Hubert Denise

Cysteine Protease B of Leishmania mexicana Inhibits Host Th1 Responses and Protective Immunity

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Δcpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Δcpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Δcpb-infected IL-12p40−/− and STAT4−/− mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-γ response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.

Resistance to Pentamidine in Leishmania mexicana Involves Exclusion of the Drug from the Mitochondrion

ABSTRACT The uptake of [3H]pentamidine into wild-type and drug-resistant strains of Leishmania mexicana was compared. Uptake was carrier mediated. Pentamidine-resistant parasites showed cross-resistance to other toxic diamidine derivatives. A substantial decrease in accumulation of the drug accompanied the resistance phenotype, although the apparent affinity for pentamidine by its carrier was not altered when initial uptake velocity was measured. The apparent Vmax, however, was reduced. An efflux of pentamidine could be measured in both wild-type and resistant cells. Only a relatively small proportion of the total accumulated pentamidine was available for efflux in wild-type cells, while in resistant cells the majority of loaded pentamidine was available for release. Pharmacological reagents which diminish the mitochondrial membrane potential reduced pentamidine uptake in wild-type parasites, and the mitochondrial membrane potential was shown to be reduced in resistant cells. A fluorescent analogue of pentamidine, 4′,6′-diamidino-2-phenylindole, accumulated in the kinetoplast of wild-type but not resistant parasites. These data together indicate that diamidine drugs accumulate in the Leishmania mitochondrion and that the development of the resistance phenotype is accompanied by lack of mitochondrial accumulation of the drug and its exclusion from the parasites.

A diamidine-resistant Trypanosoma equiperdum clone contains a P2 purine transporter with reduced substrate affinity

Following the demonstration that the transport of melaminophenyl arsenical drugs in Trypanosoma brucei is dependent upon an unusual adenosine nucleoside transporter (Carter and Fairlamb, Nature 361 (1993) 173-175) we have investigated adenosine transport in the related parasite Trypanosoma equiperdum (Botat1.1) and a cloned derivative resistant to the diamidine drug berenil (diminazene aceturate) with limited cross-resistance to the melaminophenyl arsenical cymelarsen. The parental strain possesses a bipartite adenosine transport system consisting of one component which is inhibited in a dose-dependent and saturable manner with increasing concentrations of inosine and a second component which is similarly inhibited by adenine. Uptake of adenosine on this second transporter is also inhibited in a dose-dependent fashion by berenil and cymelarsen. Both transporters have high affinity for adenosine (apparent Km values of 0.60 and 0.70 mM and Vmax values of 8.4 and 6.9 pmol (s (10(8) trypanosomes))-1 at 25 degrees C, respectively). Thus T. equiperdum shares with T. brucei a system comprising two adenosine transporters named P1 and P2, respectively. The P1 transporter is similar in the sensitive and resistant T. equiperdum clones, whereas the P2 transporter has reduced transport capacity at physiological adenosine concentration and decreased affinity for adenosine in the drug-resistant clone.

EBI metagenomics—a new resource for the analysis and archiving of metagenomic data

Metagenomics is a relatively recently established but rapidly expanding field that uses high-throughput next-generation sequencing technologies to characterize the microbial communities inhabiting different ecosystems (including oceans, lakes, soil, tundra, plants and body sites). Metagenomics brings with it a number of challenges, including the management, analysis, storage and sharing of data. In response to these challenges, we have developed a new metagenomics resource (http://www.ebi.ac.uk/metagenomics/) that allows users to easily submit raw nucleotide reads for functional and taxonomic analysis by a state-of-the-art pipeline, and have them automatically stored (together with descriptive, standards-compliant metadata) in the European Nucleotide Archive.

Expression of Multiple CPB Genes Encoding Cysteine Proteases Is Required for Leishmania mexicana Virulence In Vivo

ABSTRACT Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.

EBI metagenomics in 2016--an expanding and evolving resource for the analysis and archiving of metagenomic data.

EBI metagenomics (https://www.ebi.ac.uk/metagenomics/) is a freely available hub for the analysis and archiving of metagenomic and metatranscriptomic data. Over the last 2 years, the resource has undergone rapid growth, with an increase of over five-fold in the number of processed samples and consequently represents one of the largest resources of analysed shotgun metagenomes. Here, we report the status of the resource in 2016 and give an overview of new developments. In particular, we describe updates to data content, a complete overhaul of the analysis pipeline, streamlining of data presentation via the website and the development of a new web based tool to compare functional analyses of sequence runs within a study. We also highlight two of the higher profile projects that have been analysed using the resource in the last year: the oceanographic projects Ocean Sampling Day and Tara Oceans.

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.

BackgroundVisceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced.ResultsWe have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (ΔLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of ΔLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving ΔLicpa::CPA) was sufficient to complement the reduced infectivity of both ΔLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone ΔLicpa C1::CPA compared with the CPA-deficient mutant ΔLicpa C1.ConclusionThe data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the ΔLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

HPMCD: the database of human microbial communities from metagenomic datasets and microbial reference genomes

The Human Pan-Microbe Communities (HPMC) database (http://www.hpmcd.org/) provides a manually curated, searchable, metagenomic resource to facilitate investigation of human gastrointestinal microbiota. Over the past decade, the application of metagenome sequencing to elucidate the microbial composition and functional capacity present in the human microbiome has revolutionized many concepts in our basic biology. When sufficient high quality reference genomes are available, whole genome metagenomic sequencing can provide direct biological insights and high-resolution classification. The HPMC database provides species level, standardized phylogenetic classification of over 1800 human gastrointestinal metagenomic samples. This is achieved by combining a manually curated list of bacterial genomes from human faecal samples with over 21000 additional reference genomes representing bacteria, viruses, archaea and fungi with manually curated species classification and enhanced sample metadata annotation. A user-friendly, web-based interface provides the ability to search for (i) microbial groups associated with health or disease state, (ii) health or disease states and community structure associated with a microbial group, (iii) the enrichment of a microbial gene or sequence and (iv) enrichment of a functional annotation. The HPMC database enables detailed analysis of human microbial communities and supports research from basic microbiology and immunology to therapeutic development in human health and disease.

TrypanoCyc : a community-led biochemical pathways database for Trypanosoma brucei

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.