Transcription Dependent Loss of an Ectopically Expressed Variant Surface Glycoprotein during Antigenic Variation in Trypanosoma brucei

International audienceThe single-cell parasite Trypanosoma brucei causes the fatal disease human African trypanosomiasis and is able to colonize the blood, fat, skin, and central nervous system. Trypanosomes survive in the mammalian host owing to a dense protective protein coat that consists of a single-variant surface glycoprotein species

RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Posté le 17 mars 2020 sur BioRxiv https://www.biorxiv.org/content/10.1101/2020.03.17.994905v1.full.pdf+htmlHomologous recombination dominates as the major form of DNA repair in Trypanosoma brucei , and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 is required for RAD51-dependent homologous recombination, phosphorylation of histone H2A and controlled resection following a DNA double strand break (DSB). Perhaps surprisingly, DSB resection in the rad50 nulls was not impaired and appeared to peak earlier than in the parental strains. Finally, we show that RAD50 suppresses DNA repair using donors with short stretches of homology at a subtelomeric locus, with null strains producing a greater diversity of expressed VSG variants following DSB repair. We conclude that RAD50 promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation

Single locus phosphoproteomics reveals phosphorylation of RPA-1 is required for generation of single-strand DNA following a break at a subtelomeric locus

Damage to the genetic material of the cell poses a universal threat to all forms of life. Central to the DNA damage response (DDR) is a phosphorylation signalling cascade that leads to the co-ordination of the cellular response to a DNA break. Identifying the proteins that are phosphorylated is crucial to understanding the mechanisms underlying this DDR. We have used SILAC-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following a single double strand break (DSB) at a chromosome internal locus and the subtelomeric bloodstream form expression site in Trypanosoma brucei . We report >6500 phosphorylation sites, including a core set of 211 DSB responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we find that there is a striking distinction between the proteins phosphorylated in response to a chromosome internal DSB and one at the active BES and describe a single phosphorylation event on Replication factor A (RPA) 1 that is required for efficient resection at a bloodstream form expression site

SHERLOCK4HAT: A CRISPR-based tool kit for diagnosis of Human African Trypanosomiasis

International audienceBackgroundTo achieve elimination of Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense (gHAT), the development of highly sensitive diagnostics is needed. We have developed a CRISPR based diagnostic for HAT using SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) that is readily adaptable to a field-based setting.MethodsWe adapted SHERLOCK for the detection of T. brucei species. We targeted 7SLRNA, TgSGP and SRA genes and tested SHERLOCK against RNA from blood, buffy coat, dried blood spots (DBS), and clinical samples.FindingsThe pan-Trypanozoon 7SLRNA and T. b. gambiense-specific TgSGP SHERLOCK assays had a sensitivity of 0.1 parasite/μL and a limit of detection 100 molecules/μL. T. b. rhodesiense-specific SRA had a sensitivity of 0.1 parasite/μL and a limit of detection of 10 molecules/μL. TgSGP SHERLOCK and SRA SHERLOCK detected 100% of the field isolated strains. Using clinical specimens from the WHO HAT cryobank, the 7SLRNA SHERLOCK detected trypanosomes in gHAT samples with 56.1%, 95% CI [46.25–65.53] sensitivity and 98.4%, 95% CI [91.41–99.92] specificity, and rHAT samples with 100%, 95% CI [83.18–100] sensitivity and 94.1%, 95% CI [80.91–98.95] specificity. The species-specific TgSGP and SRA SHERLOCK discriminated between the gambiense/rhodesiense HAT infections with 100% accuracy.InterpretationThe 7SLRNA, TgSGP and SRA SHERLOCK discriminate between gHAT and rHAT infections, and could be used for epidemiological surveillance and diagnosis of HAT in the field after further technical development

VEX1 Influences mVSG Expression During the Transition to Mammalian Infectivity in Trypanosoma brucei

International audienceThe Trypanosoma (T) brucei life cycle alternates between the tsetse fly vector and the mammalian host. In the insect, T. brucei undergoes several developmental stages until it reaches the salivary gland and differentiates into the metacyclic form, which is capable of infecting the next mammalian host. Mammalian infectivity is dependent on expression of the metacyclic variant surface glycoprotein genes as the cells develop into mature metacyclics. The VEX complex is essential for monoallelic variant surface glycoprotein expression in T. brucei bloodstream form, however, initiation of expression of the surface proteins genes during metacyclic differentiation is poorly understood. To better understand the transition to mature metacyclics and the control of metacyclic variant surface glycoprotein expression we examined the role of VEX1 in this process. We show that modulating VEX1 expression leads to a dysregulation of variant surface glycoprotein expression during metacyclogenesis, and that following both in vivo and in vitro metacyclic differentiation VEX1 relocalises from multiple nuclear foci in procyclic cells to one to two distinct nuclear foci in metacyclic cells - a pattern like the one seen in mammalian infective bloodstream forms. Our data suggest a role for VEX1 in the metacyclic differentiation process and their capacity to become infectious to the mammalian host

The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

International audienceHomologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation

DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice

International audienceAntigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes

Phosphoproteomic analysis of the response to DNA damage in Trypanosoma brucei

Damage to the genetic material of the cell poses a universal threat to all forms of life. The DNA damage response is a coordinated cellular response to a DNA break, key to which is the phosphorylation signalling cascade. Identifying which proteins are phosphorylated is therefore crucial to understanding the mechanisms that underly it. We have used SILAC-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following double stranded DNA breaks, at two distinct loci in the genome of the single cell eukaryote Trypanosoma brucei. Here, we report on the Trypanosoma brucei phosphoproteome following a single double strand break at either a chromosome internal or subtelomeric locus, specifically the Bloodstream form expression site. We detected >6500 phosphorylation sites, of which 211 form a core set of double strand break responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we have identified two novel phosphorylation events on Histone H2A and find that in response to a chromosome internal break, proteins are predominantly phosphorylated, while a greater proportion of proteins dephosphorylated following a DNA break at a subtelomeric bloodstream form expression site. Our data represents the first DNA damage phosphoproteome and provides novel insights into repair at distinct chromosomal contexts in Trypanosoma brucei