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

Abstract

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.

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei

“…VSG is central to T. brucei pathogenicity . Surprisingly, VSG is essential even in vitro in BF T. brucei , and blocking its synthesis with RNAi results in cells arresting at a very precise cell‐cycle stage, immediately before cell division .…”

Infections With Extracellular Trypanosomes Require Control by Efficient Innate Immune Mechanisms and Can Result in the Destruction of the Mammalian Humoral Immune System

“…Additional data suggest that the presence of an inflammatory environment aids this process, most likely due to the fitness reducing effects of TNF and NO, either by themselves or when combined. Interestingly, by artificially blocking VSG synthesis using an RNAi approach, it was shown that the process of macrophage phagocytosis was much more efficient (68). This finding suggests that VSG recycling is a key process in the defense of the parasite against the attack by the mammalian antibody response.…”

High-speed multifocal plane fluorescence microscopy for three-dimensional visualisation of beating flagella

“…Furthermore, they can invade tissues 8 . Together, this speaks to multiple functions of flagellum motility, now known to include complex collective motion of densely packed cells [9][10][11] and surface antibody clearance 12,13 . The motility of Trypanosoma cruzi and Leishmania have been less extensively analysed, however flagellum motility is likely similarly multifunctional 14 .…”