Nathalie Moiré

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.

Sequencing and gene expression of hypodermins A, B, C in larval stages of Hypoderma lineatum

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum, were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N-linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.

Enzymatic effect of hypodermin A, a parasite protease, on bovine lymphocyte membrane antigens

The protease hypodermin A (HA) is produced by the parasitic warble‐fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti‐lymphocyte receptor antibodies tested. Anti‐BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti‐MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 μg/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)‐induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

Expression of functional hypodermin A, a serine protease from Hypoderma lineatum (Diptera, Oestridae), in Schneider 2 cells.

Hypodermin A (HA) is a serine protease secreted by first-instar Hypoderma lineatum larvae (Oestridae, Diptera). It plays a crucial role in induced immunosuppression during hypodermosis. This report describes the production of recombinant HA in Drosophila melanogaster Schneider 2 cells, its purification and its characterization, and compares it with the protease extracted form parasite larvae. The recombinant protein and the native HA have similar biochemical and biological features. Activity of the recombinant protease on the lymphocyte proliferation inhibition and on membrane antigen cleavage was tested and shown to be similar to the native one. Tunicamycin treatment of the recombinant HA shows that the two putative glycosylation sites carry glycan residues. Unglycosylated recombinant HA has the same enzymatic activity as the fully glycosylated protein, indicating that glycosylation is not important for the protease activity of HA.

Effects of dexamethasone on distribution and function of peripheral mononuclear blood cells in pneumonic calves.

Experimental pneumonic pasteurellosis was used in 30 Holstein calves to compare the effects on the immune response of different treatments: antibiotics alone or antibiotics in combination with either steroidal (SAID) or non-steroidal anti-inflammatory drugs (NSAID). Clinical parameters and the effect of treatments on the mitogen-induced proliferation of peripheral mononuclear blood cells (PMBCs) and on their distribution were evaluated. Calves in all three treated groups showed rapid improvement, but clinical signs were less marked in both groups receiving anti-inflammatory drugs. Limited difference in the effect on the immune system of treatments was observed. No inhibition of lymphocytes proliferation was detected in these experimental conditions in the dexamethasone (DM)-treated group. There was no variation among groups for the CD4+, CD5+, CD8+ and CD21+ populations. The only noteworthy change was a transient increase in the percentage of the monocyte population (CD14+) in the DM-treated group compared to the group treated with NSAID.

Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2.

Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes.

Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions.

Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm.

Profile of the mosaic element BTMR1 in the genome of the bumble bee Bombus terrestris (Hymenoptera: Apidae)

Co‐evolution involving a mariner transposon, Botmar1 and the other repeats contained in the Bombus terrestris genome was investigated. We found that the 5′‐region of Botmar1 forms one of the components of a mosaic element, known as B. terrestris mosaic repeat 1 (BTMR1), which is also composed of inner segments originating from two different retrotransposons and a pseudogene corresponding to an RNA methyltransferase cDNA. The fact that BTMR1 is interspersed within chromosomes and the differences in its abundance in different species indicate that it is very probably a mobile element. Nevertheless, the absences of direct or inverted repeats at its ends and of target site duplication indicate that its mobility is not ensured by a cardinal transposable element, but putatively by a Crypton‐like element.

Imidazo[1,2- b ]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1 decrease the parasite burden in mice with acute toxoplasmosis

The current therapeutic arsenal for toxoplasmosis is restricted to drugs non-specific to the parasite which cause important side effects. Development of more efficient and specific anti-Toxoplasma compounds is urgently needed. Imidazo[1,2-b]pyridazines designed to inhibit the calcium-dependent protein kinase 1 of Toxoplasma gondii (TgCDPK1) and effective against tachyzoite growth in vitro at submicromolar ranges were modified into hydrochloride salts to be administered in vivo in a mouse model of acute toxoplasmosis. All protonated imidazo[1,2-b]pyridazine salts (SP230, SP231 and SP232) maintained their activity on TgCDPK1 and T. gondii tachyzoites. Rat and mouse liver microsomes were used to predict half-life and intrinsic clearance, and the pharmacokinetic profile of the most rapidly degraded imidazo[1,2b]pyridazine salt (SP230) was determined in serum, brain and lungs of mice after a single administration of 50 mg/kg. Compounds were then tested in vivo in a murine model of acute toxoplasmosis. Mice infected with tachyzoites of the ME49 strain of T. gondii were treated for 4, 7 or 8 days with 25 or 50 mg/kg/day of SP230, SP231 or SP232. The parasite burdens were strongly diminished (>90% reduction under some conditions) in the spleen and the lungs of mice treated with imidazo[1,2-b]pyridazine salts compared with untreated mice, without the need for pre-treatment. Moreover, no increases in the levels of hepatic and renal toxicity markers were observed, demonstrating no significant signs of short-term toxicity. To conclude, imidazo[1,2-b]pyridazine salts have strong efficacy in vivo on acute toxoplasmosis and should be further tested in a model of mouse congenital toxoplasmosis.

Targeted Delivery of Toxoplasma gondii Antigens to Dendritic Cells Promote Immunogenicity and Protective Efficiency against Toxoplasmosis

Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment single-chain fragment variable (scFv) directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, interleukin-2, IgG2a, and nasal IgA. This study provides evidence of the potential of DC targeting for the development of new vaccines against a range of Apicomplexa parasites.