Ultrastructural studies on parasitic flagellates

Summary available: p.4

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 Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalises with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasise differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite

The use of nano polymeric self-assemblies based on novel amphiphilic polymers for oral hydrophobic drug delivery.

Purpose: To investigate the use of nano self-assemblies formed by polyallylamine (PAA) modified with 5 or 10% mole fluorenylmethoxy carbonyl (Fmoc5/10), dimethylamino-1-naphthalenesulfonyl (Dansyl5/10) and 5% mole cholesteryl group (Ch5) for oral hydrophobic drug delivery. Methods: Propofol, griseofulvin and prednisolone were loaded into amphiphilic PAAs. Particle size and morphology of drug-loaded self-assemblies were determined using photon correlation spectroscopy and transmission electron microscopy. Solubilising capacity, in vitro drug release and formulation stability were analysed by HPLC, and in vitro biocompatibility studies (haemolysis and cytotoxicity) were carried out on bovine erythrocytes and Caco-2 cells, respectively. Dansyl10 and Ch5 griseofulvin formulations were administered intra-gastrically to rats, and drug plasma levels were analysed by HPLC. Results: Drug-encapsulated self-assemblies typically have hydrodynamic size of 300–400 nm. Dansyl10 exhibited universal drug solubiliser property and had significantly improved prednisolone, griseofulvin and propofol solubility by 145, 557 and 224-fold, respectively. Fmoc polymers resulted in modest drug solubility improvement. These polymers were non-haemolytic, did not enhance cytotoxicity compared to unmodified PAA, and demonstrated significant increase in griseofulvin plasma concentration compared to griseofulvin in water after oral administration. Conclusions: Ch5 and Dansyl10 showed promising potential as nano-carriers for oral hydrophobic drug delivery

Antitumor activity of the tea polyphenol epigallocatechin-3-gallate encapsulated in targeted vesicles after intravenous administration

The therapeutic potential of epigallocatechin gallate, a green tea polyphenol with anti-cancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study is to determine whether a tumor-targeted vesicular formulation of epigallocatechin gallate would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo. Transferrin-bearing vesicles encapsulating epigallocatechin gallate were intravenously administered to mice bearing subcutaneous A431 and B16-F10 tumors. The intravenous administration of epigallocatechin gallate encapsulated in transferrin-bearing vesicles resulted in tumor suppression for 40% of A431 and B16-F10 tumors. Animal survival was improved by more than 20 days compared to controls. Encapsulation of epigallocatechin gallate in transferrin-bearing vesicles is a promising therapeutic strategy

A unique Kelch domain phosphatase in Plasmodium regulates ookinete morphology, motility and invasion

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl(-) ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores

Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)-cyclin complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively

Novel tocotrienol-entrapping vesicles can eradicate solid tumors after intravenous administration

The therapeutic potential of tocotrienol, a vitamin E extract with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration. In this work, we demonstrated that novel transferrin-bearing, tocopheryl-based multilamellar vesicles entrapping tocotrienol significantly improved tocotrienol uptake by cancer cells overexpressing transferrin receptors. This led to a dramatically improved therapeutic efficacy in vitro, ranging from 17-fold to 72-fold improvement depending on the cell lines, compared to the free drug. In vivo, the intravenous administration of this novel tocotrienol formulation led to complete tumor eradication for 40% of B16F10 murine melanoma tumors and 20% of A431 human epidermoid carcinoma tumors. Animal survival was improved by more than 20 days compared to controls, for the two tumor models tested. These therapeutic effects, together with the lack of toxicity, potentially make transferrin-bearing vesicles entrapping tocotrienol a highly promising therapeutic system as part as an anti-cancer therapeutic strategy