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Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in linking desmosomal adhesion with epidermal differentiation.

Background: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell–cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. Methods: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell–cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVβ6 antibody. Results: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVβ6 and subsequent transforming growth factor–β signaling as driver of cardiac fibrosis. Blocking integrin-αVβ6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. Conclusions: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVβ6 and transforming growth factor–β signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ~30 amino acids. They are found in metazoans and plants where they localize to mitochondria or chloroplasts. In higher plants, the mTERF family comprises ~30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

Double-stranded RNA binding (DRB) proteins are generally considered as promoting cofactors of Dicer or Dicer-like (DCL) proteins that ensure efficient and precise production of small RNAs, the sequence-specificity guide of RNA silencing processes in both plants and animals. However, the characterization of a new clade of DRB proteins in Arabidopsis has recently challenged this view by showing that DRBs can also act as potent inhibitors of DCL processing. This is achieved through sequestration of a specific class of small RNA precursors, the endogenous inverted-repeat (endoIR) dsRNAs, thereby selectively preventing production of their associated small RNAs, the endoIR-siRNAs. Here, we concisely summarize the main findings obtained from the characterization of these new DRB proteins and discuss how the existence of such complexes can support a potential, yet still elusive, biological function of plant endoIR-siRNAs.

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-β). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis., Author summary Protozoan parasites of the genus Leishmania are the causative agents of leishmaniases, a group of diseases that range from cutaneous to potentially lethal visceral forms. Unfortunately, no efficient vaccine has been developed yet and drug resistance is rapidly increasing. Hence, the current situation reflects an urgent need for a better understanding of the molecular underpinnings of the interactions between Leishmania and its host in order to identify novel regulatory nodes for therapeutic intervention. During infectious diseases, fine-tuning the efficiency of mRNA translation into proteins allows cells to tailor their anti-microbial responses but can also be exploited by the invading pathogen. Using a global-scale translatome-based approach in macrophages, herein we report a profound perturbation in host mRNA translation during L. donovani infection. Our computational analyses reveal that subsets of host mRNAs encoding functionally related proteins share the same directionality of translational regulation, suggesting that key metabolic and microbicidal functions are hijacked by L. donovani via modulation of mRNA translation. We also show enrichment of reported mTOR- and eIF4A-sensitive mRNAs in the translationally activated dataset, including several immune-related transcripts. Finally, we report that in contrast to mTOR blockade, pharmacological inhibition of eIF4A hinders intramacrophage L. donovani survival. Thus, our study warrants further investigation on the potential of targeting eIF4A-dependent host mRNA translation to treat L. donovani infections.

CXCL16 is a multifunctional chemokine that is highly expressed by macrophages and other immune cells in response to bacterial and viral pathogens; however, little is known regarding the role of CXCL16 during parasitic infections. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. Even though chemokine production is a host defense mechanism during infection, subversion of the host chemokine system constitutes a survival strategy adopted by the parasite. Here, we report that L. donovani promastigotes upregulate CXCL16 synthesis and secretion by bone marrow-derived macrophages (BMDM). In contrast to wild-type parasites, a strain deficient in the virulence factor lipophosphoglycan (LPG) failed to induce CXCL16 production. Consistent with this, cell treatment with purified L. donovani LPG augmented CXCL16 expression and secretion. Notably, the ability of BMDM to promote migration of cells expressing CXCR6, the cognate receptor of CXCL16, was augmented upon L. donovani infection in a CXCL16- and LPG-dependent manner. Mechanistically, CXCL16 induction by L. donovani required the activity of AKT and the mechanistic target of rapamycin (mTOR) but was independent of Toll-like receptor signaling. Collectively, these data provide evidence that CXCL16 is part of the inflammatory response elicited by L. donovani LPG in vitro. Further investigation using CXCL16 knockout mice is required to determine whether this chemokine contributes to the pathogenesis of visceral leishmaniasis and to elucidate the underlying molecular mechanisms.

A Publisher Correction to this article was published on 26 February 2019; International audience; Leishmania are ancient eukaryotes that have retained the exosome pathway through evolution. Leishmania RNA virus 1 (LRV1)-infected Leishmania species are associated with a particularly aggressive mucocutaneous disease triggered in response to the double-stranded RNA (dsRNA) virus. However, it is unclear how LRV1 is exposed to the mammalian host cells. In higher eukaryotes, some viruses are known to utilize the host exosome pathway for their formation and cell-to-cell spread. As a result, exosomes derived from infected cells contain viral material or particles. Herein, we investigated whether LRV1 exploits the Leishmania exosome pathway to reach the extracellular environment. Biochemical and electron microscopy analyses of exosomes derived from LRV1-infected Leishmania revealed that most dsRNA LRV1 co-fractionated with exosomes, and that a portion of viral particles was surrounded by these vesicles. Transfer assays of LRV1-containing exosome preparations showed that a significant amount of parasites were rapidly and transiently infected by LRV1. Remarkably, these freshly infected parasites generated more severe lesions in mice than non-infected ones. Moreover, mice co-infected with parasites and LRV1-containing exosomes also developed a more severe disease. Overall, this work provides evidence that Leishmania exosomes function as viral envelopes, thereby facilitating LRV1 transmission and increasing infectivity in the mammalian host.

Leishmania are ancient eukaryotes that have retained the exosome pathway through evolution. Leishmania RNA virus 1 (LRV1)-infected Leishmania species are associated with a particularly aggressive mucocutaneous disease triggered in response to the double-stranded RNA (dsRNA) virus. However, it is unclear how LRV1 is exposed to the mammalian host cells. In higher eukaryotes, some viruses are known to utilize the host exosome pathway for their formation and cell-to-cell spread. As a result, exosomes derived from infected cells contain viral material or particles. Herein, we investigated whether LRV1 exploits the Leishmania exosome pathway to reach the extracellular environment. Biochemical and electron microscopy analyses of exosomes derived from LRV1-infected Leishmania revealed that most dsRNA LRV1 co-fractionated with exosomes, and that a portion of viral particles was surrounded by these vesicles. Transfer assays of LRV1-containing exosome preparations showed that a significant amount of parasites were rapidly and transiently infected by LRV1. Remarkably, these freshly infected parasites generated more severe lesions in mice than non-infected ones. Moreover, mice co-infected with parasites and LRV1-containing exosomes also developed a more severe disease. Overall, this work provides evidence that Leishmania exosomes function as viral envelopes, thereby facilitating LRV1 transmission and increasing infectivity in the mammalian host.

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles1-3. In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)4, whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.

International audience; In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

International audience; Erratum for : Exploitation of the Leishmania exosomal pathway by Leishmania RNA virus 1

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.