The aminopeptidase inhibitor CHR-2863 is an orally bioavailable inhibitor of murine malaria

Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an everincreasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria

Cathelicidin-like Helminth Defence Molecules (HDMs) Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects. © 2013 Thivierge et al

Host proteins involved in «turnip mosaic virus» life cycle

All viruses are gene poor relative to their host, thus, most steps in virus infection involve interactions between viral components and host factors. Identification of these factors represents one of the major frontiers in current virus research. In this study, protein-protein interaction methodologies were used to find host interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRP), VPg-protease (VPg- Pro) and P3 protein.First, eukaryotic elongation factor 1A (eEF1A) was shown to interact with TuMV RdRp and VPg-Pro using tandem affinity purification in Arabidopsis thaliana and/or in vitro assays. Interaction of eEF1A with both viral proteins was shown to take place within 6K-VPg-Pro-induced vesicles. The same vesicles were also shown to contain poly(A)-binding (PABP) and heat shock cognate 70-3 proteins (Hsc70), two previously identified RdRp interactors. To further characterize the content of these vesicles upon TuMV infection, a fluorescently labeled 6K-GFP TuMV infectious clone was constructed and used in confocal microscopy experiments. The inclusion of eEF1A, PABP, Hsc70, eukaryotic initiation factor (iso)4E and VPg-Pro in TuMV-induced vesicles was demonstrated. It is well establish that positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes, usually in association with vesicle formation. For TuMV, our data suggest that it is the 6K-induced vesicles that house the viral replication complex (VRC). Moreover, the presence of replication and translation elements in these vesicles indicates that both processes might be coupled in TuMV VRC.Secondly, the yeast two-hybrid system was used to identify plant P3-interacting proteins in a cDNA library from A. thaliana. A lipase was recovered from the screen and shown to interact with P3 in vitro. Both proteins were also demonstrated to partially co-localize in the cytoplasm of the cell. Given that lipases play important roles in the plant response to biotic stress, this interaction reinforce the role of TuMV P3 in plant resistance and/or pathogenesis.Les virus ont de petits génomes qui codent pour un nombre limité de protéines et dépendent conséquemment des facteurs de l'hôte pour compléter leur cycle de réplication. Dans ce projet, nous avons utilisé différentes méthodes pour identifier des partenaires protéiques de la polymérase virale à ARN (RdRp), de la VPg-Pro et de la protéine P3 du virus de la mosaïque du navet (TuMV).Premièrement, nous avons trouvé que le facteur eucaryote d'élongation de la traduction 1A (eEF1A) interagit avec la RdRp et la VPg-Pro en utilisant une stratégie de purification en tandem in planta et/ou des essais in vitro. Nous avons montré que ces interactions se produisent en association avec les membranes du réticulum endoplasmique, plus précisément dans les vésicules induites par le polypeptide 6K-VPg-Pro. Nous avons aussi démontré que ces mêmes vésicules contiennent les protéines Hsc70-3 et PABP, deux partenaires connus de la RdRp. Afin de poursuivre la caractérisation du contenu de ces vésicules, nous avons créé un vecteur infectieux du TuMV permettant d'étiqueter les vésicules avec la GFP et d'être utilisé en microscopie confocale. À l'aide de ce vecteur, nous avons observé la présence du facteur eEF1a, de la PABP, de la Hsc70, du facteur eucaryote d'initiation de la traduction (iso) 4E et de la VPg-Pro dans les vésicules induites par le TuMV. Il est bien établit que le complexe de réplication des virus à ARN positif est associé aux membranes cytoplasmiques, généralement sous forme de vésicules. Pour le TuMV, nos données semblent indiquer que les vésicules induites par la protéine 6K contiennent le complexe de réplication viral (VRC). De plus, la présence d'éléments participants à la réplication ainsi qu'à la traduction dans ces vésicules suggère que ces deux processus sont possiblement couplés dans le VRC du TuMV.Deuxièmement, le système du double-hybride en levure a été utilisé pour rechercher des partenaires protéiques de P3. Le criblage de P3 contre une banque d'ADNc d'Arabidopsis thaliana a révélé une interaction entre P3 et une lipase. Lorsque exprimées ensembles dans Nicotiana benthamiana, les deux protéines co-localisent au cytoplasme. Étant donné le rôle des lipases dans les réponses des plantes aux attaques pathogènes, cette interaction renforce le rôle suggéré de la protéine P3 dans la pathogenèse et les mécanismes de résistance des plantes

Protein-protein interactions in turnip mosaic potyvirus replication complex

Interactions between plant and virus proteins play pivotal roles in many processes during the viral infection cycle. Analysis of protein-protein interactions is crucial for understanding virus and host protein functions and the molecular mechanisms underlying viral infection. Several interactions between virus-encoded proteins have been reported. However, few interactions between viral and plant proteins have been identified so far. To examine interactions between Turnip mosaic potyvirus (TuMV) proteins and plant proteins, recombinant proteins were produced and used in ELISA-type assays and in in vitro co-immunoprecipitation experiments. An interaction between TuMV P1 proteinase and wheat poly(A)-binding protein (PABP) was identified. An interaction between P1 protein and the plant Arabidopsis thaliana eukaryotic initiation factor (iso)4E [eIF(iso)4E] was also found. Finally, potential interactions between both TuMV CI and P1 proteins and between TuMV CI protein and eIF(iso)4E were identified

Schistosoma mansoni immunomodulatory molecule Sm16/SPO-1/SmSLP is a member of the trematode-specific helminth defence molecules (HDMs)

Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34-117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34-117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34-117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34-117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to inflammatory responses. These results offer new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, and places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities.JS was supported by a fellowship provided by the Department for the Economy through Queen’s University Belfast, Northern Ireland, UK. KT, SC, BGS and JPD were supported by a Canadian Institute of Health Research (CIHR) Chair (Tier 1) in Infectious Diseases awarded to JPD (https://cihr-irsc.gc.ca/e/193.html). KC and JPD were funded by the Science Foundation Ireland (SFI, Republic of Ireland) Research Professorship grant 17/RP/5368 (https://www.sfi.ie/). RA, JT and SD were supported by an NHMRC project grant APP1142006 (https://www.nhmrc.gov.au/). CCT and SW are supported by the Medical Research Council, UK (https://mrc.ukri.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.peer-reviewe

Multi-Scale Clustering of Lyme Disease Risk at the Expanding Leading Edge of the Range of Ixodes scapularis in Canada

Since its detection in Canada in the early 1990s, Ixodes scapularis, the primary tick vector of Lyme disease in eastern North America, has continued to expand northward. Estimates of the tick’s broad-scale distribution are useful for tracking the extent of the Lyme disease risk zone; however, tick distribution may vary widely within this zone. Here, we investigated I. scapularis nymph distribution at three spatial scales across the Lyme disease emergence zone in southern Quebec, Canada. We collected ticks and compared the nymph densities among different woodlands and different plots and transects within the same woodland. Hot spot analysis highlighted significant nymph clustering at each spatial scale. In regression models, nymph abundance was associated with litter depth, humidity, and elevation, which contribute to a suitable habitat for ticks, but also with the distance from the trail and the type of trail, which could be linked to host distribution and human disturbance. Accounting for this heterogeneous nymph distribution at a fine spatial scale could help improve Lyme disease management strategies but also help people to understand the risk variation around them and to adopt appropriate behaviors, such as staying on the trail in infested parks to limit their exposure to the vector and associated pathogens

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP)

AbstractThe Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0–8.0), although PfM1AAP also possesses some activity (&lt; 20%) at the lower pH range of 5.0–5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.</jats:p

Turnip Mosaic Virus RNA Replication Complex Vesicles Are Mobile, Align with Microfilaments, and Are Each Derived from a Single Viral Genome ▿ †

Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K2GFP and the other expressed 6K2mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle

Plant Virus RNAs. Coordinated Recruitment of Conserved Host Functions by (+) ssRNA Viruses during Early Infection Events

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5′ and 3′ ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5′ and 3′ ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication

Molecular characterization of Cryptosporidium isolates from humans in Ontario, Canada

Abstract Background Cryptosporidiosis is a gastrointestinal disease with global distribution. It has been a reportable disease in Canada since 2000; however, routine molecular surveillance is not conducted. Therefore, sources of contamination are unknown. The aim of this project was to identify species and subtypes of Cryptosporidium in clinical cases from Ontario, the largest province in Canada, representing one third of the Canadian population, in order to understand transmission patterns. Methods A total of 169 frozen, banked, unpreserved stool specimens that were microscopy positive for Cryptosporidium over the period 2008–2017 were characterized using molecular tools. A subset of the 169 specimens were replicate samples from individual cases. DNA was extracted directly from the stool and nested PCR followed by Sanger sequencing was conducted targeting the small subunit ribosomal RNA (SSU) and glycoprotein 60 (gp60) genes. Results Molecular typing data and limited demographic data were obtained for 129 cases of cryptosporidiosis. Of these cases, 91 (70.5 %) were due to Cryptosporidium parvum and 24 (18.6%) were due to Cryptosporidium hominis. Mixed infections of C. parvum and C. hominis occurred in four (3.1%) cases. Five other species observed were Cryptosporidium ubiquitum (n = 5), Cryptosporidium felis (n = 2), Cryptosporidium meleagridis (n = 1), Cryptosporidium cuniculus (n = 1) and Cryptosporidium muris (n = 1). Subtyping the gp60 gene revealed 5 allelic families and 17 subtypes of C. hominis and 3 allelic families and 17 subtypes of C. parvum. The most frequent subtype of C. hominis was IbA10G2 (22.3%) and of C. parvum was IIaA15G2R1 (62.4%). Conclusions The majority of isolates in this study were C. parvum, supporting the notion that zoonotic transmission is the main route of cryptosporidiosis transmission in Ontario. Nonetheless, the observation of C. hominis in about a quarter of cases suggests that anthroponotic transmission is also an important contributor to cryptosporidiosis pathogenesis in Ontario