VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection

Abstract

Trypanosoma brucei relies on antigenic variation of its variant surface glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10(-4) per division. We find a highly reproducible hierarchy of VSG activation, which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterized switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi-based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro.

Fig. 1

Induction of VSG221 RNAi results in selection of new VSG switch variants. Above is a Coomassie stained SDS/PAGE gel with T. brucei total protein lysates, with the prominent band observed corresponding to VSG. Known VSGs are indicated with arrows, with VSGX putatively corresponding to an unknown VSG. Control protein lysates are from a T. brucei strain expressing VSGV02: T. brucei 221GP1(VO2+) (Sheader et al., 2004) (Lane 1), or a T. brucei strain expressing VSG221: T. brucei HNI(221+) (Rudenko et al., 1998) (Lane 2). Protein lysates isolated from T. brucei 221VG2.1 where VSG RNAi was induced with tetracycline for 0, 65, 112, 135 or 157 hours are loaded in lanes 3–7 respectively. Below are panels from the Western blot of the same gel reacted with anti-VSG221 or VSG1.8 antibodies. As a control for protein loading, the blot was reacted with an antibody against BiP (Bangs et al., 1993). The lane with protein markers is indicated with M.

Fig. 2

Experimental procedure for selection of independent VSG switch events using VSG RNAi rather than an immune system. A culture of T. brucei 221VG1.1 or T. brucei 221VG2.1 was serially diluted, and each dilution was distributed over a 96-well plate at a concentration of 100–800 cells ml⁻¹. As the VSG switching frequency is about 10⁻⁴ in our strain using this procedure, individual wells would be highly unlikely to already contain a VSG switch variant. The wells were then expanded for typically 3 generations to allow a VSG switch to occur. Tetracycline was added to all of the wells to induce VSG221 RNAi. This results in the cell-cycle arrest and subsequent death of all trypanosomes which had not switched to a new VSG. After incubation for 6–8 days clonal T. brucei variants were scored and analysed. See Experimental Procedures for detailed protocol.

Fig. 3

Strategy for categorisation of switched T. brucei clones according to the mechanism used for VSG switching. The large coloured boxes indicate trypanosomes, with the parental strain indicated above and the switch variants below. The VSG ES promoters are indicated with flags, transcription with arrows, and various genes with solid coloured boxes. After the induction of VSG RNAi, cells that had switched away from VSG221 were characterised further. A comparison of the genotypes and phenotypes of the swiched T. brucei clones allowed the extrapolation of which VSG switching mechanism had mediated the switch. GFP expression was determined by microscopy. Cells which had retained GFP expression were categorised as having switched via gene conversion or telomere exchange, which could be differentiated by establishing the presence of VSG221. The presence (+) or absence (−) of GFP or VSG221 sequences was determined using PCR. Cells which had retained GFP and VSG221 but did not express either gene were established to have switched via an in situ switch. In the event that all marker genes in the inactivated ES were lost, the switched clone was categorised as having switched via an in situ switch plus deletion of the old ES (Cross et al., 1998; Rudenko et al., 1998)

Fig. 4

Induction of VSG221 RNAi allows the isolation of T. brucei which has switched its VSG coat. A. Transcriptional switching between ESs (in situ switch) is the preferred switching mechanism in T. brucei 427. A total of 127 T. brucei VSG221 RNAi resistant variants were characterised, with the percentage calculated to have switched by each mechanism indicated with bars. The mechanisms indicated are in situ switch (IS), in situ switch coupled with delection of the old ES (IS-del), gene conversion (GC) and telomere exchange (TX). T. brucei clones that were resistant to VSG RNAi but had retained the VSG221 coat are indicated in the category “Broken RNAi”. B. Induction of VSG RNAi in the presence of puromycin selection for activity of the VSG221 ES results in the selective isolation of trypanosomes which have switched via DNA rearrangements. In addition, there is increased isolation of clones which have inactivated the RNAi machinery. A total of 41 clones was analysed.

Fig. 5

Preferential hierarchies of VSG activation in T. brucei 427. A. Frequency of activation of different VSGs after induction of VSG RNAi. Results were compared between experiments performed in the presence or absence of drug selection on the active VSG221 ES. Results from experiments performed without drug selection for the active ES (light blue bars) are shown using a total of 127 T. brucei switch variants. These results are compared with those from experiments performed in the presence of drug selection for the active ES (dark purple bars) (41 variants analysed). The VSG type identified by cDNA sequencing is indicated below. Cells still expressing VSG221 after induction of VSG RNAi appeared to have inactivated the VSG RNAi machinery. B. Preferential hierarchies of VSG activation plotted per experiment performed in the absence of puromycin selection for the active VSG221 ES. The number of the experiment is indicated on the left. The number (No.) of switch variants isolated per experiment is indicated on the right. The relative percentage of the different activated VSGs per experiment are indicated with coloured bars.

Fig. 6

RNA analysis of T. brucei VSG switch variants which have switched via an in situ switch (A), telomere exchange (B) or gene conversion (C). A. VSG switch variants which have switched away from VSG221 via a transcriptional in situ switch. Total RNA from the parental T. brucei VG2.1 (lane 1) is compared with that from switch variants T.b.N19–2R2 (lane 2), T.b.N9–1R12 (lane 3), T.b.W3-1R2 (lane 4) and T.b.N9-1R15 (lane 5). B. Variants which have switched via telomere exchange. Parental T. brucei VG2.1 RNA is compared with that from switch variant T.b.N9-1R1 (lane 2), T.b.N17-1R6 (lane 3), T.b.S2-1R2 (lane 4) and T.b.N17-1R11 (lane 5). C. Transcriptional analysis of variants which have switched via gene conversion. Parental T. brucei VG2.1 RNA is compared with that from switch variant T.b.N17-1R2 (lane 2), T.b.S3-1R3 (lane 3), T.b.N17-1R3 (lane 4) and T.b.N17-1R4 (lane 5).

Fig. 7

Pulsed Field Gel analysis of selected VSG switch events away from VSG221. (A) Switches from VSG221 to VSGbR2. The ethidium stained gel is shown in panel E with the location of VSG221 hybridising bands marked with arrows. The VSG221 ES is located on a 3 MB chromosome, and the VSG221 RNAi construct hybridising with the VSG221 probe on a minichromosome (221 MC). The three VSGbR2 genes separated under these conditions in the parental T. brucei line are indicated with arrowheads on the right. Lane P has DNA from the parental T. brucei VG1.1 strain. Lane 1 has DNA from a switched clone which has activated VSGbR2 via an in situ switch (IS) (T.b.N9-1R10). Lane 2 has DNA from a clone which has activated VSGbR2 via a telomere exchange (TX) (T.b.N17-1R6). Lane 3 has DNA from a clone which has switched via gene conversion (GC) (T.b.N17-1R5). The blots in the different panels are probed with the VSG indicated below. (B) Switches from VSG221 to VSG121. Lane P has DNA from the parental T. brucei VG1.1. The three VSG121 genes separated under these conditions in the parental T. brucei line are indicated with arrowheads on the right. Lane 1 has DNA from a clone which has switched to VSG121 via an in situ switch (T.b.N9-1R7), Lane 2 has DNA from a clone which has activated VSG121 gene via a telomere exchange (T.b.N9-1R1), and Lane 3 has DNA from a clone which has switched via gene conversion (T.b.S3-1R5). (C) Switches from VSG221 to VSGJS1. Lane P has DNA from the parental T. brucei VG1.1. The two copies of VSGJS1 separated under these conditions in the parental T. brucei line are indicated with arrowheads on the right. Lane 1 shows DNA from a clone which has activated VSGJS1 via an in situ switch (T.b.W5-2R9), Lane 2 shows DNA from a switched clone which has activated VSGJS1 gene via telomere exchange (T.b.N17-1R11), and Lane 3 shows DNA from a clone which has switched via gene conversion (T.b.S3-1R3). Note that the VSGJS1 ES appears to be located on the same chromosomal band as the VSG221 ES.

Fig. 8

Additional DNA rearrangements can occur during duplication of the donor VSG by gene conversion. During activation of VSGbR2 by gene conversion, there is often evidence for loss of copies of VSGbR2 in addition to duplication of VSGbR2 into the VSG221 ES. The ethidium stained gel (Eth) shows separation of T. brucei chromosomes, whereby bands containing the VSG221 ES on a 3 MB chromosome and the VSG221 construct on a minichromosome (221 MC) are indicated with arrows. The three VSGbR2 genes separated under these conditions in the parental T. brucei line are indicated with arrowheads on the right. Lane P shows DNA from the parental T. brucei VG 1.1 line. Lanes 1–5 contains DNA from clones which have all switched via gene conversion of VSGbR2 into the VSG221 ES. In all of these cases VSGbR2 is duplicated into the VSG221 ES, and there is loss of VSG221. However, in many clones there is loss of additional VSGbR2 copies which appear unrelated to the switch. Lane 1 shows DNA from T.b.N17-2R1, Lane 2 from T.b.N17-2R4, Lane 3 from T.b.N17-2R5, Lane 4 from T.b.S2-1R7 and Lane 5 from T.b.S2-1R3. The chromosomal bands that miss copies of VSGbR2 are indicated with arrows.