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Las enzimas proteolíticas, conocidas como peptidasas o proteasas, son críticas en todos los organismos vivos. Pueden actuar como exo– o endopeptidasas. Las peptidasas se segregan en clases que dependen en gran medida de la natu-raleza química de los grupos involucrados en la catálisis. Controlan la activación, síntesis y recambio de proteínas, y regulan la mayoría de los procesos bioquímicos y fisiológicos. En consecuencia, son los principales reguladores de la homeostasis, el envejecimiento, las enfermedades y la muerte. Las proteasas también son esenciales para la propa-gación de agentes infecciosos, ya que son los principales contribuyentes de la patogénesis en varias enfermedades infecciosas, incluida la actual pandemia de COVID-19. Las exopeptidasas son enzimas que catalizan la escisión de los aminoácidos N-terminal o C-terminal de las proteínas o sustratos peptídicos. Están ampliamente distribuidos en mu-chos filos y desempeñan papeles críticos en fisiología y fisiopatología. Son principalmente metalopeptidasas pertene-cientes a las familias M1, M2 y M17, entre otras. Algunas de ellas, las aminopeptidasas M1 N, A y la ectoenzima que degrada la hormona liberadora de tirotropina, la enzima convertidora de angiotensina M2 y la leucil aminopeptidasa M17 son blancos actuales para el desarrollo de nuevos agentes terapéuticos para diferentes enfermedades huma-nas, que incluyen el cáncer, la hipertensión, trastornos del sistema nervioso central, inflamación, desordenes del sis-tema inmune y enfermedades infecciosas como la malaria. La relevancia de las exopeptidasas ha impulsado la bús-queda e identificación de inhibidores potentes y selectivos, como una herramienta importante para controlar la pro-teólisis con impacto en la bioquímica, la biotecnología y la biomedicina. Por estas razones, los inhibidores específicos de estas peptidasas son foco de estudio para el desarrollo de herramientas terapéuticas prometedoras en el trata-miento de diferentes trastornos. En particular, el presente trabajo centra su atención en la biodiversidad marina co-mo una fuente importante y promisoria de inhibidores de serino y metalo exopeptidasas de diferentes familias, con aplicaciones biomédicas en enfermedades humanas.

Additional file 1: Figure S1. Phylogeny of Alveolata. Proteomes from 89 alveolates genomes and transcriptome assemblies from the MMETSP project ( https://zenodo.org/record/257026/files/ ) were used to create orthologous groups using orthofinder v2.2 with the diamond BLAST similarity search. Single ortholog alignments were pruned using PhyloTreePruner v.1.0 (minimum taxa to keep 44 and support value 0.9) and realigned using mafft v7 and filtered with Gblocks v.0.91b (−b5 = a -p = n). Filtered alignments were concatenated using seqCat.pl and a phylogenetic tree was produced under Maximum Likelihood framework using RAxML v8.2.9 with the PROTGAMMALGF model of sequence evolution and 101 bootstraps. Asterics represent support values of 95 and above. A detailed method can be found in Kayal et al. 2018 BMC Evol. Biol. ( https://doi.org/10.1186/s12862-018-1142-0 ). The full tree can be found at http://mmo.sb-roscoff.fr/jbrowseAmoebophrya/ . Figure S2. SSU rDNA sequence identity (in percentage, relative to A25 and A120 compared to other species). Figure S3. Distribution of k-mer in A25 and A120 genomes. Figure S4. Classification of repeated elements in 3 Amoebophrya genomes (AT5, A25, and A120) using REPET. The x-axis represents the cumulated number of bases of repeated elements in the genome. Figure S5. Conserved motif of the putative splice leader (SL) in A25 and A120. Figure S6. Alignments of gene encoding the putative spliced leader (SL) gene in A25 and A120. Figure S7. Gene orientation change rate in 3 Amoebophrya genomes. Figure S8. Number of orthologs genes shared by selected taxa. Figure S9. Boxplot of the dN/dS ratios of orthologous genes between A25 and A120, calculated using the model average method (MA). Figure S10. Synteny dot-plot obtained by comparison between Amoebophrya A25 and AT5 genomes. Figure S11. Synteny dot-plot obtained by comparison between Amoebophrya A120 and AT5 genomes. Figure S12. Intron length distribution. Figure S13. GC content distribution. Figure S14. Multiple alignments of U2 snRNAs. Figure S15. Multiple alignments of U4 snRNAs. Figure S16. Multiple alignments of U5 snRNAs. Figure S17. Multiple alignments of U6 snRNAs. Figure S18. Secondary structure of Amoebophrya snRNA. Figure S19. Example of introner elements (IEs) in Amoebophrya. Figure S20. Distribution the direct repeats with size ranging between 3 and 8 nucleotides in A25. Figure S21. Distribution of the direct repeats with size ranging between 3 and 8 nucleotides in A120. Figure S22. Composition of direct repeats in introners elements. The diversity in composition of the three (a, b, c) most abundant of direct repeats in introner elements in A25 (up) and A120 (down). Figure S23. Terminal inverted repeat locations around the splicing sites in A25 and A120. The position of inverted repeats according to the location of the splice sites in A25 and A120. Left, the inverted repeats of A120 are located at 1–5 the nucleotides upstream and downstream of the splice sites. Right, the inverted repeats of A25 are located at the 1–6 nucleotides in upstream and downstream of the splice sites. Figure S24. The flowchart for the in silico search of introner elements. Figure S25. Hierarchical clustering analysis (pairwise similarity and OrthoMCL) of all intron families and of the inverted repeats in A25 and A120. Figure S26. Percentage of genes with assigned functions in relation with introns composition. Figure S27. Difference in the proportion of IEs-containing-genes compared to their KEGG assignment in A25 and A120. Figure S28. Distribution of conserved introns. Table S1. RCC number, date and site of isolation of strains considered in this study. Table S2. Metrics of Nanopore runs for the two Amoebophrya strains. Table S3. Search for pathways involved in plastidial functions that are entirely independent of plastid-encoded gene content. Table S4. Number of the different types of introns identified in A25 and A120 genomes. Table S5. Search for RNA editing in A25 and A120 introns. Table S6. Putative Amoebophrya A25 and A120 snRNP homologs. Table S7. Classification into families of non-canonical introns in A25 and A120. Table S8. RNAseq read assembly statistics of Amoebophrya A25 and A120 corresponding samples from the different time of infection and to the free-living stage (dinospore only). Table S9. Total number of contigs belonging to samples from different stages of infection and the proportion of them that were aligned against the genomes of both Amoebophrya A25 and A120. ND corresponds to “not determined” when no measurement was done. Table S10. Metabolic pathway screened in A25 and A120 proteomes.

The analysis of immune responses in diverse malaria endemic regions provides more information to understand the host’s immune response to Plasmodium falciparum. Several plasmodial antigens have been reported as targets of human immunity. PfAMA1 is one of most studied vaccine candidates; PfRH5 and Pf113 are new promising vaccine candidates. The aim of this study was to evaluate humoral response against these three antigens among children of Lastourville (rural area) and Franceville (urban area). Malaria was diagnosed using rapid diagnosis tests. Plasma samples were tested against these antigens by enzyme-linked immunosorbent assay (ELISA). We found that malaria prevalence was five times higher in the rural area than in the urban area (p < 0.0001). The anti-PfAMA1 and PfRh5 response levels were significantly higher in Lastourville than in Franceville (p < 0.0001; p = 0.005). The anti-AMA1 response was higher than the anti-Pf113 response, which in turn was higher than the anti-PfRh5 response in both sites. Anti-PfAMA1 levels were significantly higher in infected children than those in uninfected children (p = 0.001) in Franceville. Anti-Pf113 and anti-PfRh5 antibody levels were lowest in children presenting severe malarial anemia. These three antigens are targets of immunity in Gabon. Further studies on the role of Pf113 in antimalarial protection against severe anemia are needed.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families, it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

Understanding factors that generate, maintain, and constrain host-parasite associations is of major interest to biologists. Although little studied, many extremely virulent micro-eukaryotic parasites infecting microalgae have been reported in the marine plankton. This is the case for Amoebophrya, a diverse and highly widespread group of Syndiniales infecting and potentially controlling dinoflagellate populations. Here, we analyzed the time-scale gene expression of a complete infection cycle of two Amoebophrya strains infecting the same host (the dinoflagellate Scrippsiella acuminata), but diverging by their host range (one infecting a single host, the other infecting more than one species). Over two-thirds of genes showed two-fold differences in expression between at least two sampled stages of the Amoebophrya life cycle. Genes related to carbohydrate metabolism as well as signaling pathways involving proteases and transporters were overexpressed during the free-living stage of the parasitoid. Once inside the host, all genes related to transcription and translation pathways were actively expressed, suggesting the rapid and extensive protein translation needed following host-cell invasion. Finally, genes related to cellular division and components of the flagellum organization were overexpressed during the sporont stage. In order to gain a deeper understanding of the biological basis of the host-parasitoid interaction, we screened proteins involved in host-cell recognition, invasion, and protection against host-defense identified in model apicomplexan parasites. Very few of the genes encoding critical components of the parasitic lifestyle of apicomplexans could be unambiguously identified as highly expressed in Amoebophrya. Genes related to the oxidative stress response were identified as highly expressed in both parasitoid strains. Among them, the correlated expression of superoxide dismutase/ascorbate peroxidase in the specialist parasite was consistent with previous studies on Perkinsus marinus defense. However, this defense process could not be identified in the generalist Amoebophrya strain, suggesting the establishment of different strategies for parasite protection related to host specificity.

International audience; Giardia duodenalis is a protozoan parasite responsible for giardiasis, a disease characterized by intestinal malabsorption, diarrhea and abdominal pain in a large number of mammal species. Giardiasis is one of the most common intestinal parasitic diseases in the world and thus a high veterinary, and public health concern. It is well-established that some probiotic bacteria may confer protection against this parasite in vitro and in vivo and we recently documented the implication of bile-salt hydrolase (BSH)-like activities from strain La1 of Lactobacillus johnsonii as mediators of these effects in vitro. We showed that these activities were able to generate deconjugated bile salts that were toxic to the parasite. In the present study, a wide collection of lactobacilli strains from different ecological origins was screened to assay their anti-giardial effects. Our results revealed that the anti-parasitic effects of some of the strains tested were well-correlated with the expression of BSH-like activities. The two most active strains in vitro, La1 and Lactobacillus gasseri CNCM I-4884, were then tested for their capacity to influence G. duodenalis infection in a suckling mice model. Strikingly, only L. gasseri CNCM I-4884 strain was able to significantly antagonize parasite growth with a dramatic reduction of the trophozoites load in the small intestine. Moreover, this strain also significantly reduced the fecal excretion of Giardia cysts after 5 days of treatment, which could contribute to blocking the transmission of the parasite, in contrast of La1 where no effect was observed. This study represents a step toward the development of new prophylactic strategies to combat G. duodenalis infection in both humans and animals.

Background Plasmodium falciparum M1 family aminopeptidase is currently considered as a promising target for anti-malarial chemotherapy. Several series of inhibitors developed by various research groups display IC50/Ki values down to nM range on native PfA-M1 or recombinant forms and block the parasite development in culture at µM to sub-µM concentrations. A handful of these inhibitors has been tested on murine models of malaria and has shown anti plasmodial in vivo activity. However, most of these inhibitors do also target the other neutral malarial aminopeptidase, PfA-M17, often with lower Ki values, which questions the relative involvement and importance of each enzyme in the parasite biology. Results An amino-benzosuberone derivative from a previously published collection of chemicals targeting specifically the M1-aminopeptidases has been identified; it is highly potent on PfA-M1 (Ki = 50 nM) and devoid of inhibitory activity on PfA-M17 (no inhibition up to 100 µM). This amino-benzosuberone derivative (T5) inhibits, in the µM range, the in vitro growth of two P. falciparum strains, 3D7 and FcB1, respectively chloroquino-sensitive and resistant. Evaluated in vivo, on the murine non-lethal model of malaria Plasmodium chabaudi chabaudi, this amino-benzosuberone derivative was able to reduce the parasite burden by 44 and 40% in a typical 4-day Peters assay at a daily dose of 12 and 24 mg/kg by intraperitoneal route of administration. Conclusions The evaluation of a highly selective inhibitor of PfA-M1, over PfA-M17, active on Plasmodium parasites in vitro and in vivo, highlights the relevance of PfA-M1 in the biological development of the parasite as well as in the list of promising anti-malarial targets to be considered in combination with current or future anti-malarial drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2032-4) contains supplementary material, which is available to authorized users.

Neutral metallo-aminopeptidase (APN) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of protein or peptide substrates. APN expression is dysregulated in inflammatory diseases as well as in several types of cancer. Therefore, inhibitors of APN may be effective against cancer and inflammation. By virtual screening and enzymatic assays, we identified three non-competitive inhibitors (α > 1) of the porcine and human APN with Ki values in the μM range. These non-peptidic compounds lack the classical zinc-binding groups (ZBG) present in most of the APN inhibitors. Molecular docking simulations suggested the novel inhibitors suppress APN activity by an alternative mechanism to Zn coordination: they interacted with residues comprising the S1 and S5′ subsites of APN. Of note, these compounds also inhibited the porcine aminopeptidase A (pAPA) using a competitive inhibition mode. This indicated differences in the binding mode of these compounds with APN and APA. Based on sequence and structural analyses, we predicted the significance of targeting human APN residues: Ala-351, Arg-442, Ala-474, Phe-896 and Asn-900 for improving the selectivity of the identified compounds. Remarkably, the intraperitoneal injection of compounds BTB07018 and JFD00064 inhibited APN activity in rat brain, liver and kidney indicating good bio-distribution of these inhibitors in vivo. These data reinforce the idea of designing novel APN inhibitors based on lead compounds without ZBG., Graphical abstract Image 1, Highlights • We identified three non-competitive inhibitors of the human and porcine APN. • These compounds lack the classical zinc-binding groups of the APN inhibitors. • We proposed these molecules block APN by an alternative mechanism to Zn chelation. • All the inhibitors interact with APN residues comprising the S1 and S5′ subsites. • Two compounds blocked the APN activity in the brain, liver and kidney of rats.

Background:\ud \ud The several-micrometer-sized Toxoplasma gondii protozoan parasite invades virtually any type of nucleated cell from a warm-blooded animal within seconds. Toxoplasma initiates the formation of a tight ring-like junction bridging its apical pole with the host cell membrane. The parasite then actively moves through the junction into a host cell plasma membrane invagination that delineates a nascent vacuole. Recent high resolution imaging and kinematics analysis showed that the host cell cortical actin dynamics occurs at the site of entry while gene silencing approaches allowed motor-deficient parasites to be generated, and suggested that the host cell could contribute energetically to invasion. In this study we further investigate this possibility by analyzing the behavior of parasites genetically impaired in different motor components, and discuss how the uncovered mechanisms illuminate our current understanding of the invasion process by motor-competent parasites.\ud Results:\ud \ud By simultaneously tracking host cell membrane and cortex dynamics at the site of interaction with myosin A-deficient Toxoplasma, the junction assembly step could be decoupled from the engagement of the Toxoplasma invasive force. Kinematics combined with functional analysis revealed that myosin A-deficient Toxoplasma had a distinct host cell-dependent mode of entry when compared to wild-type or myosin B/C-deficient Toxoplasma. Following the junction assembly step, the host cell formed actin-driven membrane protrusions that surrounded the myosin A-deficient mutant and drove it through the junction into a typical vacuole. However, this parasite-entry mode appeared suboptimal, with about 40 % abortive events for which the host cell membrane expansions failed to cover the parasite body and instead could apply deleterious compressive forces on the apical pole of the zoite.\ud Conclusions:\ud \ud This study not only clarifies the key contribution of T. gondii tachyzoite myosin A to the invasive force, but it also highlights a new mode of entry for intracellular microbes that shares early features of macropinocytosis. Given the harmful potential of the host cell compressive forces, we propose to consider host cell invasion by zoites as a balanced combination between host cell membrane dynamics and the Toxoplasma motor function. In this light, evolutionary shaping of myosin A with fast motor activity could have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.