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1.
J Biol Chem ; : 107657, 2024 Aug 09.
Article in English | MEDLINE | ID: mdl-39128729

ABSTRACT

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.

2.
EBioMedicine ; 85: 104308, 2022 Nov.
Article in English | MEDLINE | ID: mdl-36374773

ABSTRACT

BACKGROUND: To 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. METHODS: We 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. FINDINGS: The 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. INTERPRETATION: The 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. FUNDING: Institut Pasteur (PTR-175 SHERLOCK4HAT), French Government's Investissement d'Avenir program Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (LabEx IBEID), and Agence Nationale pour la Recherche (ANR-PRC 2021 SherPa).


Subject(s)
Trypanosomiasis, African , Humans , Animals , Trypanosomiasis, African/diagnosis , Trypanosoma brucei gambiense/genetics
3.
Front Cell Dev Biol ; 10: 851475, 2022.
Article in English | MEDLINE | ID: mdl-35450294

ABSTRACT

The 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.

4.
mBio ; 13(2): e0384721, 2022 04 26.
Article in English | MEDLINE | ID: mdl-35229632

ABSTRACT

In the mammalian host, Trypanosoma brucei is coated in a single-variant surface glycoprotein (VSG) species. Stochastic switching of the expressed VSG allows the parasite to escape detection by the host immune system. DNA double-strand breaks (DSB) trigger VSG switching, and repair via gene conversion results in an antigenically distinct VSG being expressed from the single active bloodstream-form expression site (BES). The single active BES is marked by VSG exclusion 2 (VEX2) protein. Here, we have disrupted monoallelic VSG expression by stably expressing a second telomeric VSG from a ribosomal locus. We found that cells expressing two VSGs contained one VEX2 focus that was significantly larger in size than the wild-type cells; this therefore suggests the ectopic VSG is expressed from the same nuclear position as the active BES. Unexpectedly, we report that in the double VSG-expressing cells, the DNA sequence of the ectopic copy is lost following a DSB in the active BES, despite it being spatially separated in the genome. The loss of the ectopic VSG is dependent on active transcription and does not disrupt the number or variety of templates used to repair a BES DSB and elicit a VSG switch. We propose that there are stringent mechanisms within the cell to reinforce monoallelic expression during antigenic variation. IMPORTANCE The 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. Stochastic switching of one VSG for an immunologically distinct one enables the parasite to escape recognition by the host immune system. We have disrupted monoallelic antigen expression by expressing a second VSG and report that following DSB-triggered VSG switching, the DNA sequence of the ectopic VSG is lost in a transcription-dependent manner. We propose that there are strict requirements to ensure that only one variant antigen is expressed following a VSG switch, which has important implications for understanding how the parasite survives in the mammalian host.


Subject(s)
Trypanosoma brucei brucei , Trypanosomiasis, African , Animals , Antigenic Variation , Gene Conversion , Humans , Mammals , Membrane Glycoproteins , Trypanosoma brucei brucei/genetics
5.
PLoS Pathog ; 17(11): e1010038, 2021 11.
Article in English | MEDLINE | ID: mdl-34767618

ABSTRACT

Antigenic 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.


Subject(s)
Antigenic Variation , DNA Breaks, Double-Stranded , DNA, Protozoan/genetics , Protozoan Proteins/genetics , Trypanosoma/immunology , Trypanosomiasis/immunology , Variant Surface Glycoproteins, Trypanosoma/genetics , Animals , DNA Repair , Gene Conversion , Protozoan Proteins/immunology , Repetitive Sequences, Nucleic Acid , Trypanosoma/genetics , Trypanosomiasis/genetics , Trypanosomiasis/parasitology , Variant Surface Glycoproteins, Trypanosoma/immunology
6.
Nucleic Acids Res ; 49(3): 1436-1454, 2021 02 22.
Article in English | MEDLINE | ID: mdl-33450001

ABSTRACT

Homologous 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.


Subject(s)
Antigenic Variation , DNA-Binding Proteins/physiology , MRE11 Homologue Protein/physiology , Protozoan Proteins/physiology , Recombinational DNA Repair , Trypanosoma brucei brucei/genetics , DNA Breaks, Double-Stranded , Trypanosoma brucei brucei/immunology
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