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Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The “9+2” axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked if there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana, have an extensive RSP complement including two divergent RSP9 orthologs, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither ortholog is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 ortholog. deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked with the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα-/-/Il-5-/-) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα-/-/Il-5-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα-/-/Il-5-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80intermediate macrophages from infected Il-4rα-/-/Il-5-/- mice failed to mature into resident F4/80high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα-/-/Il-5-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

Giardia intestinalis is a protist intestinal parasite responsible for giardiasis, a disease whose impact is recognized in public health. After ingestion of Giardia cysts from either contaminated food or water, the trophozoite proliferative form, responsible for pathogenic effects, develops in the proximal small intestine of the host where it coexists with gut microbiota. Several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. intestinalis infections through partially known mechanisms (Travers et al. Journal of Parasitology Research, 2011). In the last years, our team has shown, using biological and biochemical approaches, that some probiotic strains of Lactobacillus, in particular L. johnsonii La1 and L. gasseri CNCM-I 4884, display anti-Giardia effects both in vitro and in vivo (Travers et al. Frontiers in Microbiology 2016; Allain et al. Frontiers in Microbiology 8:2707, 2018a, Frontiers in Microbiology 9:98, b). Our investigations have demonstrated that the supernatant of these strains contains Bile-Salt-Hydrolase (BSH)-like activities mediating toxic effects on Giardia. This effect is not directly, but by converting non-toxic components of bile (conjugated bile salts) into bile salts deconjugates proved to be highly toxic to the parasite. These anti-Giardia effects could be mimicked in vitro by treating Giardia cultures with either commercially available BSH bacterial enzymes (Travers et al. 2016) or two L. johnsonii La1 BSH enzymes produced and purified from recombinant Escherichia coli strains (Allain et al. 2018a), in the presence of bile, or even directly with some deconjugated bile salts (Travers et al. 2016). Currently, we are focusing on understanding the mechanism of action (MoA) of toxic metabolites generated by these BSH activities on the parasite itself using imaging and RNA sequencing methods in order to explore the changes in gene expressions in Giardia. Altogether, these data pave the way for new approaches for the treatment of this widespread neglected infectious disease.

SummarySexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses is unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal “9+2” structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water), Giardia excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, Giardia cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. duodenalis infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH)-like activities from the probiotic strain of Lactobacillus johnsonii La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three bsh genes present in the L. johnsonii La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. In vitro assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against Giardia trophozoites in vitro. Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain Lactobacillus johnsonii La1 may yield anti-giardial effects in vitro and in vivo. These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

International audience; Cyanobacterial blooms threaten human health as well as the population of other living organisms in the aquatic environment, particularly due to the production of natural toxic components, the cyanotoxin. So far, the most studied cyanotoxins are microcystins (MCs). In this study, the hepatic alterations at histological, proteome and transcriptome levels were evaluated in female and male medaka fish chronically exposed to 1 and 5 μg L−1 microcystin-LR (MC-LR) and to the extract of MC-producing Microcystis aeruginosa PCC 7820 (5 μg L−1 of equivalent MC-LR) by balneation for 28 days, aiming at enhancing our understanding of the potential reproductive toxicity of cyanotoxins in aquatic vertebrate models. Indeed, both MC and Microcystis extract adversely affect reproductive parameters including fecundity and egg hatchability. The liver of toxin treated female fish present glycogen storage loss and cellular damages. The quantitative proteomics analysis revealed that the quantities of 225 hepatic proteins are dysregulated. In particular, a notable decrease in protein quantities of vitellogenin and choriogenin was observed, which could explain the decrease in reproductive output. Liver transcriptome analysis through Illumina RNA-seq reveals that over 100–400 genes are differentially expressed under 5 μg L−1 MC-LR and Microcystis extract treatments, respectively. Ingenuity pathway analysis of the omic data attests that various metabolic pathways, such as energy production, protein biosynthesis and lipid metabolism, are disturbed by both MC-LR and the Microcystis extract, which could provoke the observed reproductive impairment. The transcriptomics analysis also constitutes the first report of the impairment of circadian rhythm-related gene induced by MCs. This study contributes to a better understanding of the potential consequences of chronic exposure of fish to environmental concentrations of cyanotoxins, suggesting that Microcystis extract could impact a wider range of biological pathways, compared with pure MC-LR, and even 1 μg L−1 MC-LR potentially induces a health risk for aquatic organisms.

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

Numerous strategies are under development for the correction of deleterious effects of mutations in muscular dystrophies, and these strategies must be validated in compelling models. Cellular models seem straightforward to set up; however, the proliferative capacity of muscle cells isolated from dystrophic patients is limited, and in addition it is difficult to envisage the use of large muscle biopsies from patients to obtain enough cells for ex vivo assessments. To overcome these problems, we have devised a strategy to obtain, from a patient with Duchenne muscular dystrophy (DMD), an inexhaustible source of myogenic progenitor cells with a deletion of exons 49 and 50 in the dystrophin gene. Starting material consisted of dermal fibroblasts isolated from a skin biopsy taken in a noninvasive way. These fibroblasts were first immortalized by telomerase gene transfer. Subsequent cell lines were converted into myogenic cells by means of a lentiviral vector encoding an inducible MyoD construct. Before myogenic induction, engineered DMD fibroblasts were able to proliferate infinitely. Under induction conditions, they were converted into myogenic cells, which differentiated into large multinucleated myotubes. We used these DMD fibroblast cell lines to assess dystrophin rescue by using engineered U7 small nuclear RNAs harboring antisense sequences required to restore an in-frame dystrophin mRNA by skipping exon 51. Further molecular analyses showed dystrophin rescue ex vivo as well as in vivo after engrafting of treated cells into regenerating muscles in immunodeficient mice.

In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle-derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2(-/-) gammaC(-/-) C5(-/-) mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle-derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.

International audience; The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.