Date Published: November 28, 2016
Publisher: Public Library of Science
Author(s): Jackie L. Y. Cheung, Nadina V. Wand, Cher-Pheng Ooi, Sophie Ridewood, Richard J. Wheeler, Gloria Rudenko, Kent L. Hill.
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 African trypanosome Trypanosoma brucei is a unicellular parasite uniquely adapted to parasitize the mammalian bloodstream after inoculation by tsetse flies. Here, T. brucei establishes chronic infections leading to devastating diseases such as Human African Trypanosomiasis (HAT) or ‘nagana’ in livestock. T. brucei can also infect a broad range of African mammals, which tolerate low grades of trypanosome infection and serve as reservoirs for disease [1, 2]. Within the blood, as an extracellular pathogen T. brucei is confronted with continuous attack from the innate and adaptive arms of the host immune system. These include the complement system, antibodies and phagocytic cells such as macrophages. An essential trypanosome survival feature within this hostile environment, is a protective coat of Variant Surface Glycoprotein (VSG) [3, 4].
Here, we investigate the role of the VSG coat and its recycling in the phagocytosis of T. brucei by macrophages. We find that blocking VSG synthesis results in a significant increase in macrophage phagocytosis of trypanosomes, but only in the presence of anti-VSG antibodies. Blocking VSG synthesis for 8 hours resulted in reduced rates of clearance of anti-VSG antibodies. This was particularly pronounced in G2/ M stage cells, where the half-life of the anti-VSG antibody increased about two and a half fold from 39.3 ± 4.2 seconds to 99 ± 15.9 seconds. There was specifically a perturbation in the removal of VSG-antibody complexes, as rates of internalisation of total biotinylated surface VSG were not significantly changed after blocking VSG synthesis. There was no evidence for a restriction operating around the flagellar pocket of the stalled cells, as VSG antibody still reached the lysosomal compartment after a VSG synthesis block, although at slower rates than normal. Rates of endocytosis of various endocytic markers including tomato lectin, dextran or transferrin were not significantly reduced in the stalled cells. Motility was altered in the arrested cells, resulting in cells which swam persistently rather than tumbling. We postulate that these motility changes play a direct role in the reduced clearance of anti-VSG antibodies that we observe. These experiments shed light on T. brucei evasion of macrophages. In addition, they highlight the essentiality of high rates of VSG synthesis and internalisation coupled with VSG quality control in removing host cell opsonins, thereby enabling trypanosome escape from host phagocytes.