Phosphorylation of eIF2α on Threonine 169 is not required for Trypanosoma brucei cell cycle arrest during differentiation.

The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in Trypanosoma brucei occurs in two steps in vivo. First proliferating 'slender' bloodstream forms differentiate to non-dividing 'stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.This work was supported by Fapesp grants 09/52047-5 and 11/51973-3 to B.A.C. and S. S., respectively, and CNPq grants 309860/2011-3 and 478903/2012-0 to B.A.C. and 477143/2011-3 and 445655/2014-3 to S.S.. C.C.A. was supported by a Fapesp doctoral fellowship (2007/59753-7) and a CAPES-PSDE fellowship. Work in Cambridge and Bristol was supported by Wellcome Trust Project, Grants 085956 and 088099 respectively.This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.molbiopara.2016.03.00

Proteome-wide analysis of Trypanosoma cruzi exponential and stationary growth phases reveals a subcellular compartment-specific regulation

Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation

Development of a <i>Trypanosoma cruzi</i> strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2)

<div><p>Background</p><p>Chagas disease also known as American trypanosomiasis is caused by the protozoan <i>Trypanosoma cruzi</i>. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. <i>T</i>. <i>cruzi</i> strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to <i>T</i>.<i>cruzi</i> genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a <i>Trypanosoma cruzi</i> Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2.</p><p>Methods/Principal findings</p><p>The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate <i>T</i>. <i>cruzi</i> strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped <i>T</i>. <i>cruzi</i> reference strains. For identification, the MS/MS peptide spectra of unknown <i>T</i>. <i>cruzi</i> cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms.</p><p>Conclusions and significance</p><p>This is the first study that develops a MS-based platform for <i>T</i>. <i>cruzi</i> strain typing. Indeed, the Tc-STAMS2 method allows <i>T</i>. <i>cruzi</i> strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current <i>T</i>. <i>cruzi</i> genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.</p></div

Genotype discrimination based on spectral similarity searches.

<p>Six <i>T</i>.<i>cruzi</i> strains (Sylvio X10 cl1, Y, M6241 cl6, CanIII cl1, MN cl2 and CL Brener) belonging to six DTUs were selected to test the Tc-STAMS2 method. The unique dot product SDSS score is reported along with the number of MS/MS spectra matches (unique/total). Genotypes identified with the highest score are highlighted in gray.</p

MS/MS spectral matching comparison using different growth phases of Sylvio X10 cl1 (DTU-I) using SpectraST software.

<p>St_1—Stationary phase; St_2—Biological replicate stationary phase; Ex—Exponential phase; Ex_2—Biological replicate exponential phase.</p

Tc-STAMS2 approach tested against: 1) the CL14 <i>T</i>.<i>cruzi</i> strain, 2) <i>T</i>. <i>vivax</i> dataset and 3) LC-MS/MS datasets from <i>E</i>.<i>coli</i> and human and mouse placental tissues.

<p>The sensitivity of Tc-STAMS2 approach was tested for the detection of intra-DTU strains such as CL14 and CLBrener strains belonging to DTU-VI. Moreover, the specificity of Tc-STAMS2 approach was tested for the assignment of MS/MS spectra derived from phylogenetically distant organisms such as mouse and human. In particular, the MS/MS spectral library was built using seven strains belonging to six DTUs such as Sylvio X10 cl1 (DTU-I), Y (DTU-II), M6241 cl6 (DTU-III), CanIII cl1 (DTU-IV), MN cl2 (DTU-V), CL Brener and CL14 (DTU-VI) strains and MS/MS data from <i>T</i>. <i>vivax</i> (epimastigote, metacyclic and bloodstream forms) were added to the spectral library [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006351#pntd.0006351.ref022" target="_blank">22</a>]. Independent LC-MS/MS runs of the different <i>T</i>.<i>cruzi</i> strains, <i>T</i>.<i>vivax</i> life stages, human and mouse placenta tissue and <i>E</i>.<i>coli</i> were compared against the MS/MS spectral library using SpectraST software. Intra-DTU discrimination was achieved for CL14 and CL Brener and no assignment was made for the E.coli, mouse and human samples. MS/MS spectra from <i>T</i>.<i>vivax</i> were assigned specifically to <i>T</i>.<i>vivax</i> without identification of <i>T</i>.<i>cruzi</i>.</p

Spectral matching of different <i>T</i>.<i>cruzi</i> strains using the DiagnoProt software.

<p>Seven <i>T</i>.<i>cruzi</i> strains (Sylvio X10 cl1, Y, M6241 cl6, CanIII cl1, MN cl2, CL Brener and CL14) belonging to six DTUs were selected to test the Tc-STAMS2 method. The database was constructed with six strains (Sylvio X10 cl1, Y, M6241 cl6, CanIII cl1, MN cl2 and CL Brener) and it was used for comparison of the different strains including CL14.</p

Tc-STAMS2 was tested for its robustness towards technical and experimental variations.

<p>Initially, the Tc-STAMS2 approach was tested for inter-laboratory comparison. Two unknown <i>T</i>.<i>cruzi</i> strains (A and B) were processed as described in the step B of <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006351#pntd.0006351.g001" target="_blank">Fig 1</a> and acquired using the EasynLC coupled to LTQ-Orbitrap Velos mass spectrometer located in the CEFAP mass spectrometry facility at the University of Sao Paulo, Sao Paulo, Brazil. The MS/MS spectral library was built using Sylvio X10 cl1 (DTU-I), Y (DTU-II), M6241 cl6 (DTU-III), CanIII cl1 (DTU-IV), MN cl2 (DTU-V), CL Brener (DTU-VI) and acquired in the PR group, Odense, Denmark using a similar LC-MS/MS setup (EasynLC coupled to LTQ-Orbitrap Velos). A1 and A2 indicate a biological duplicate of <i>T</i>.<i>cruzi</i> M6241 cl6 (DTU-III). B is the <i>T</i>.<i>cruzi</i> Sylvio X10 cl1 (DTU-I). Different sample preparation strategies were used to test the robustness of the Tc-STAMS2 approach such as changing the pH for peptide desalting. B/acid refers to peptides derived from sample B were purified using acidic conditions (0.1% TFA). B/basic refers to peptides derived from sample B were purified using basic conditions (0.1% ammonia). Moreover, different analytical parameters were changed in order to test the robustness of the Tc-STAMS2 approach such as the MS/MS fragmentation type, CID—Collision-Induced Dissociation and HCD—Higher-energy collisional dissociation. Different sample amounts were loaded onto the nano LC column. High and Low indicate 1 and 0.5 ug, respectively. The Tc-STAMS2 approach was robust towards different analytical and experimental challenges.</p

<i>T</i>.<i>cruzi</i> strain discrimination based on spectral similarity searches.

<p>Fourteen <i>T</i>.<i>cruzi</i> strains (Sylvio X10 cl1, Sylvio X10/4, G, Y, Esmeraldo, M6241 cl6, 3869, CanIII cl1, José Júlio, MN cl2, NR cl3, CL Brener and CL14) belonging to six DTUs were selected to test the Tc-STAMS2 method. The MS/MS spectral library was built using two biological replicates for each strain and one independent replicate was used to search against the library. The unique dot product SDSS score is reported.</p

Genotype discrimination based on spectral similarity searches.

<p>Fourteen <i>T</i>.<i>cruzi</i> strains (Sylvio X10 cl1, Sylvio X10/4, G, Y, Esmeraldo, M6241 cl6, 3869, CanIII cl1, José Júlio, MN cl2, NR cl3, CL Brener and CL14) belonging to six DTUs were selected to test the Tc-STAMS2 method. In order to build the MS/MS spectral library, each strain was assigned to the corresponding DTU. Two biological replicates for each strain were used to build the library and one independent replicate was used to match against the library. The unique dot product SDSS score.</p