Biology subjects

Varet, H.

Publications and source records attributed to Varet, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Integrin beta 1 and mannose receptor 2 are involved in the antifungal activity of bronchial epithelial cells through Aspergillus fumigatus lectin FleA interactions

Aspergillus fumigatus is a world-wide saprophyte filamentous fungus which released conidia, its infectious morphotype, in the atmosphere. These conidia are inhaled daily by humans and can colonize the respiratory tract, where they may develop into hyphae, the invasive morphotype. We previously showed that bronchial epithelial cells (BECs) restrict A. fumigatus virulence by inhibiting conidial germination and filament formation through a process requiring PI3K signaling and the conidial fucose-specific lectin FleA. In the present study, we are looking to identify host factors and cellular partners involved in the BEC antifungal response and to define the molecular interactions underpinning FleA recognition. For this, we analyzed transcriptome of BECs infected with A. fumigatus in the presence or absence of the PI3K inhibitor LY294002. Functional involvement of candidate genes was assessed by siRNA knockdown and readouts of fungal filamentation (microscopic scoring and galactomannan release). FleA-interacting host proteins were identified by biotin-FleA affinity co-precipitation coupled to Tandem mass spectrometry, and validated by surface plasmon resonance and biolayer interferometry. The spatiotemporal dynamics of FleA and candidate partners were analyzed by confocal microscopy and proximity ligation assay We demonstrated that BEC antifungal activity involves at least two complementary pathways: a PI3K/laminin-332 axis promoting conidial adhesion, and a FleA-dependent pathway engaging ITGB1 and MRC2 consistent with lectin uptake and trafficking toward LAMP1-positive compartments. These findings nominate FleA-host receptor interactions as attractive targets for anti-adhesive strategies against A. fumigatus. Author summaryFungal pathogens are an increasing threat to public health, as they are becoming more common and harder to treat due to rising drug resistance. Among them, Aspergillus fumigatus has been classified as a critical pathogen by the World Health Organization (WHO). This filamentous fungus delivers spores in the air daily, which are constantly inhaled by humans. In people with weakened immunity, these spores can cause a range of lung diseases known as aspergillosis, with severity ranging from mild to life-threatening. Lung epithelial cells are the first cells of the respiratory tract to encounter inhaled spores. In a previous study, we showed that bronchial cells can prevent spore from developing into filaments, the invasive form of A. fumigatus that is responsible for tissue damage. This protective effect depends of on the recognition of a fungal protein called FleA. In the present study, we identified host cell proteins that bind to FleA and transport it into intracellular compartments. Our findings suggest that these proteins help bronchial epithelial cells to internalize fungal spores, thereby blocking their transformation into the invasive filamentous form.

cell biology↗

bioRxiv2025

RABV L protein plays a role in immune escape through its methyltransferase activity

Viruses in the Mononegavirales order encode a large protein that orchestrates replication, transcription, and the capping of viral RNA. This protein, comprising over 2.000 amino acids, contains an RNA-dependent RNA polymerase, a capping domain, and a methyltransferase (MTase) domain involved in methylating the cap structure. The MTase domain features a conserved K-D-K-E catalytic tetrad -typical of 2'O-methyltransferases-which is essential for methylating viral mRNA caps at both the N7 and 2'O positions. However, the role of these residues in other epitranscriptomic modifications of rabies virus (RABV) RNAs remains poorly characterized. To further explore the role of mRNA cap methylation in the immune evasion strategies of RABV, we investigated the functional contribution of the K-D-K-E motif within the MTase domain, using the Thai isolate as a model. Using reverse genetics, we demonstrated that the mutation K1830R in the K-D-K-E tetrad of the Tha MTase domain induces changes in the methylation landscape of viral mRNAs and, intriguingly, of host mRNAs. In addition, viruses harbouring the K1830R mutation are more sensitive to interferon- and exhibit a less pathogenic phenotype in vitro and in vivo compared to the wild-type virus. Overall, these results suggest that the regulation of viral and cellular RNA methylation landscapes plays a crucial role in controlling RABV infection. Although the exact role of these epitranscriptomic modifications is not yet fully understood, some of these methylations appear to have proviral effects and enhance viral propagation by allowing RABV to efficiently evade the hosts antiviral response. ImportanceThis study highlights the pivotal role of the K-D-K-E catalytic domain included in the methyltransferase domain of the large protein of Rabies virus, by modelling viral RNAs with epitranscriptomic changes. For the first time, we identify specific methylations on the viral RNA, such as 2-O and m6A methylations, which seem to enable the virus to mask its RNA and evade detection by the hosts pattern recognition receptors. These epitranscriptomic modifications affect not only viral RNAs but also cellular RNAs, underscoring a complex interplay between viral and host mechanisms. We further demonstrate that RABV harbouring an altered K-D-R-E catalytic domain, exhibit differential methylation patterns correlated with increased sensitivity to IFN and lower pathogenicity. This emphasizes the importance of this domain in virulence and immune evasion.

microbiology↗

bioRxiv2025

Serotype swapping in Klebsiella spp. by plug-and-play

Understanding how complex, multi-gene systems evolve and function across genetic backgrounds is a central question in molecular evolution. While such systems often impose costs through epistatic interactions, some may behave as modular, "plug-and-play" units that retain function with minimal disruption. We used the polysaccharide capsule locus of Klebsiella pneumoniae, a highly exchangeable and fast-evolving locus, as a model. We genetically engineered capsule exchanges (swaps) across diverse genetic backgrounds and combined transcriptomics, fitness assays, and evolution experiments, to show that capsule exchange has negligible effects on global expression and only marginal fitness costs, regardless of serotype. Adaptation to capsule-costly environments consistently reduced capsule production, regardless of serotype, revealing shared adaptive trajectories rather than serotype-specific pathways. Moreover, serotype-specific traits involved in bacterial virulence, such as biofilm formation and hypermucoviscosity, were conserved across genetic backgrounds. This reveals that capsule swapping can directly shape host-pathogen interactions and influence within-patient evolution. Our findings provide strong evidence that capsule loci display plug-and-play dynamics: they are transferable, functional across contexts, and minimally disruptive to the host genome. This allows capsules to be seamlessly swapped, and help explain the evolutionary success, ecological versatility, and pervasive exchangeability of capsules in K. pneumoniae.

evolutionary biology↗

bioRxiv2025

Intratumoral Heterogeneity of Vimentin Modulates Nuclear Mechanotransduction, DNA Damage Response and Cancer Cell Survival

Vimentin, a major intermediate filament protein, is essential for maintaining cellular integrity and regulating cytoskeletal dynamics. Its upregulation is a hallmark of epithelial-to-mesenchymal transition (EMT), a process that enhances cancer cell migration, invasion, and metastatic potential. However, single-cell transcriptomic analyses of glioblastoma, the most common and aggressive primary brain tumor, reveal that vimentin expression exhibits significant intratumoral heterogeneity, reflecting diverse cellular subpopulations that may contribute to tumor plasticity, therapy resistance, and disease progression. Here, we show that the absence of vimentin alters nuclear mechanotransduction in response to compression, leading to chromatin remodelling and profound changes in gene expression in cancer cells. Remarkably, we demonstrate that external compressive forces, akin to vimentin deficiency, disrupt DNA damage response pathways. This impairment compromises DNA damage sensing and repair, bypassing DNA damage checkpoints and apoptosis. Consequently, vimentin-negative tumor cells exhibit increased survival in response to physical stress and DNA damage, potentially driving radioresistance and further amplifying intratumoral heterogeneity.

cancer biology↗

bioRxiv2025

Mapping regulatory networks underlying Leishmania stage differentiation reveals an essential role for protein degradation in parasite development

Vector-borne, protist parasites have evolved complex developmental programs to adapt to very distinct host environments. How these important pathogens transition between insect and mammalian stages is only poorly understood. Here we investigate stage differentiation in the trypanosomatid parasite Leishmania that shows constitutive gene transcription, thus providing a unique model system to assess how development is governed by post-transcriptional mechanisms. Using a five-layer integrative systems analysis (from genome to metabolome), we examined hamster-isolated Leishmania donovani amastigotes and culture-derived, insect-stage promastigotes. This approach enabled us to rule out genomic adaptation as a key driver of parasite stage differentiation, while confirming the pivotal role of differential mRNA turnover in stage-specific gene expression. Assessing transcriptomic against proteomic expression changes uncovered an unexpectedly broad dynamic range of stage-regulated changes in protein abundance that only poorly correlated with mRNA levels. This discrepancy correlated with (i) alterations in snoRNA expression and changes in rRNA modification they guide suggesting stage-specific adaptation of the protein translation apparatus that can uncouple mRNA from protein abundancies, and (ii) differential protein degradation as revealed by quantitative proteomics of parasites treated with the proteasomal inhibitor lactacystin. Lactacystin treatment stalled the transition of spleen-derived amastigotes into promastigotes in culture, further underscoring the role of proteasomal activity in stage differentiation. Integration of our five-layer systems analysis established the first link between Leishmania development and the expression of co-regulated genetic networks encompassing mRNA turnover, protein translation, phosphorylation, and degradation. Our findings provide a powerful new resource for research programs that aim to dissect the emergent properties of regulatory networks and feedback loops underlying Leishmania stage differentiation, serving as a blueprint for other vector-borne pathogens that rely on disease-associated developmental transitions.

systems biology↗

bioRxiv2025

Impaired stem cell migration and divisions in Duchenne Muscular Dystrophy revealed by live imaging

Dysregulation of stem cell properties is a hallmark of many pathologies, but the dynamic behaviour of stem cells in their microenvironment during disease progression remains poorly understood. Using the mdx mouse model of Duchenne Muscular Dystrophy, we developed innovative live-imaging of muscle stem cells (MuSCs) in vivo, and ex vivo on isolated myofibres. We show that mdx MuSCs have impaired migration and precocious differentiation through unbalanced symmetric divisions, driven by p38 and PI3K signalling pathways, in contrast to the p38-only dependence of healthy MuSCs. Cross-grafting shows that MuSC fate decisions are governed by intrinsic cues, whereas their migration behaviour is determined by the extracellular niche. This study provides the first dynamic analysis of dystrophic MuSC properties in vivo, reconciling conflicting reports on their function. Our findings establish DMD as a MuSC disease with intrinsic defects and niche dysfunction, offering strategies to restore stem cell functions for improved muscle regeneration.

cell biology↗

bioRxiv2025

Dengue virus susceptibility in Aedes aegypti linked to natural cytochrome P450 variants

The mosquito Aedes aegypti is the primary vector for dengue virus (DENV), which infects millions of people annually. Variability in DENV susceptibility among wild Ae. aegypti populations is governed by genetic factors, but specific causal variants are unknown. Here, we identify a cytochrome P450-encoding gene (CYP4G15) whose variants drive differences in DENV susceptibility in a natural Ae. aegypti population. CYP4G15 is transiently upregulated in DENV-resistant midguts, while knockdown increases susceptibility, and transgenic overexpression enhances resistance. A naturally occurring 18-base-pair promoter deletion reduces CYP4G15 expression and confers higher DENV susceptibility. The unexpected role of a cytochrome P450 in DENV susceptibility challenges the long-standing focus on canonical immune pathways and opens new avenues for understanding antiviral defense and DENV transmission in mosquitoes.

microbiology↗

bioRxiv2025

Evaluation of the role of whiB6 and kdpDE in the dominant multidrug resistant clone Mycobacterium tuberculosis B0/W148

Multidrug resistant (MDR) strains of Mycobacterium tuberculosis represent an obstacle to eradicating tuberculosis (TB) due to the low treatment success rate of MDR TB. Among them, the MDR B0/W148 clone has recently evolved from the M. tuberculosis Beijing lineage 2 and widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of the MDR M. tuberculosis B0/W148 clone in addition to environmental and patient-related features, we focused on two mutations specific from this clone that are found in the transcriptional regulators WhiB6 and KdpDE and investigated in a H37Rv strain background the transcriptional profile associated with these mutations and their impact on the in vitro and in vivo growth characteristics. Through the construction and use of H37Rv{Delta}whiB6, H37Rv{Delta}kdpDE, and complemented strains, we found that both mutations did not impair the in vitro growth of M. tuberculosis in standard mycobacterial growth media. The mutation T51P in whiB6 prevented the upregulation of 9 genes in the esx-1 core region and 44 genes elsewhere in the genome, while the deletion of two nucleotides in kdpD leads to a fusion protein of KdpD with KdpE that inhibits the transcriptional activity of KdpE. Both mutations did not lead to hypervirulence in a mouse infection model. These results point to the role of other MDR B0/W148 specific mutations in the wide geographic diffusion of this clone, and/or put in question a hypothesized hypervirulence as driving factor for this large dissemination. ImportanceHuman tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, remains a global public health issue estimated to have been responsible for 1.25 million deaths in 2023. Multidrug resistant (MDR) strains of M. tuberculosis, resistant to rifampicin and isoniazid, lead to lower treatment success. Among them, the MDR B0/W148 clone has widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of this clone, we investigated two strain-specific mutations found in the transcriptional regulators WhiB6 and KdpDE. By constructing and analysing laboratory M. tuberculosis strains carrying these specific mutations, we found numerous changes in their transcriptional profiles, whereas we observed only little impact of these mutations on virulence of M. tuberculosis in a mouse infection model. Our study provides new insights into the transcriptional landscape of the selected MDR strains, although no direct connection to virulence could be established.

microbiology↗

bioRxiv2024

Limited impact of the siRNA pathway on transposable element expression in Aedes aegypti

Transposable elements (TEs) are DNA sequences that can change their position within a genome. In the germline of arthropods, post-transcriptional regulation of TE expression is mainly mediated by the Piwi-interacting RNA (piRNA) pathway. piRNAs are small RNAs of 24-30 nucleotides (nt) in length produced from genomic precursor transcripts as well as through a ping-pong amplification cycle. In somatic tissues, certain insects, such as Drosophila, instead rely on the small interfering RNA (siRNA) pathway as a key regulator of TE expression. siRNAs are 21nt small RNAs produced from double-stranded RNA by the endonuclease Dicer2, which guides an RNA-induced silencing complex to degrade a complementary RNA. However, whether the siRNA pathway also regulates TE expression in the mosquito Aedes aegypti, a medically significant vector species with abundant somatic piRNAs, is unknown. To address this question, we investigated the expression of TEs and small RNAs in both somatic and gonadal tissues of a Dicer2 mutant line of Ae. aegypti and its wild-type counterpart. Our results show a modified pattern of TE expression and a decrease in TE-derived 21nt small RNAs in the Dicer2 mutant, but no major shift of TE transcript abundance. The lack of a functional siRNA pathway also causes perturbations in piRNA ping-pong signatures and the expression of certain piRNA-associated genes, but without clear evidence for compensation by increased piRNA pathway activity. We conclude that the mosquito Ae. aegypti produces siRNAs targeting TEs but these lack a critical role in the regulation of TE expression both in somatic and in gonadal tissues.

genomics↗

bioRxiv2024

A Vago-like gene enhances dengue and Zika virus dissemination in Aedes aegypti

Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control the spread of arboviruses focus on harnessing the antiviral immune system of their primary vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. We conducted a phylogenetic analysis of the Vago gene family in Diptera, which led us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti, which revealed a broad impact of VLG-1 on the mosquito transcriptome, affecting several biological processes potentially related to viral replication, including the oxidative stress response. Surprisingly, experimental viral challenge of the VLG-1 mutant line indicated a modest proviral role for this gene during DENV and ZIKV infections in vivo. In the absence of VLG-1, virus dissemination throughout the mosquitos body was slightly impaired, albeit not altering virus transmission rates. Our results challenge the conventional understanding of Vago-like genes as antiviral factors and underscore the need for further in vivo research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.

immunology↗

bioRxiv2024

MAPPING CHANGES OF MIRNA-MRNA NETWORKS IN LEISHMANIA-INFECTED MACROPHAGES PREDICTS REGULATORY MIRNA-TF LOOPS AS NOVEL TARGETS OF PARASITE IMMUNE SUBVERSION

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and play a crucial role in numerous disease processes, including infections. Although intracellular microbial pathogens are known to modulate host cell gene expression to establish permissive conditions for infection, the specific role of host-encoded miRNAs underlying such subversion remains poorly understood. In this study, we employed the protozoan parasite Leishmania amazonensis as a model system to investigate how infection of macrophages modifies the host cell miRNA profile to evade antimicrobial functions and to establish permissive conditions for intracellular proliferation. Dual RNA-seq analyses using matched mRNA and miRNA-enriched samples from uninfected and L. amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed 102 differentially expressed miRNAs (padj<0.05), with 18 miRNAs showing reduced and 84 miRNAs showing increased abundance in infected BMDMs. Mapping putative networks of miRNA-mRNA interactions based on the observed expression changes, combined with Gene Ontology enrichment analyses, allowed us to identify potential miRNA target genes involved in key biological processes and metabolic pathways that permit parasite intracellular survival and proliferation. Our analyses predict the existence of a large miRNA-mRNA network affecting the expression level of numerous transcription factors that indicates inhibition of the NF-{kappa}B-dependent inflammatory response or the promotion of cholesterol biosynthesis during infection. In particular, the over 10e3-fold increase in the abundance of mmu-miR-686 in infected BMDMs was correlated with a reduced abundance of putative target transcripts implicated in miRNA biogenesis itself, in RNA binding, and in regulation of apoptosis, such as Caspase 12, the mRNA decay activator protein Zfp36l1 or Leukemia Inhibitory Factor Receptor Alpha. Likewise, the over 200-fold increase in abundance of mmu-miR-6546-3p was associated with a reduced abundance of putative target mRNAs implicated in cytokine-mediated signaling, positive regulation of apoptotic process and regulation of gene expression, affecting, for example, the MADS box transcription enhancer factor 2, the transformation related protein 53 inducible nuclear protein 1, or the G protein-coupled receptor 35. Interestingly, both miRNAs are predicted to simultaneously target 32 mRNAs that showed reduced abundance in infected BMDMs, including Maturin Neural Progenitor Differentiation Regulator (Mturn), a regulator of NF-{kappa}B transcription factor activity. In conclusion, our approach provides novel insight into molecular mechanisms that may govern macrophage subversion and intracellular Leishmania survival. Our results shed new light on the complex relationship among miRNAs, macrophage gene expression and Leishmania infection, proposing regulatory feed-forward loops (FFLs) and feedback loops (FBLs) between miRNAs and TFs as a novel target of Leishmania immune subversion. These findings open exciting new avenues for the development of intervention strategies aimed at disrupting such crucial interactions, for example using an anti-miR (antagomir) approach against mmu-miR-686 and mmu-miR-6546-3p.

microbiology↗

bioRxiv2023

Inhibition of the macrophage demethylase LSD1 reverses Leishmania amazonensis-induced transcriptomic changes and causes a decrease in parasite load

Intracellular pathogens exploit host cell functions to favor their own survival. In recent years, the subversion of epigenetic regulation has emerged as a key microbial strategy to modify host cell gene expression and evade antimicrobial immune responses. Using the protozoan parasite Leishmania as a model system, we have recently demonstrated that infection causes histone H3 hypomethylation, which is associated with the establishment of an anti-inflammatory phenotype, suggesting that host cell demethylases may play a role in the intracellular survival of these parasites. In this study, we combined pharmacological inhibition with RNA sequencing and quantitative immune-precipitation analysis to investigate the role of the macrophage lysine demethylase LSD1 (KDM1a) in Leishmania intracellular infection in vitro. Treatment of infected macrophages with validated, LSD1-specific inhibitors resulted in a significant reduction in parasite burden. We confirmed the impact of these inhibitors on LSD1 activity within macrophage nuclear extracts using an in vitro demethylase assay and established their LSD1 target engagement in situ by cellular thermal shift assay. RNA-seq analysis of infected and inhibitor-treated macrophages linked parasite killing to a partial reversion of infection-dependent expression changes, restoring the macrophage anti-microbial response and limiting cholesterol biosynthesis. While we ruled out any impact of Leishmania on LSD1 expression or localization, we uncovered significant alterations in LSD1 complex formation within infected macrophages, involving unique interactions with host cell regulatory proteins such as Rcor-1. Our study sheds important new light on the epigenetic mechanisms of macrophage immuno-metabolic subversion by intracellular Leishmania and identifies LSD1 as a potential candidate for host-directed, anti-leishmanial therapy.

cell biology↗

bioRxiv2023

Prosaposin maintains adult neural stem cells in a state associated with deep quiescence

In most vertebrates, adult neural stem cells (NSCs) continuously give rise to neurons in discrete brain regions. A critical process for maintaining NSC pools over long periods of time in the adult brain is NSC quiescence, a reversible and tightly regulated state of cell cycle arrest. Recently, lysosomes were identified to regulate the NSC quiescence-proliferation balance. However, it remains controversial whether lysosomal activity promotes NSC proliferation or quiescence, and a finer influence of lysosomal activity on NSC quiescence duration or depth remains unexplored. Using RNA-sequencing and pharmacological manipulations, we show that lysosomes are necessary for NSC quiescence maintenance. Additionally, we reveal that expression of psap, encoding the lysosomal regulator Prosaposin, is enriched in quiescent NSCs (qNSCs) that reside upstream in the NSC lineage and display a deep/long quiescence phase in the adult zebrafish telencephalon. We show that shRNA-mediated psap knock-down increases the proportion of activated NSCs (aNSCs) as well as NSCs that reside in shallower quiescence states (signed by ascl1a and deltaA expression). Collectively, our results identify the lysosomal protein Psap as a (direct or indirect) quiescence regulator and unfold the interplay between lysosomal function and NSC quiescence heterogeneities.

neuroscience↗

bioRxiv2023

Transcriptome profiling of human colonic cells exposed to the gut pathobiont Streptococcus gallolyticus subsp. gallolyticus.

Streptococcus gallolyticus sp. gallolyticus (SGG) is a gut pathobiont involved in the development of colorectal cancer (CRC). To decipher the contribution of SGG in tumor initiation and/or acceleration respectively, a global transcriptome was performed in normal colonic cells (FHC) and in tumoral colonic cells (HT29). To identify SGG-specific alterations, we chose the phylogenetically closest relative, Streptococcus gallolyticus subsp. macedonicus (SGM) as the control bacterium. We show that SGM, a bacterium generally considered as safe, did not induce any transcriptional changes on the two human colonic cells. The transcriptional reprogramming induced by SGG was significantly different in FHC and HT29 cells, with most of the up- and down-regulated genes associated with cancer disease. Top up-regulated genes related to cancer were: (i) IL-20, CLK1, SORBS2, ERG1, PIM1, SNORD3A for normal FHC cells and (ii) TSLP, BHLHA15, LAMP3, ZNF27B, KRT17, ATF3 for cancerous HT29 cells. SGG induces much stronger transcriptional changes in cancerous than in normal colonic cells (2,090 vs 128 genes being affected, respectively). Gene set enrichment analysis reveals that SGG-induced strong ER- (endoplasmic reticulum) stress and UPR- (unfolded protein response) activation in colonic epithelial cells. Our results suggest that SGG induces a pro-tumoral shift in human colonic cells, particularly in transformed cells potentially accelerating tumor development in the colon.

microbiology↗

bioRxiv2022

Leishmania amazonensis controls macrophage-regulated cell death to establish chronic infection in vitro and in vivo

Pathogenic protists of the genus Leishmania have evolved various strategies to exploit macrophages as host cells and subvert their immuno-metabolic functions to favour intracellular parasite survival. Surprisingly little is known on how Leishmania affects regulated cell death (RCD) pathways of its host cell, even though increased survival of in vitro infected macrophages has been reported, and chronic macrophage infection in vivo causes the devastating immunopathologies of leishmaniasis. To overcome this limitation and gain first systems-level insight into the interaction between intracellular Leishmania and the host cell RCD pathways, including apoptosis, pyroptosis and necroptosis, we applied transcriptomic analyses on L. amazonensis-infected, primary macrophages (termed LIMs) and used YO-PRO-1 to monitor cell death by fluorescent microscopy. RNAseq analyses at day 3 post-infection (PI) revealed dichotomic dysregulation of more than 60% of RCD-related genes in LIMs, characterized by up-regulation of anti-RCD and down-regulation of pro-RCD markers, including key regulators common to the three forms of cell death such as casp8, fadd, tradd, tnfaip3, tax1bp1, birc3, and itch. This profile correlated with expression changes of transcription factors known to regulate RCD, including AP1 and NF-{kappa}B family members, ppar{gamma} and cebp{beta}. Consequently, LIMs showed remarkable longevity in culture for at least 50 days, despite a constant increase of parasite burden to about 100 parasites per cell, while non-infected cells were cleared from the culture in just a few days. Longitudinal expression analysis of LIMs at days 0, 3, 15, and 30 PI by RT-qPCR confirmed stable maintenance of this high longevity profile with the dichotomic decrease and increase of RCD-activators and -inhibitors, respectively. LIMs further showed significant resistance to RCD-inducing signals compared to non-infected cells, including CSF-1 deprivation (intrinsic apoptosis), actinomycin D treatment (extrinsic apoptosis), LPS/ATP stimulation (pyroptosis). Significantly, we extended the anti-RCD expression pattern and RCD resistance phenotype to L. amazonensis-infected macrophages recovered from lesions, thus validating our long-term in vitro infection system as an easily accessible model to study chronic macrophage infection. In conclusion, our analyses firmly document the pan-anti RCD effect of L. amazonensis on its macrophage host cell in vitro and in vivo and shed important new light on mechanisms underlying Leishmania chronic infection.

microbiology↗

bioRxiv2022

Immunometabolic profiling of in vitro and ex vivo Leishmania-infected macrophages (LIMs) reveals unique polarization and bioenergetic signatures

Macrophages are the major host cells of the protozoan parasite Leishmania in mammalian infection. These key innate immune cells display remarkable phenotypic plasticity ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages that can control infection and tissue homeostasis, respectively. It has been recognized that Leishmania exploits macrophage phenotypic plasticity to establish chronic infection. However, the current notion that these parasites simply trigger an M2-like phenotype seems over-simplified considering the immunopathology observed during leishmaniasis - in particular in response to Leishmania amazonensis - which is often characterized by a mixed Th1/Th2 immune response. Here we combined a series of systems-level analyses to shed new light on the phenotype of Leishmania-infected macrophages (LIMs) during short- and long-term infection, in vitro and in vivo. Immuno-metabolic profiling by RNA-seq, RT-qPCR, cytokine immunoassays, and real-time bioenergetic flux analysis of L. amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed a highly complex and unique phenotypic and bioenergetic signature. In vitro LIMs were characterized by co-expression of both M1 and M2 markers at RNA and protein levels and increased expression of glycolytic genes that matched a progressive metabolic switch from a M2-like respiratory to a M1-like glycolytic energy production observed for both long-term in vitro and in vivo infected macrophages. Unlike in M1 macrophages, glycolytic gene expression did not correlate with increased expression of its key regulatory HIF-1. In contrast, siRNA knock down experiments in primary BMDMs uncovered an essential role of the m6A reader protein IGF2BP2 in stabilizing m6A modified transcripts of the glycolytic pathway, contributing to HIF-1-independent induction of glycolysis. In conclusion, L. amazonensis establishes a complex and unique phenotypic shift in infected macrophages in vitro and in vivo that combines M1-like and M2-like immuno-metabolomic characteristics and implicates differential mRNA stability in induction of aerobic glycolysis. Our data thus uncover epi-transcriptomic regulation as a novel target for Leishmania immune subversion to establish a host cell phenotype beneficial for intracellular parasite development and chronic infection.

immunology↗

bioRxiv2022

Recognition of copy-back defective interfering rabies virus genomes by RIG-I triggers the antiviral response against vaccine strains

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year around the world. A typical feature of RABV infection is the suppression of type I and III interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) to initiate IFN signaling remain elusive. Here, we showed that RABV RNAs are recognized by RIG-I (retinoic acid-inducible gene I) sensor resulting in an IFN response of the infected cells but that this global feature was differently modulated according to the type of RABV used. RNAs from pathogenic RABV strain, THA, were poorly detected in the cytosol by RIG-I and therefore mediated a weak antiviral response. On the opposite, we revealed a strong interferon activity triggered by the RNAs of the attenuated RABV vaccine SAD strain mediated by RIG-I. Using next-generation sequencing (NGS) combined with bioinformatics tools, we characterized two major 5copy-back defective interfering (5cb DI) genomes generated during SAD replication. Furthermore, we identified a specific interaction of 5cb DI genomes and RIG-I that correlated with a high stimulation of the type I IFN signaling. This study indicates that RNAs from a wild-type RABV poorly activate the RIG-I pathway, while the presence of 5cb DIs in vaccine SAD strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection and therefore strongly stimulates the IFN response. HighlightsO_LIRABV pathogenic strain replication in vitro is characterized by the absence of defective interfering genomes thus induces a weak RLR-mediated innate immunity antiviral response. C_LIO_LIRABV vaccine attenuated strain shows a high release of 5 copy-back defective interfering genomes during replication in vitro and therefore enhances a strong antiviral response upon infection. C_LIO_LIRIG-I is the main sensor for RABV RNA detection within cells. C_LI

microbiology↗

bioRxiv2021

Mosquito-bacteria interactions during larval development trigger metabolic changes with carry-over effects on adult fitness

In animals with distinct life stages such as holometabolous insects, adult phenotypic variation is often shaped by the environment of immature stages, including their interactions with microbes colonizing larval habitats. Such carry-over effects were previously observed for several adult traits of the mosquito Aedes aegypti after larval exposure to different bacteria, but the mechanistic underpinnings are unknown. Here, we investigated the molecular changes triggered by gnotobiotic larval exposure to different bacteria in Ae. aegypti. We initially screened a panel of 16 bacterial isolates from natural mosquito breeding sites to determine their ability to influence adult life-history traits. We subsequently focused on four bacterial isolates (belonging to Flavobacterium, Lysobacter, Paenibacillus, and Enterobacteriaceae) with significant carry-over effects on adult survival and found that they were associated with distinct transcriptomic profiles throughout mosquito development. Moreover, we detected carry-over effects at the level of gene expression for the Flavobacterium and Paenibacillus isolates. The most prominent transcriptomic changes in gnotobiotic larvae reflected a profound remodeling of lipid metabolism, which translated into phenotypic differences in lipid storage and starvation resistance at the adult stage. Together, our findings indicate that larval exposure to environmental bacteria trigger substantial physiological changes that impact adult fitness, uncovering a mechanism underlying carry-over effects of mosquito-bacteria interactions during larval development.

microbiology↗