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

Comprehensive transcriptome of the maize stalk borer, Busseola fusca, from multiple tissue types, developmental stages, and parasitoid wasp exposures

International audienc

Dissecting the pathogenesis of Chagas disease by deep glycomics and glycoproteomics approaches.

Trypanosoma cruzi é um protozoário unicelular responsável pela doença tropical negligenciada (DTN) denominada doença de Chagas (tripanossomíase americana), uma doença endêmica em 21 países da América do Sul e Central. A doença de Chagas também se apresenta como uma preocupação emergente de saúde global com casos reportados na América do Norte, Europa, Japão e Austrália. A doença pode apresentar diferentes formas clínicas e as opções de tratamentos disponíveis, Benznidazol e Nifurtimox, são limitadas devido à alta toxicidade, efeitos colaterais e reduzida eficácia do tratamento devido à resistência apresentada pelos parasitas. As cepas de T. cruzi são geneticamente diversas com implicações na patogenicidade e virulência, progressão e desfecho da doença, além da susceptibilidade/resistência a drogas. Nesta tese, será apresentada a aplicação da espectrometria de massas para elucidar os principais aspectos moleculares de T. cruzi, incluindo: 1) modulação sistemática do proteoma entre estágios e entre diferentes cepas e espécies de parasitas e 2) modulação de modificações pós-traducionais (PTMs) de T. cruzi. Relativo à parte proteômica, será apresentado A) A modulação de proteínas de membrana da cepa CL14 de T. cruzi durante a progressão da fase exponencial para a fase estacionária a fim de elucidar as mudanças moleculares durante os estágios iniciais de metaciclogênese e B) análise global de perfis de expressão de proteínas entre cepas de T. cruzi e espécies de tripanossomas intimamente relacionadas. A análise de PTMs por espectrometria de massas dará enfoque A) na modulação da S-nitrosilação de proteínas em tripomastigotas seguida pela incubação com a matriz extracelular do hospedeiro, B) na análise global das mudanças conformacionais de glicoproteínas e C) no mapeamento do glicoproteoma (N- e O-ligados), glicoma (N- e O-glicanos) e suas respectivas expressões diferenciais entre as cepas de T. cruzi e espécies de tripanossomas intimamente relacionadas. Considerando todos os achados, esta tese mostra a importância da proteômica no estudo das mudanças moleculares na expressão de proteínas e modificações pós-traducionais durante a interação patógeno-hospedeiro. Especificamente, os métodos desenvolvidos e implementados neste estudo serão úteis para a comunidade científica para estudar, além de infecções pelo T. cruzi, outros sistemas biológicos. Finalmente, este trabalho elucida o papel de proteínas específicas e modificações pós-traducionais, alvos para o diagnóstico e terapia da doença de Chagas.Trypanosoma cruzi is a unicellular protozoan parasite responsible for the neglected tropical disease (NTD) termed Chagas disease (American Trypanosomiasis), a disease endemic in 21 South and Central American countries. Chagas disease is also an emerging global health concern with cases reported in Northern America, Europe, Japan, and Australia. The disease presents in variable clinical forms, and the treatment options available, Benznidazole and Nifurtimox are limited by high toxicities, side effects and decreasing treatment efficiency due to resistance by T. cruzi parasites. T. cruzi strains are genetically diverse, with implications on pathogenicity and virulence, disease progression and outcome, and drug susceptibility/resistance. In this thesis, the application of mass spectrometry to elucidate key T. cruzi molecular aspects will be presented, including: 1) system-wide modulation of the proteome between growth stages and between different parasite strains and species, and 2) modulation of T. cruzi posttranslational modifications (PTMs). For the proteomics part, A) the modulation of membrane proteins of T. cruzi CL14 strain during progression from exponential to stationary growth phases to elucidate the molecular changes during the early stages of metacyclogenesis, and B) the systems-wide protein expression profiles between T. cruzi strains and closely related trypanosome species will be presented. Analysis of PTMs by mass spectrometry will focus on: A) the modulation of T. cruzi trypomastigote protein S-nitrosylation following incubation with host extracellular matrix, B) the systems-wide analysis of glycoprotein conformational changes, and C) the mapping of intact N- and O-linked glycoproteomes and N- and O-glycomes, and their differential expression between T. cruzi strains and closely related trypanosome species. Taken together, this thesis shows the importance of proteomics in studying the molecular changes in protein expression and PTMs during host-pathogen interaction. Specifically, the methods developed and implemented in this thesis will be useful for the scientific community to study not only T. cruzi infections but also other biological systems. Finally, this thesis sheds new lights on the role of specific protein and PTMs targets for Chagas disease diagnostic and therapy

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

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