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[Preprint]. 2023 May 30:rs.3.rs-2960526.
doi: 10.21203/rs.3.rs-2960526/v1.

Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target

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Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target

Norman van Rhijn et al. Res Sq. .

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Abstract

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

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Figures

Figure 1
Figure 1. Generation of the kinase knockout collection in A. fumigatus.
A) A phylogenetic tree (Maximum-likelihood) of the kinases found in Af293. The kinase family (first colour ring), the Kinomer score (barchart), the occurrence of each kinase within the pangenome of A. fumigatus (grey circle size), if a homokaryon null was constructed (green squares) and if the kinase is fungal specific (purple square) is shown as annotations around the phylogenetic tree. B) Four potential essential kinases were validated using a Tet-OFF construct to replace the native promoter. Shutting down expression via addition of doxycycline to the medium resulted in no growth after 72 hours at 37 Celsius, adding further evidence to the essentiality of these kinase for viability.
Figure 2
Figure 2. Bar-seq of A. fumigatus identifies YakA as a potential novel antifungal target.
A) Heatmap of fitness relative to the control condition for each kinase (n = 5). Fitness was calculated by DESeq2 on raw counts. Heatmap was generated by Pheatmap, full linkage clustering on rows and columns (cutree_cols=2). B) Dry weight was measured after overnight culture in HMM, with and without 0.5 mg/L itraconazole, at 37 Celsius shaking 130 rpm. Statistical difference was assessed by using one-way ANOVA. C) 1000 spores of MFIG001, ΔyakA and the reconstituted isolate (yakA+) were spot inoculated on HMM without iron and HMM with 1 μM iron added and incubated for 72 hours at 37 Celsius. D) Survival curves in a Galleria mellonella model of aspergillosis. Statistical significance was assessed by Kaplan–Meier survival analysis. E) Survival in a leukopenic murine model of aspergillosis. Statistical difference was assessed by Kaplan–Meier survival analysis.
Figure 3
Figure 3. In detail phenotypic analysis of ΔyakA.
A) 1000 spores of MFIG001, ΔyakA, yakA+ were spotted on HMM + 30 μM Fe and imaged after 72 hours at 37 Celsius. An increasing agarose concentration (0.5–6%) was used to make up the HMM medium. B) Representative histological sections of lesions found in murine lungs infected for 3 days with MFIG001, ΔyakAand yak+. H&E and GMS stains were performed on neighbouring sections. C) The hyphal length within histological sections (n = 15) was measured. Statistical difference was assessed by one-way ANOVA. D) Recovery from cytorrhysis after addition of glycerol to the medium. The % cytoplasm compared to the total cell size was measured for individual septal compartments.
Figure 4
Figure 4. Phosphoproteomics in response to iron limitation.
A) Heatmap of phosphopeptides upon iron limitation in MFIG001 and ΔyakA. K-means clustering was performed to identify clusters of phosphopeptides differentially phosphorylated dependent upon yakA or independent of yakA. B) GO-term analysis in Revigo of yakA-dependent phosphopeptides in response to iron limitation. Size of circles is the size of each enrichment category. Colour represents the p-value. C) A detailed analysis of phosphopeptides in components of the septal pore. Only phosphopeptides that are differentially regulated in at least one condition are shown (> 2-fold up or down).
Figure 5
Figure 5. Microscopic analysis of yakA localisation.
A) YakA-GFP localisation in control, iron limiting and conditions adding rapamycin. After overnight growth medium was shifted for 1 hour before imaging. Localisation to the septal pore can be observed. B) Quantification of signal through a cross-section of the septum shows higher fluorescent intensity in the central region of the septum, indicating septal pore localisation of YakA-GFP.
Figure 6
Figure 6. Microscopic characterisation of septal pore components, HexA and Lah.
A) Microscopy of single hyphae of HexA-GFP and LacC-GFP upon iron deleted conditions show yakA-dependent localisation to the septum or the septal pore. B) Quantification of fluorescent intensity measured across the septum for HexA-GFP and LahC-GFP in the parental isolate or upon deletion of yakA.
Figure 7
Figure 7. YakA is a druggable target in A. fumigatus.
A) An alphafold2 model of the YakA protein docked to a library of small molecules. Druggable pockets P1-P3 are shown. Pocket P1 is shown in further detail containing the 1-ECBC molecule. B) Microscopic evaluation of YakA-GFP upon iron limitation and addition of 1-ECBC. Hyphae were followed for one hour upon 1-ECBC addition. C) Checkerboard assay (n=3) for 1-ECBC and voriconazole to assess synergism of these drugs to inhibit growth of A. fumigatus MFIG001. The FICI (top-left) was calculated and shown synergism (<0.5).

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