SummaryIn 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.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

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 host's 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.

SummaryPolo-like kinases (PLKs) are conserved eukaryotic cell cycle regulators, which play multiple roles, particularly during mitosis. The function of Trypanosoma brucei PLK was investigated in procyclic and bloodstream-form parasites. In procyclic trypanosomes, RNA interference (RNAi) of PLK, or overexpression of TY1-epitope-tagged PLK (PLKty), but not overexpression of a kinase-dead variant, resulted in the accumulation of cells that had divided their nucleus but not their kinetoplast (2N1K cells). Analysis of basal bodies and flagella in these cells suggested the defect in kinetoplast division arose because of an inhibition of basal body duplication, which occurred when PLK expression levels were altered. Additionally, a defect in kDNA replication was observed in the 2N1K cells. However, the 2N1K cells obtained by each approach were not equivalent. Following PLK depletion, the single kinetoplast was predominantly located between the two divided nuclei, while in cells overexpressing PLKty, the kinetoplast was mainly found at the posterior end of the cell, suggesting a role for PLK kinase activity in basal body and kinetoplast migration. PLK RNAi in bloodstream trypanosomes also delayed kinetoplast division, and was further observed to inhibit furrow ingression during cytokinesis. Notably, no additional roles were detected for trypanosome PLK in mitosis, setting this protein kinase apart from its counterparts in other eukaryotes.

The involvement of macromolecules in the formation of biological and other membranes has important implications for structural biology and nanoengineering. Using cetyl polyethylenimines of varying molecular weight and hydrophobicity, it was found that polymer hydrophobicity (mol % cetylation) controlled the nature of the self-assembly, giving micellar (cetyl groups < 23 mol %), vesicular (cetyl groups = 23−42 mol % or cetyl groups = 3−42 mol % with cholesterol), and dense nanoparticle (cetyl groups ≥ 49 mol %) aggregates. Thick (up to 15 nm) membranes due to the polyelectrolyte coating with the amphiphile were observed with low levels of cetylation only, and both dn/dc (indirectly) and vesicle/nanoparticle size (directly) varied linearly with mol % cetylation (r = 0.96−0.99).

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 cpb genes of Leishmania mexicana encode stageregulated, 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 COOHterminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 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 the cpb 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 (Asn 18 , Asp 60 , Asn 61 , Ser 64 , and Tyr 84 ) 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 Llike cysteine proteinases to diverse functions in parasitic protozoa.

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. Glycol chitosan modified by attachment of a strategic number of fatty acid pendant groups (11-16 mol%) assembles into unilamellar polymeric vesicles in the presence of cholesterol. These polymeric vesicles are found to be biocompatible and haemocompatible and capable of entrapping water-soluble drugs. By use of an ammonium sulphate gradient bleomycin (MW 1400), for example, can be efficiently loaded on to these polymeric vesicles to yield a bleomycin-to-polymer ratio of 0.5 units mg(-1). Previously polymers were thought to assemble into vesicles only if the polymer backbone was separated from the membrane-forming amphiphile by a hydrophilic side-arm spacer. The hydrophilic spacer was thought to be necessary to decouple the random motion of the polymer backbone from the ordered amphiphiles that make up the vesicle membrane. However, stable polymeric vesicles for use in drug delivery have been prepared from a modified carbohydrate polymer, palmitoyl glycol chitosan, without this specific architecture. These polymeric vesicles efficiently entrap water-soluble drugs.