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

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

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

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

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

Inhibition of VSG synthesis in <i>T</i>. <i>brucei</i> leads to an increase in phagocytosis by macrophages in the presence of anti-VSG221 antibodies.

<p><b>(A)</b> Phagocytosis of <i>T</i>. <i>brucei</i> by mouse RAW264.7 macrophages. The cells are visualised by differential interference contrast (DIC) or after immunofluorescence microscopy using a monoclonal antibody against the <i>T</i>. <i>brucei</i> paraflagellar rod protein PFR2 (L8C4). This allows visualisation of <i>T</i>. <i>brucei</i> after phagocytosis by the macrophage. Scale bar: 5 μm. <b>(B)</b> Quantitation of the percentage of macrophages with one or more internalised <i>T</i>. <i>brucei</i> flagella. VSG synthesis was blocked by the induction of <i>VSG221</i> RNAi in <i>T</i>. <i>brucei</i> 221VG1.1 for 0 or 8 hours (h). Parasites were then either left untreated, or were opsonised with normal mouse serum (NMS), polyclonal anti-VSG221 antibody (Ab) or both NMS and anti-VSG221 antibody. Phagocytosis of <i>T</i>. <i>brucei</i> was quantified by immunofluorescence microscopy, and was found to be significantly increased in the presence of anti-VSG antibodies and NMS (single asterisk), or after induction of a VSG synthesis block in the presence of anti-VSG antibodies (asterisks over brackets). The results are the mean from three independent experiments (n = 200) with the standard deviation indicated with error bars. Statistical analysis was performed using the Student’s t-test **P<0.001, *P<0.05.</p

Proceedings of The HKIE Geotechnical Division 43rd Annual Seminar: Towards a Smart-Green-Resilient Geo-Future for World-class City

This seminar proceedings contain articles on the various research ideas of the academic community and practitioners presented at The HKIE Geotechnical Division 43rd Annual Seminar (GDAS2023). This seminarprovides a platform for policymakers, practitioners, and academia to share their insights and brainstorm ideas with a view to seizing future opportunities and shaping the new future of Hong Kong. GDAS2023 was organized by the Geotechnical Division, The Hong Kong Institution of Engineers on 19th May 2023. Seminar Title: The HKIE Geotechnical Division 43rd Annual SeminarSeminar Acronym: GDAS2023Seminar Date: 19 May 2023Seminar Location:  Hong KongSeminar Organizers: Geotechnical Division, The Hong Kong Institution of Engineers Link to the GDAS2021 Proceedings: Proceedings of The HKIE Geotechnical Division 41st Annual Seminar Link to the GDAS2022 Proceedings: Proceedings of The HKIE Geotechnical Division 42nd Annual Semina