Genetic interrogation of putative proteases in Plasmodium falciparum

Malaria remains a major cause of morbidity and mortality around the world. The emergence and spread of parasites resistant to all frontline antimalarials means that the identification of novel targets is vital for the development of drugs with novel mechanisms of action. The protozoan parasites of the Plasmodium genus cause malaria through the continual infection of and egress from red blood cells (RBCs). Plasmodium spp. encode uncharacterised orthologues to approved protease drug targets, namely HIV-1 protease and DPP4, for the treatment of AIDS and diabetes, respectively. In this project, DiCre-mediated conditional truncation of four putative proteases was performed in the malaria parasite, P. falciparum. Truncation of two of four proteins resulted in growth defects in the asexual blood stages. One putative serine protease, named S9C, was found to be important, but not essential, for intraerythrocytic development. The delay in growth was paired with stunted early parasite morphology and abnormal structure of the parasitophorous vacuole, in which the parasite resides within the RBC. A putative aspartyl protease that is conserved in eukaryotes, DNA-damage inducible protein 1 (Ddi1), is essential in the asexual blood stages, and its loss results in an almost complete block of erythrocyte invasion. We found evidence that Ddi1 was implicated in cellular proteostasis, potentially interacting with proteins involved in protein trafficking, sorting and quality control. We also find a potentially conserved interaction between Ddi1 and a subset of ubiquitinated proteins. For both S9C and Ddi1, a conditional allelic replacement with wildtype and mutant alleles demonstrated the importance of the putative proteolytic activity. The importance of the Ddi1 catalytic activity makes it a novel druggable antimalarial target. Furthermore, studying these putative proteases for the first time has uncovered new aspects of parasite biology in P. falciparum.Open Acces

Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies

The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection

The role of genomic location and flanking 3′UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I‐transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16‐mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I‐transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell‐cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell‐cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I‐transcribed ES, as well as conserved VSG 3′UTR 16‐mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei

Identification of the ISWI chromatin remodeling complex of the early branching eukaryote Trypanosoma brucei

ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution

Blocking VSG synthesis results in reduced clearance of surface-bound anti-VSG221 antibodies.

<p>Immunofluorescence microscopy analysis of <i>T</i>. <i>brucei</i> 221VB1.2 where VSG221 synthesis was blocked by the induction of <i>VSG221</i> RNAi for 0 or 8 hours (h). Cells were next coated with an anti-VSG221 antibody at 4°C (to stop endocytosis), and subsequently transferred to 37°C for the time indicated in minutes to reinitiate endocytosis. Cells were fixed, and the anti-VSG221 antibody was visualised using an anti-rabbit Alexa 488-conjugated secondary antibody. DNA is stained with DAPI (blue). Scale bar = 10 μm. As a lethality control, a comparable experiment was performed after the induction of RNAi against the essential TDP1 chromatin protein.</p

Blocking VSG synthesis results in impairment of internalisation of anti-VSG antibodies.

<p>Reduced rates of internalisation and lysosomal degradation of anti-VSG221 antibody after induction of a VSG221 synthesis block. VSG221 synthesis was blocked in <i>T</i>. <i>brucei</i> 221VB1.2 cells by the induction of <i>VSG221</i> RNAi for 8 hours (h). Cells were coated with an anti-VSG221 antibody at 4°C to stop endocytosis, and then transferred to 37°C to activate endocytosis for the time shown in minutes. The lysosomal thiol protease inhibitor FMK-024 was used to inhibit degradation of anti-VSG antibody in the lysosomal compartment, and facilitate its visualisation. Cells were then fixed and stained with an Alexa Fluor 488 coupled secondary antibody. The lysosomal compartment was visualised with an antibody against the p67 lysosomal protein stained with an Alexa Fluor 594 coupled secondary antibody. The merge of the two signals is shown in yellow. Below are shown panels of the trypanosomes visualised using differential interference contrast. Scale bar: 5 μm.</p

Motility analyses show an increase in swim velocity in cells where VSG synthesis has been inhibited.

<p><b>(A)</b><i>T</i>. <i>brucei</i> swims more persistently in the same direction after VSG synthesis is blocked. Particle motion tracks of cells where <i>VSG221</i> RNAi was induced for 0 or 8 hours (h). Each track represents the distance travelled by an individual cell over the course of measurement. One image was captured every 200 ms (5 Hz) for 1.7 minutes. Scale bar: 200 μm. <b>(B)</b> There is a significant increase in swim velocity (or directional swimming) after the induction of a VSG synthesis block for 8 hours. Velocity is shown in μm per second (s). The whole population (2500 cells) is compared with 50 bi-flagellated cells from the induced or uninduced population. Statistical significance was determined with Student’s t-test (***P<0.0001). <b>(C)</b> Increase in angular persistence in cells where VSG synthesis has been blocked. The average (av.) angular persistence is shown in either the whole cell population or bi-flagellated cells. Angular persistence is denoted on an arbitrary continuous scale of -1 to 1 where a value of 1 indicates continuous swimming in the same direction as observed after a 2 second period, a value of 0 represents cells moving in a random direction after this time period, and a value of -1 represents a trypanosome observed swimming in the reverse direction after 2 seconds. 1500 cells from the whole population (pop.) and 50 bi-flagellated cells are analysed. The observed change in motility after the induction of a VSG synthesis block was highly significant (***P<0.0001). <b>(D)</b> Histogram distribution showing an increase in persistent directional swimming in cells where VSG synthesis was blocked. The data is from 1500 cells from the whole population (either untreated or where <i>VSG221</i> RNAi has been induced for 8 hours) which are also analysed in panel <b>(C)</b>.</p

Induction of a VSG synthesis block leads to a decrease in clearance of surface-bound anti-VSG221 antibodies.

<p><b>(A)</b> Surface clearance of anti-VSG221 antibodies was analysed by flow cytometry in <i>T</i>. <i>brucei</i> 221VB1.2 where <i>VSG221</i> RNAi was induced for 0 or 8 hours (h). The cells were subsequently transferred to 4°C to stop endocytosis, and then coated with anti-VSG221 antibodies. Cells were next incubated at 37° to reinitiate endocytosis for the time indicated in minutes. The reaction was subsequently stopped, and the cells were fixed and stained with an AlexaFluor 488 coupled secondary antibody and propidium iodide (to visualise DNA content). The amount of surface anti-VSG221 antibody was determined at the G1 (orange), S (blue) or G2/ M (purple) cell cycle stages. <b>(B)</b> Quantitation of the reduced clearance of anti-VSG221 antibody after blocking VSG221 synthesis. Surface anti-VSG221 antibody was detected using an Alexa 488-conjugated secondary antibody, and total fluorescence was quantitated by flow cytometry. Mean fluorescence intensity values are expressed as a percentage of the value at 0 minutes. Results shown are the mean of three independent biological replicates with the standard deviation indicated with error bars. <b>(C)</b> Quantitation of the half-life of anti-VSG221 antibodies after blocking VSG221 synthesis. Results shown are the mean of three independent biological replicates with the standard deviation indicated with error bars. After fitting each data set to the non-linear regression model, statistical analysis was performed using the Student’s t-test *P<0.05.</p

The rate of internalisation of biotinylated VSG is unaffected by the induction of a block in VSG synthesis.

<p>VSG synthesis was arrested in <i>T</i>. <i>brucei</i> 221VB1.2 cells by the induction of <i>VSG221</i> RNAi for 8 hours (h). Endocytosis was stopped by cooling the cells to 4°C. Flow cytometry analysis of internalisation of biotinylated VSG. Cells were labelled with biotin, and transferred to 37°C for 0, 30, 60, 90, 180, 360 or 720 seconds to activate endocytosis. Remaining surface biotin was removed by the addition of ice cold stripping buffer. Cells were next fixed, permeabilised and incubated with Alexa Fluor 488 conjugated streptavidin. Mean fluorescence intensity values were normalised against that of time point 0 seconds. Error bars show the standard deviation of three independent biological replicates.</p