GoKawiilMaterials availability
New plasmids associated with this study were deposited to Addgene under Mugnier Lab and McCulloch Lab and are associated with this publication.
Statistics and reproducibility
Sample size was not predetermined and experiments were not randomized or blinded. In vitro experiments were derived from two independently generated biological replicates, arising from different parentals except for the truncation experiments (same parental, but biologically unique clones), and offered high congruence sufficient for analysis. Mouse experimental size was selected based upon previous experiments with sufficient effect size observed1,9. WT versus μMT experiments were performed three independent times with independent vials of T. brucei stock using five mice of each genotype at the start of the infection. ΔRAD51 and Tb427VSG-8 infections in μMT mice were performed once from the same vial of stock, infecting four and five mice, respectively. Flow cytometry experiments were performed twice for Fig. 5b, with one sample, mosaic 5, repeated across both experiments, and once for Fig. 5c. Data presented are technical replicates. Antisera collected from mice represent four biological replicates.
A Shapiro–Wilk normality test was performed for all statistics calculated from mouse experiments, followed by the specified Wilcoxon test. Significance from FACS experiments was determined from a one-way ANOVA with post hoc Tukey HSD. All graphs include mean if n = 2 or mean ± s.d. if n ≥ 3. Calculations were performed in R (v.4.0.2). Details with exact P values and statistical test results can be found in Supplementary Data 2.
Parasites
Pleiomorphic EATRO1125 AnTat1.1 90-13 T. brucei parasites were maintained in HMI-9 media with 10% heat-inactivated FBS (F0960, 500 ml, Sigma-Aldrich) and 10% Serum Plus (14008C, 500 ml, Sigma-Aldrich) or in HMI-11 with 10% FBS where specified60. These parasites originated from K. Matthews. Parasites were passaged when they reached approximately 5 × 105 cells per ml unless otherwise specified. Monomorphic Single Marker Lister427 VSG221 TetR T7RNAP bloodstream form (NR42011; LOT: 61775530)61 T. brucei were maintained in HMI-9 up to 1 × 106 parasites per ml. VSG221 has since been renamed to VSG-2. Monomorphic Single Marker 427 1339 Cas9 TetR T7RNAP (bloodstream form) (NR-56793; LOT: 70056027) T. brucei subsp. brucei were maintained in HMI-9 up to 1 × 106 parasites per ml. This was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). EATRO1125 AnTat1.1 J1339 pleiomorphic parasites were a gift from K. Matthews62. Parasites were verified for expected VSG expression via amplicon sequencing. Parasite cultures were not tested for mycoplasma contamination.
Plasmids
Plasmids were synthesized with Gibson Assembly with a custom master mix63 unless otherwise specified. Whole plasmid sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology and their custom analysis and annotation pipelines. Detailed description of plasmid construction, using published publicly available plasmids64,65,66,67,68,69, can be found in the Supplementary Methods.
Transgenic parasites
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