Fig 1.
Cas9-induced DNA breaks trigger antigenic variation.
(A) The schematic shows two examples of VSGs with salient features highlighted; the N-terminal signal sequence, Cys residues (red), predicted N-glycosylation sites (black) and C-terminal GPI anchor signal. Length, in amino acids, and class (B3 or C2) are indicated. See S2 Fig for more examples. (B) The schematic shows the active telomeric VSG (VSG-2) and the sites targeted by Cas9-sgRNAs to introduce DNA double-strand breaks. Recombination within 70-bp repeats triggers gene conversion and duplicative replacement of VSG-2 with a new VSG. (C) The protein blot shows robust inducible expression of Cas9. EF1α was used as loading control. (D) Immunofluorescence microscopy reveals switched and intermediate switching cells following Cas9-induction. Two switched sub-clones assessed by immunofluorescence microscopy are also shown. (E) An immunofluorescence microscopy time-course assay, with continuous Cas9 induction. Two biological replicates for each sgRNA. n > 90 cells for each sample at each time-point.
Fig 2.
RNA-seq confirms that switched cells retain common ESAGs.
(A) An immunofluorescence microscopy time-course assay, beginning on day-5; after three days of growth in tetracycline, to induce Cas9 with sgRNA-3, and a further two days after removal of tetracycline. Three technical replicates. n > 90 cells for each sample at each time-point. RNA was extracted from these samples for RNA-seq analysis. (B) The schematic shows the active telomeric VSG-ES (BES1) and the site targeted by sgRNA-3. Recombination with a silent bloodstream ES triggers replacement of VSG-2 with a new ES-VSG, while retaining the BES1 ESAGs. (C) RNA-seq analysis of VSG-ESs. Only the VSGs (3, 6 and 8) are activated, but not their respective ESAGs (grey boxes below each read-mapping profile, in BES7, 3 and 12), as illustrated in B. Values expressed in RPMs (reads per million). Flags indicate VSG-ES promoters; grey blocks indicate the extent of the 70-bp repeats. (D) The graph shows VSG expression levels as determined by RNA-seq on day-5 as jittered dots; averages of three replicates. The number of VSGs is: metacyclic ES-VSGs (M-ES) = 5, bloodstream ES-VSGs (B-ES) = 11, minichromosomal VSGs (MC) = 20. The horizontal bars indicate the mean for each group. Values from S1 Data. (E) The graph shows the length of the activated VSG set as jittered dots. ES-VSGs, n = 16; MC-VSGs n = 20. The summary of the data is shown as a boxplot, with the box indicating the IQR, the whiskers showing the range of values that are within 1.5*IQR and a horizontal line indicating the median. The notches represent for each median the 95% confidence interval (approximated by 1.58*IQR/sqrt(n)). The p value was derived using a two-tailed t-test.
Fig 3.
VSG dynamics are related to VSG template location and VSG length.
(A) A principal component analysis of read-counts (S1 Data) for the activated VSGs (n = 36) in each replicate during the RNA-seq time-course. (B) Relative read-counts for ES-VSGs (n = 16) and MC-VSGs (n = 20) over the RNA-seq time-course. Error bars, SD; three replicates. (C) Read-counts for individual ES-VSGs and MC-VSGs over the RNA-seq time-course. The upper panels show bloodstream ES-VSGs, red; metacyclic ES-VSGs, black; MC-VSGs, blue. Error bars, SD; three replicates. The lower panels show the same dataset but displayed relative to the day-5 values to emphasize the different behavior for the two sets of VSGs. (D) The graphs show fold-change in read-counts for either ES-VSGs or MC-VSGs between the day-5 and day-9 time-points relative to VSG length. R2 and p values were derived using regression analysis in Excel.
Fig 4.
VSG dynamics and relationship to other features of VSGs.
(A) The graphs show fold-change in read-counts for VSGs between the day-5 and day-9 time-points relative to VSG mass. (B) As in A but relative to predicted N-glycosylation sites. (C) As in A but relative to number of Cys residues. (D) The graphs show read-counts for VSGs at the day-5 time-point (a measure of VSG activation rate) relative to VSG length. (E) The graphs show fold-change in read-counts for VSGs between the day-9 and day-13 time-points relative to VSG length. Further comparisons between the day-13/17 and day-17/25 time-points and VSG length yielded R2 values <0.1 and p values >0.3. All R2 and p values were derived using regression analysis in Excel.
Fig 5.
Differential growth is associated with wider transcriptome differences.
(A) A principal component analysis of log2 RPKM (reads per kilobase per million) values (S1 Data) for the transcriptome (n = 7255) during the RNA-seq time-course. The closed datapoints represent averages while the open datapoints represent each replicate. (B) The scatterplots show transcriptome differences between the day-5 and either day-9 or day-25 samples, highlighting transcripts encoding nucleolar proteins (n = 35), tRNA synthetases (n = 23) or glycolytic enzymes (n = 11). p values were derived using a χ2 test for increased v decreased expression of these 69 genes. The upper panels indicate data distribution. (C) The graphs show cell numbers for 36 clones and six of those clones selected for RNA-seq (right-hand panel), also indicating the VSG expressed by each clone. The data are shown as jittered dots and, when n>1, the horizontal line indicates the mean and the box indicates the range of values. (D) A principal component analysis of log2 RPKM values (S1 Data) for the transcriptomes of the six selected clones. (E) The scatterplots show transcriptome differences between the pairs of clones indicated. Other details as in B above.