Novel Fungal Lipids AMinoacylation Systems – N-FLAMS

For the subcellular localization of ErdS and ErdH proteins, two approaches are used. The first is to construct a recombinant Afm strain expressing a GFP-tagged ErdS and a mCherry-taged ErdH (GFP-erdS-mCherry-erdH). The second approach and fallback approach will to use a polyclonal antibody directed against the ErdS protein and immunofluorescence microscopy on fixed cells. These two approaches will allow us to detect and localize ErdS at different stages of the development of the fungus. These complementary approaches will also allow us to check whether ErdS co-localizes with components of the fungal cell wall and/or membranes (b-glucan, chitin, Erg) using compartment-specific markers. This will also give us an indication of the role of Erg-Asp in the development of the fungus. Transcriptional changes induced by the absence of Erg-Asp will be studied by analyzing the transcriptome of WT, ?erdS and clinical strains under standard growth conditions. For this, cultures of the different strains are performed on sterile cellophane membranes laid on a rich agar medium, then the cultures are collected after 72 hours and the total RNA extracted (TRIzol® method). The samples (technical and biological triplicates) are then analyzed by the IGBMC RNA-seq platform (Illumina HiSeq 4000 sequencer). The role of Erg-Asp in the immune response and resistance to antifungal agents will also be studied. Indeed, Erg is the main target of currently available antifungal drugs. We hypothesized that Erg-Asp may play a role in the susceptibility of Afm to antifungal agents used in medical treatments. In order to test this hypothesis, susceptibility tests to antifungal drugs used in hospitals such as azoles and amphotericin B are performed with WT, ?erdS strains and clinical isolates. For this, a cell viability test with resazurin is used. We also want to show the possible involvement of Erg-Asp in the host immune response, including immune escape and virulence. For this purpose, phagocytosis and virulence of WT, ?erdS and clinical strains will be studied using respectively an ex vivo phagocytosis assay (mouse macrophages infected with the fungus) and an insect infection model Galleria mellonella. Infection kinetics and different doses of dormant or swollen spores are used. Phagocytosis is quantified after 3 hours and survival of larvae after infection is monitored over 24 and 48 hours to determine 90 and 50% lethal doses. Finally, production of cytokines and other molecules of the immune system will be measured.

We already obtained, using immunofluorescence microscopy, preliminary indications on ErdS intracellular localization at various developmental stages of Aspergillus fumigatus (Afm) : spores, tube germ and mycelium. The protein seems to be located in membrane compartments including the plasma membrane. This observation has to be confirmed by other approaches but are in agreement with Western Blots performed on extracts from yeast cells expressing ErdS where ErdS is mainly found in membrane fractions. In parallel, we constructed an Afm recombinant strain expressing GFP-erdS-mCherry-ErdH and obtained 50 clones on selective media that were not confirmed by PCR. A second attempt is currently attempted. In order to understand the Erg-Asp role in Afm, phenotypic analyses have been undertaken under various conditions (stress, nutrients deprivation, …). Preliminary results show i) a modification of membrane and cell wall components (chitin, Erg, ?-glucan,…), ii) a growth defect in presence of congo red stress and iii) a better resistance to osmotic stress for the ?erdS. These preliminary results suggest that Erg-Asp might be implicated in cell wall and membrane remodeling. In order to test if Erg-Asp is involved in transcriptional regulation of gene expression, an RNAseq approach has been started with WT, ?erdS and two clinical isolates at the IGBMC platform. To validate a role for Erg-Asp in antifungal drugs susceptibility, viability tests have been done. Our results suggest that Erg-Asp is not implicated in the latter process. A similar result has been obtained for clinical isolates, one having Erg-Asp and the other not. Fungal virulence tests using Galleria melonella have also shown that there is no impact on the survival of larvae infected with WT or ?erdS spores (presence or absence of Erg-Asp). However, first experiments using mouse macrophages and WT or ?erdS spores show a clear difference in the level of spore internalization by macrophages. ?erdS spores are less well internalized than WT ones, suggesting that Erg-Asp might be involved in the interaction between the fungus and the immune system.

In order to decipher the role of Erg-Asp in the physiopathology of Aspergillus fumigatus (Afm), we intend to pursue our analyses of the localization of ErdS/ErdH and Erg-Asp and to identify the interacting partner of ErdS/ErdH and Erg-Asp. To do so, we are currently engineering Afm recombinant strains expressing GFP-erdS-mCherry-erdH. Once we obtain this strain, immunofluorescence and pull-down/Co-IP experiments will be made to get insights on (i) the localization of ErdS/ErdH (confirmation or not of the immunofluorescence microscopy results with anti-ErdS antibodies); (ii) the different partner interacting with ErdS/ErdH by pull-down/Co-Ips followed by mass spectrometry analyses. We also intend to study the localization of ErdS/ErdH at various stages of Afm development by colocalization experiments with known components of the membrane or of the cell wall. In addition western blots on sub-cellularly fractionated Afm extracts treated or not with detergents will be made to confirm in which compartment(s) ErdS/ErdH are located. Once we get the results of the RNASeq transcriptional analyses, we’ll first confirm the results by RT-QPCR and Afm strains mutated in genes for which transcription is regulated by ErdS/erdH/Erg-Asp will be generated and subjected to an in-depth phenotypic screening. The development of the mass spectrometry analyses aiming at measuring Erg-Asp content in patient clinical samples will be pursued and is currently tested on lipids extracted from WT ?erdS and clinical strains. Our first results about the connection between Erg-Asp and Afm virulence do not seem to confirm such a connection. However, an in-depth analysis of the immune response of Galleria melonella against infection by Afm spores will be done by looking at infection kinetics, by varying the spore doses used to infect the larvae, by quantifying hemocytes (cells from the insect’s immune system) before and after infection, by measuring anti-microbial peptide production using mass spectrometry, etc. Finally, our preliminary results on the involvement of Erg-Asp in the host immune response suggest that the absence of Erg-Asp decreases internalization and phagocytosis of the spores by murine macrophages. We intend to undertake a thorough analysis of the host immune response upon challenge with WT, ?ErdS and clinical strain spores using murine macrophages. We will (i) analyze the 4 steps of phagocytosis (receptor-ligand binding, internalization, mature phagosome formation and spore remediation), (ii) quantify production of cytokines and (iii) production of reactive oxygen species.

Publications

Yokokawa, D., Tatematsu, S., Takagi, R., Sga, R., Roy, H., Fischer, F., Becker, H. D. & Kushiro, T. (2021) Synthesis of aminoacylated ergosterols: a new lipid component of fungi. Steroïds 169, 108823 (doi:10.1016/j.steroids.2021.108823)

Darnet S, Blary A, Chevalier Q, Schaller H. (2021) Phytosterol profiles, genomes and enzymes. Front. Plant. Sci. 12, 665206. (doi: 10.3389/fpls.2021.665206)

Yakobov N, Mahmoudi N, Grob G, Yokokawa D, Saga Y, Kushiro T, Worrell D, Roy H, Schaller H, Senger B, Huck L, Gascon GR, Becker H & Fischer F. (2022) RNA-dependent synthesis of ergosteryl-3ß-O-glycine in Ascomycota expands the diversity of steryl-amino acids. Journal of Biological Chemistry. 298(3):101657. (doi: 10.1016/j.jbc.2022.101657)

Grob G, Hemmerle M, Yakobov N, Mahmoudi N, Fischer F, Senger B, Becker HD. (2022) tRNA-dependent addition of amino acids to cell wall and membrane components. Biochimie. 30:S0300-9084(22)00253-X;(doi: 10.1016/j.biochi.2022.09.017)

Meeting and Symposia presentations

G. Grob – 16th GERLI meeting, Bordeaux, France, Deciphering ergosteryl-amino acid synthases’ evolutionary history (26-29/09/2021). Poster

Dr. N. Mahmoudi 16th GERLI meeting, Bordeaux, France, tRNA-dependent lipid modification by aminoacylation in Aspergillus fumigatus (26-29/09/2021). Poster.

N. Yakobov – Société de Biologie de Strasbourg, Strasbourg, France, oral presentation at the PhD prize ceremony: Discovery of tRNA-dependent ergosterol aminoacylation in fungi (22/04/2021).

N. Yakobov – GERLI 16th International Lipidomics Meeting, Bordeaux, France. oral presentation at the PhD prize ceremony: RNA-dependent synthesis of 3ß-O-aminoacylated sterols in Fungi (26-29/09/2021).

Pr. H.D. Becker – Polish Academy of Science, Institute of Biochemistry and Biophysics, Warsaw, Poland, Filamentous fungi use aminoacyl-tRNAs to reshape sterols (29/06/2021). External invited seminar.

Pr. H.D. Becker – 28th tRNA conference, Colombus, USA, Using aminoacyl-tRNA to conjugate cell wall components in microbes. Invited speaker (14/06/2022).

Pr. H.D. Becker – Yale Systems Biology Institute, New Haven, USA, RNA-dependent remodeling of cell wall components in microbes. External invited seminar (17/06/2022).

Prizes

Dr. N. Yakobov: PhD prize awarded by the Groupe d’étude et de recherche en lipidomique (GERLI) and by the Société de Biologie de Strasbourg (SBS) sponsored by the Eurométropole de Strasbourg) in 2021.

M. G. Grob: best poster prize awarded by GERLI during the 16th International Lipidomics Meeting (2021)

The JBC publication describing the discovery of a new aminoacylated ergosterol, ergosterol-glycine, and the enzyme responsible for its synthesis: Ergosteryl-glycine Synthase was selected as the Editor's pick of the journal's issue in which the study was published (2022).

Aspergillus fumigatus (Afm) is an opportunistic pathogenic fungus responsible for invasive aspergillosis whose mortality, despite treatment, remains very high in immunocompromised patients or patients with pulmonary pathologies and/or lesions. As with other fungal pathogens, the emergence of resistance to antifungal agents partly explains this finding, but also a lack of knowledge on the molecular aspects that trigger pathogenesis. In pathogenic bacteria, where these mechanisms have been well studied, it has been observed that a family of enzymes called MprF are able to divert amino acids from protein synthesis and transfer the amino acid moiety onto membrane lipids, glycerolipids (GLs); this GLs "aminoacylation" of GLs results in changes of the cell membrane properties, increasing both antibiotic resistance and immune system escape. This type of membrane modification has never before been described outside the bacterial branch of the tree of life.
We discovered that, in fact, the majority of higher fungi (Dikarya) have similar enzymes, which we called ErdS, capable of modifying membrane lipids with an amino acid, aspartic acid (Asp). We have shown that, unlike bacteria, fungi do not use Asp to modify GLs, but ergosterol (Erg), a lipid essential for membrane integrity, pathogenesis, virulence and resistance to antifungal agents. The ergosterol-aspartate (Erg-Asp) produced by ErdS enzymes is a new form of modified lipid, specific to fungi, that had never been detected before. The function of Erg-Asp, remains unknown, but it is produced by many pathogenic fungi, such as Afm. By analogy with bacteria, we assume that Erg-Asp could have a role similar to that of modified GLs in bacteria: antimicrobial resistance, virulence/pathogenesis. Our preliminary results suggest that Erg-Asp influences the growth of Afm, and that it would participate in many central molecular and cellular processes in fungi, such as sporulation (conidiation), autophagy, maintenance of cell wall integrity, sterol metabolism, etc. Erg-Asp could also be involved in a lipid signaling cascade leading to modulation of Afm virulence and antimicrobial susceptibility.
The objective of this research project is to decipher the role and function(s) of this new lipid, Erg-Asp, by studying its role in the physiology and virulence of Afm, including clinical strains from patients with aspergillosis, which we are using as a model. As Erg is involved in many antimicrobial resistance processes in fungi, we want to study the impact of Erg-Asp on Afm resistance to antifungal agents but also on its pathogenesis. Our work will also provide a better understanding of the general biology of fungi, including pathogens such as Afm and other health impacting opportunists. This could allow, in the longer term, the exploration of new therapeutic strategies.