Laurence Tetley

Biology of African trypanosomes in the tsetse fly

African trypanosomes present several features of interest to cell biologists. These include: a repressible single mitochondrion with a large mass of mitochondrial DNA, the kinetoplast; a special organelle, the glycosome, which houses the enzymes of the glycolytic chain; a surface coat of variable glycoprotein which enables the parasite to evade the mammalian hosts immune response; and a unique flagellum‐to‐host attachment mechanism associated with novel cytoskeletal elements. Trypanosome development during the life cycle involves cyclical activation and repression of genes controlling these activities. Understanding the complexity of parasite development in the tsetse fly vector is especially challenging but may help to suggest new methods for the control of trypanosomiasis.

Polymeric Chitosan‐based Vesicles for Drug Delivery

A simple carbohydrate polymer glycol chitosan (degree of polymerization 800 approx.) has been investigated for its ability to form polymeric vesicle drug carriers. The attachment of hydrophobic groups to glycol chitosan should yield an amphiphilic polymer capable of self‐assembly into vesicles. Chitosan is used because the membrane‐penetration enhancement of chitosan polymers offers the possibility of fabricating a drug delivery system suitable for the oral and intranasal administration of gut‐labile molecules.

Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB‐deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.

The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes that differ in their stage regulation and substrate preferences.

The cpb genes ofLeishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3–cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection ofcpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into thecpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite’s interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

Bloodstream form trypanosoma brucei depend upon multiple metacaspases associated with RAB11-positive endosomes

Trypanosoma brucei possesses five metacaspase genes. Of these, MCA2 and MCA3 are expressed only in the mammalian bloodstream form of the parasite, whereas MCA5 is expressed also in the insect procyclic form. Triple RNAi analysis showed MCA2, MCA3 and MCA5 to be essential in the bloodstream form, with parasites accumulating pre-cytokinesis. Nevertheless, triple null mutants (Δmca2/3Δmca5) could be isolated after sequential gene deletion. Thereafter, Δmca2/3Δmca5 mutants were found to grow well both in vitro in culture and in vivo in mice. We hypothesise that metacaspases are essential for bloodstream form parasites, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of Δmca2/3Δmca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin D2. The metacaspases were found to colocalise with RAB11, a marker for recycling endosomes. However, variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, Δmca2/3Δmca5 and triple RNAi induced parasites. Thus, the data provide no support for the direct involvement of T. brucei metacaspases in programmed cell death and suggest that the proteins have a function associated with RAB11 vesicles that is independent of known recycling processes of RAB11-positive endosomes.

The release of model macromolecules may be controlled by the hydrophobicity of palmitoyl glycol chitosan hydrogels

A non-covalently cross-linked palmitoyl glycol chitosan (GCP) hydrogel has been evaluated as an erodible controlled release system for the delivery of hydrophilic macromolecules. Samples of GCP with hydrophobicity decreasing in the order GCP12>GCP11>GCP21 were synthesised and characterised by 1H NMR. Hydrogels were prepared by freeze-drying an aqueous dispersion of the polymer in the presence or absence of either a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran, MW 4400), and/or amphiphilic derivatives Gelucire 50/13 or vitamin E d-alpha-tocopherol polyethylene glycol succinate. Gels were analysed for aqueous hydration, FITC-dextran release, and bioadhesion, and imaged by scanning electron microscopy. The gels were highly porous and could be hydrated to up to 95x their original weight without an appreciable volume change and most gels eventually eroded. Hydration and erosion were governed by the hydrophobicity of the gel and the presence of the amphiphilic additives. GCP gels could be loaded with up to 27.5% (w/w) of FITC-dextran by freeze-drying a dispersion of GCP in a solution of FITC-dextran. The controlled release of FITC-dextran was governed by the hydrophobicity of the gel following the trend GCP21>GCP11>GCP12. GCP gels were bioadhesive but less so than hydroxypropylmethylcellulose, Carbopol 974NF (7:3) tablets.

Vesicle Size Influences the Trafficking, Processing, and Presentation of Antigens in Lipid Vesicles

Although it is accepted that particulate Ags are more immunogenic than soluble Ags in vivo, it is unclear whether this effect can be explained solely through enhanced uptake by APCs. In this study we demonstrate that vesicle size modulated the efficiency of Ag presentation by murine macrophages and that this effect was accompanied by a profound change in trafficking of Ag. Ag prepared in large particles (560 nm) was delivered into early endosome-like, immature phagosomes, whereas smaller vesicles (155 nm) and soluble Ags localized rapidly to late endosomes/lysosomes. However, peptide/class II complexes could be detected in both compartments. Phagosomes formed on uptake of large vesicles recruit Ag-processing apparatus while retaining the characteristics of early endosomes. In contrast, smaller vesicles bypassed this compartment, appeared to go more rapidly to lysosomal compartments, and exhibited reduced Ag-presenting efficiency. We conclude that the ability of phagocytosed, particulate Ag to target early phagosomes results in more efficient Ag presentation.

Ultrastructural analysis of the sporozoite of Cryptosporidium parvum.

Cryopreparation of live sporozoites and oocysts of the apicomplexan parasite Cryptosporidium parvum, followed by transmission electron microscopy, was undertaken to show the 3D arrangement of organelles, their number and distribution. Profiles of parasites obtained from energy-filtering transmission electron microscopy of serial sections provided 3D reconstructions from which morphometric data and stereo images were derived. The results suggest that sporozoites have a single rhoptry containing an organized lamellar body, no mitochondria or conventional Golgi apparatus, and one or two crystalline bodies. Micronemes were shown to be spherical, numerous and apically located, and to account for 0.8% of the total cell volume. Dense granules were less numerous, larger, accounted for 5.8% of the cell volume, and were located more posteriorly than micronemes. A structure juxtaposed to the nucleus with similarities to the plastid-like organelle reported for other members of the Apicomplexa was observed. The detailed analysis illustrates the advantages of cryopreparation in retaining ultrastructural fidelity of labile or difficult to preserve structures such as the sporozoite of Cryptosporidium.

Niosomes and polymeric chitosan based vesicles bearing transferrin and glucose ligands for drug targeting

AbstractPurpose. To prepare polymeric vesicles and niosomes bearing glucose or transferrin ligands for drug targeting. Methods. A glucose-palmitoyl glycol chitosan (PGC) conjugate was synthesised and glucose-PGC polymeric vesicles prepared by sonication of glucose-PGC/ cholesterol. N-palmitoylglucosamine (NPG) was synthesised and NPG niosomes also prepared by sonication of NPG/ sorbitan monostearate/ cholesterol/ cholesteryl poly-24-oxyethylene ether. These 2 glucose vesicles were incubated with colloidal concanavalin A gold (Con-A gold), washed and visualised by transmission electron microscopy (TEM). Transferrin was also conjugated to the surface of PGC vesicles and the uptake of these vesicles investigated in the A431 cell line (over expressing the transferrin receptor) by fluorescent activated cell sorter analysis. Results. TEM imaging confirmed the presence of glucose units on the surface of PGC polymeric vesicles and NPG niosomes. Transferrin was coupled to PGC vesicles at a level of 0.60 ± 0.18 g of transferrin per g polymer. The proportion of FITC-dextran positive A431 cells was 42% (FITC-dextran solution), 74% (plain vesicles) and 90% (transferrin vesicles). Conclusions. Glucose and transferrin bearing chitosan based vesicles and glucose niosomes have been prepared. Glucose bearing vesicles bind Con-A to their surface. Chitosan based vesicles are taken up by A431 cells and transferrin enhances this uptake.

Tumor regression after systemic administration of a novel tumor-targeted gene delivery system carrying a therapeutic plasmid DNA

The possibility of using genes as medicines to treat cancer is limited by the lack of safe and efficacious delivery systems able to deliver therapeutic genes selectively to tumors by intravenous administration. We investigate if the conjugation of the polypropylenimine dendrimer to transferrin, whose receptors are overexpressed on numerous cancers, could result in a selective gene delivery to tumors after intravenous administration, leading to an increased therapeutic efficacy. The objectives of this study are to evaluate the targeting and therapeutic efficacies of a novel transferrin-bearing polypropylenimine dendrimer. The intravenous administration of transferrin-bearing polypropylenimine polyplex resulted in gene expression mainly in the tumors. Consequently, the intravenous administration of the delivery system complexed to a therapeutic DNA led to a rapid and sustained tumor regression over one month, with long-term survival of 100% of the animals (90% complete response, 10% partial response).The treatment was well tolerated by the animals, with no apparent signs of toxicity. Transferrin-bearing polypropylenimine may thus be a promising gene delivery system for cancer therapy.