild-type A. 212 fumigatus (Fig 8C), PYGL, GLUL, GLUD1, PKA, and Rab7 were suppressed in 213 OE-Afrip3 strain (Table 1). 214 In mammalian cells, the c-Jun NH 2 -terminal kinase (JNK) is activated by TNF and

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In attempt to identify the downstream target of AfRip3, pull-down assay was carried 232 out by using GST-AfRip3 protein expressed in A. fumigatus (Fig 10A). A 25 kDa 233 protein band was detected on SDS-PAGE ( Fig 10B) and analyzed by mass spectrum.

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As a result, 23 proteins were identified (S1 Table). Among these proteins, eEF1Bγ 235 and adenylylsulfate kinase (ASK) were confirmed to interact with AfRip3 by co-IP 236 with anti-His-tag mAb-Magnetic Beads ( Fig 10C).  In the meantime, the autophagy pathway initiated by a Ca 2+ -mediated mechanism in 273 some types of cell was also unraveled [53][54]. Also, it is reported that in yeast cell  fumigatus ASK plays a role as MET14 does in yeast, which might be the way that 296 AfRip3 passes the necroptotic signal to its downstream. Somehow, further 297 investigation needs to be carried out.

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In summary, in this study we confirmed that AfRip3 was a necroptotic regulator in A.        10-20 mg/mL of 2,5-dihydroxybenzoic acid (DHB) was taken as a dot-like matrix.

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The acceleration voltage was set to 2 kV. For each strain a triplicate was tested.              Then the membrane was washed three times with TBST buffer and incubated with an 666 AP-conjugated secondary antibody for 1 h at room temperature. After washing three 667 times with TBST buffer, bands were detected with NBT/BCIP reagent.    Table.