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Abstract
Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen variant surface glycoprotein (VSG) to evade mammalian host immune responses at the bloodstream form (BF) stage. Monoallelic expression of BF Expression Site (BES)-linked VSGs and silencing of metacyclic VSGs (mVSGs) in BF cells are essential for antigenic variation, whereas silencing of both BES-linked and mVSGs in the procyclic form (PF) cells is important for cell survival in the midgut of its insect vector. We have previously shown that silencing BES-linked VSGs in BF cells depends on TbRAP1. We now show that TbRAP1 silences both BES-linked and mVSGs at both BF and PF stages. The strength of TbRAP1-mediated BES-linked VSG silencing is stronger in the PF cells than that in BF cells. In addition, Formaldehyde-Assisted Isolation of Regulatory Elements analysis and MNase digestion demonstrated that depletion of TbRAP1 in PF cells led to a chromatin structure change, which is significantly stronger at the subtelomeric VSG loci than at chromosome internal loci. On the contrary, no significant chromatin structure changes were detected on depletion of TbRAP1 in BF cells. Our observations indicate that TbRAP1 helps to determine the chromatin structure at the insect stage, which likely contributes to its strong silencing effect on VSGs.
Highlights
Human African trypanosomiasis is caused by infection of Trypanosoma brucei and is inevitably fatal without treatment
To reduce the phenotypic variations often associated with clonal cell lines, we obtained a pool of TbRAP1 RNA interference (RNAi) cells for subsequent detailed analyses, and critical phenotypes were confirmed in the PRi-C2 clone (Supplementary Figures S1B, D and F and S8B)
We observed a mild decrease in the G1 population and a mild increase in the G2/M and the sub G1 populations in PRi-pool cells (Figure 1D, Supplementary Figure S2A), which is similar to what we observed in TbRAP1-depleted bloodstream form (BF) cells in our current and earlier studies (Supplementary Figure S2B and C) [23]
Summary
Human African trypanosomiasis is caused by infection of Trypanosoma brucei and is inevitably fatal without treatment. T. brucei cells stay in extracellular spaces and express variant surface glycoprotein (VSG) as its major surface antigen that is exposed to the host immune system [1,2]. To evade the host’s immune responses, T. brucei cells undergo antigenic variation and regularly switch their VSG coat [3], which is essential for a persistent infection. There are >1000 VSG genes and pseudogenes in the T. brucei genome [4], in BF cells, VSGs are expressed exclusively from BF VSG Expression Sites (BESs), which are polycistronically transcribed by RNA Pol I [5]. The T. brucei 427 strain used in this study has $20 nearly identical BESs [7], 14 of which carry distinctive VSG genes [8]. VSG monoallelic expression ensures the effectiveness of antigenic variation and is essential for T. brucei virulence
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