Pellicular Membrane Research Articles

TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the flagellar pocket collar (FPC). TbBILBO1 is the first-described FPC protein of Trypanosoma brucei. It is essential for parasite survival, FP and FPC biogenesis. In this work, we characterize TbKINX1B, a novel TbBILBO1 partner. We demonstrate that TbKINX1B is located on the basal bodies, the microtubule quartet (a set of four microtubules) and the FPC in T. brucei. Down-regulation of TbKINX1B by RNA interference in bloodstream forms is lethal, inducing an overall disturbance in the endomembrane network. In procyclic forms, the RNAi knockdown of TbKINX1B leads to a minor phenotype with a small number of cells displaying epimastigote-like morphologies, with a misplaced kinetoplast. Our results characterize TbKINX1B as the first putative kinesin to be localized both at the basal bodies and the FPC with a potential role in transporting cargo along with the microtubule quartet.

213 - Symptoms of severe serotonin syndrome – A pooled case analysis

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function.

Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.

Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body via the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

Proteomics and theTrypanosoma bruceicytoskeleton: advances and opportunities

Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.

Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu.

Acylation-dependent and-independent membrane targeting and distinct functions of small myristoylated proteins (SMPs) in Leishmania major

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function.

Improving TEM Fixation with Additives

Attempts to fix the gregarinePterospora floridiensis(a protozoan parasite found in marine bristle worms) for TEM using a conventional glutaraldehyde and osmium protocol produced poor preservation of the outer pellicular membranes and internal membranous structures. However, fixation success was achieved using the well-known fixative additives tannic acid and potassium ferrocyanide, which improved membrane preservation, cytoplasmic integrity, and contrast in both the unencysted feeding stage ofP. floridiensisas well as the difficult-to-fix cyst stage. The longer fixation times (24 hours) needed for the cysts were compatible with the modified fixatives. Interestingly,bothadditives were needed to improve fixation.

Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane

Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms.

IMC1b Is a Putative Membrane Skeleton Protein Involved in Cell Shape, Mechanical Strength, Motility, and Infectivity of Malaria Ookinetes*

Membrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

Biogenesis of the Trypanosome Endo-Exocytotic Organelle Is Cytoskeleton Mediated

Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.

Possibility of membrane modification as a mechanism of antimony resistance in Leishmania donovani

Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resistance whose localization in the present study was confirmed on the pellicular plasma membrane of the parasite thereby indicating towards membrane modification as a mechanism of resistance in this resistant isolate.

Flagellar membrane trafficking in kinetoplastids

Trypanosomes are flagellated protozoa belonging to the order Kinetoplastidae. Three trypanosomes, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are the causative agents of the human diseases: African sleeping sickness, Chagas disease, and leishmaniasis, respectively. These organisms have complex life cycles involving bloodsucking insects and vertebrates; humans become infected when infested insects feed on them. The trypanosome contains several unique organelles, one of the most important and conspicuous of which is the flagellum (Fig. 1). The trypanosome flagellum is a unique multifunctional organelle that plays critical roles in motility, chemotaxis, cell signaling, and host cell invasion (Landfear and Ignatushchenko 2001; Gull 2003; Hill 2003). In addition to containing a classical axonemal microtubular motor, the trypanosome flagellum also includes a paracrystalline structure called the paraflagellar rod (PFR), positioned alongside the axoneme. The flagellum emerges from the cell body from an invagination known as the flagellar pocket. As the cell body is constrained by a tight corset of cross-linked subpellicular microtubules (Fig. 1b), all vesicular trafficking into and out of the cell occurs at the non-microtubule-bound flagellar pocket, as does the trafficking of flagellar membrane-associated proteins between the cytoplasm and the flagellum (Bloodgood 1990; McConville et al. 2002). Because no new protein synthesis takes place in the flagellum, all protein components must be imported to the flagellum from the cell body, also via the flagellar pocket. To enter the flagellum, a protein must first pass through a cytoskeletal “neck” region within the flagellar pocket known as the flagellar pore complex (Rosenbaum and Witman 2002; Gull 2003). This complex, which contains structures including the basal body and transition zone filaments, is believed to act as a “stopper” for the flagellum, providing a selective barrier for protein entry (Vickerman and Preston 1976; Dentler and Adams 1992). Regulated flagellar protein import creates a flagellar microenvironment that is unique from that of the rest of the organism, where some of the protein and lipid components differ in composition from those in the cell body (Bloodgood 1990; Landfear and Ignatushchenko 2001). Because of differences in composition and function, the surface membrane of a trypanosome is divided into three distinct but contiguous domains: the pellicular (cell body) membrane, flagellar membrane, and flagellar pocket membrane (Balber 1990; Landfear and Ignatushchenko 2001; Gull 2003). As the flagellum is not a “closed” membrane-bound organelle like the mitochondrion or a lysosome, it has this far Parasitol Res DOI 10.1007/s00436-006-0329-2

A Flagellum-specific Calcium Sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.

Monoclonal antibodies to Hammondia hammondi allowing immunological differentiation from Toxoplasma gondii.

Five murine monoclonal antibodies (MAbs) were developed against purified sporozoites of Hammondia hammondi. Despite a large antigenic similarity between the 2 closely related coccidia, H. hammondi and Toxoplasma gondii, these MAbs only reacted with H. hammondi. Three MAbs, ID3, 3F2, and 4C9-7, recognized antigens of 38 kDa localized in rhoptries (1D3), in rhoptries and in oocyst and cyst walls (3F2), and in rhoptries and the apical region (4C9-7). Another MAb, 4C9-10, reacted with a 27-kDa antigen in dense granules of sporozoites and tachyzoites, and MAB 11B3 labeled an antigen of >94 kDa located in the pellicular membrane of the 3 stages of the parasite. These MAbs could be used for a rapid discrimination of the 2 coccidia in epidemiological studies or for diagnostic purposes in tissues.

Cytoskeletal association is important for differential targeting of glucose transporter isoforms in Leishmania.

The major glucose transporter of the parasitic protozoan Leishmania enriettii exists in two isoforms, one of which (iso-1) localizes to the flagellar membrane, while the other (iso-2) localizes to the plasma membrane of the cell body, the pellicular membrane. These two isoforms differ only in their cytosolic NH2-terminal domains. Using immunoblots and immunofluorescence microscopy of detergent-extracted cytoskeletons, we have demonstrated that iso-2 associates with the microtubular cytoskeleton that underlies the cell body membrane, whereas the flagellar membrane isoform iso-1 does not associate with the cytoskeleton. Deletion mutants that remove the first 25 or more amino acids from iso-1 are retargeted from the flagellum to the pellicular membrane, suggesting that these deletions remove a signal required for flagellar targeting. Unlike the full-length iso-1 protein, these deletion mutants associate with the cytoskeleton. Our results suggest that cytoskeletal binding serves as an anchor to localize the iso-2 transporter within the pellicular membrane, and that the flagellar targeting signal of iso-1 diverts this transporter into the flagellar membrane and away from the pellicular microtubules.

Effects of phenothiazine neuroleptic drugs on the microtubular-membrane complex in bloodstream forms of Trypanosoma brucei.

African trypanosomes are parasitic protozoa causing sleeping sickness in humans and related diseases in domestic animals against which no entirely satisfactory forms of chemotherapy are yet available. It was previously shown that related species of trypanosomes, as well as procyclic (insect) forms of Trypanosoma brucei are extremely sensitive to the action of phenothiazine neuroleptic drugs in vitro. In this work, we have carried out a more detailed investigation of the effects of thioridazine, one of the most potent neuroleptic phenothiazine drugs known, on the morphology of the infective bloodstream forms of T. brucei, with particular reference to the parasite's prominent pellicular membrane complex. Our data show that this drug induces rapid changes in cell shape that appear to involve some reorganization of the microtubular membrane skeleton, but does not affect the structural integrity of the microtubular complex. Another early consequence of drug action involved damage to nuclear and cytoplasmic membranes and the appearance of tubular arrays of coated membrane within the flagellar pocket. It was also revealed that the drug induces a rapid release of the variant-specific glycoprotein (VSG) which makes up the surface coat protecting bloodstream forms of the parasite against the host immune system. Our evidence suggests that this release of VSG involves cleavage of the protein's glycosyl-phosphatidylinositol (GPI) membrane anchor by endogenous GPI-specific phospholipase C, probably as a consequence of minor damage to the parasite plasma membrane induced by the drug.

Cloning and structure-function analysis of the Leishmania donovani kinetoplastid membrane protein-11

This report describes the complete translated gene sequence, predicted secondary structure and lipid bilayer association of a novel kinetoplastid membrane protein (KMP-11) from Leishmania donovani promastigotes. KMP-11 was previously referred to as the lipophosphoglycan-associated protein (LPGAP). The isolation, species distribution and chemical characterization, including a partial protein sequence analysis and post-translational modifications, of this major membrane component have been described [Jardim, Funk, Caprioli and Olafson (1995) Biochem. J. 305, 307-313]. C.d. measurements of KMP-11 indicated a very high helical content estimated to be approximately 86% in trifluoroethanol. This was in agreement with computer-based secondary-structure analyses which predicted KMP-11 to be almost exclusively alpha-helical, with the protein adopting a helix-loop-helix motif. Arrangement of the residues located in the putative helical regions on an Edmundson helical wheel showed that this molecule could have a strongly amphipathic conformation and provided an explanation for how such a highly charged protein might be inserted into the plasma membrane. Evidence in support of KMP-11 association with lipid bilayers was provided by showing that KMP-11 could mediate carboxyfluorescein release from liposomes. These findings suggested that KMP-11 may function in part to increase bilayer pressure, stabilizing molecules such as lipophosphoglycan within the parasite pellicular membrane.

Development of Plasmodium berghei ookinetes to young oocysts in vitro.

The mosquito stage of Plasmodium berghei was cultivated in vitro, with special attention to ookinete transformation into early oocyst. The ookinetes were obtained by in vitro culture of gametocytes taken from infected mice, purified by density gradient of metrizoic acid or a lymphocyte separation medium, and incubated either in acellular culture or in co-cultivations with mosquito cells. In acellular culture, the ookinetes were found to aggregate with each other and transformed from banana to round shapes. Their inner pellicular membranes and subpellicular microtubules partially disappeared, indicating that development to early oocyst had occurred. Co-cultivation wtih Aedes albopictus cells (C6/36 clone) revealed that ookinetes transformed into early oocyst in the medium, or invaded the cells and then transformed to early oocysts within the cell cytoplasm as well. However all of these transformed cells failed to develop further, i.e., neither deposition of the oocyst capsule nor nuclear division was observed. Many ookinetes which failed to penetrate the Aedes cells were phagocytized within three days of culture. A significant difference between invaded and transformed oocysts and phagocytized ookinetes was seen in that the former lacked vacuole membrane. Co-cultivation with Toxorhynchites amboinensis cells (TRA-284-SFG clone) permitted transformation of ookinetes into early oocysts in the medium as in the acellular culture, but no ookinete invasion nor phagocytosis by the cell was observed.

Antiserum directed against cell surface antigens is lethal toLeishmania donovani promastigotes

The purified flagellar fraction ofLeishmania donovani promastigotes consists of 30–35 polypeptides. Antiserum raised against this fraction reacts with both flagella and pellicular membrane antigens as evident from immunoblot and immunofluorescence studies. Only 3 of these immunoreactive polypeptides are flagellum-specific. The antiserum agglutinates the cells and inhibits their growth in liquid culture medium. Moreover, glucose uptake and glucose-stimulated oxygen uptake of the promastigotes are significantly inhibited by the antiserum. The results indicate that the antiserum has a profound lethal effect on the invitro propagation of the parasite.