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

<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

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 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

Biosynthesis of nicotinic acid and NAD.

<p>Enzymes surrounded by a gray box were possibly acquired through horizontal transfer from Bacteria to trypanosomatids (see main text). <b>Metabolites - I</b>: Aspartate<b>; II:</b> Glycerone-phosphate<b>; III:</b> Iminoaspartate<b>; IV:</b> Quinolinate<b>; V:</b> Nicotinate D-ribonucleotide<b>; VI:</b> Deamino-NAD+<b>; VII:</b> Nicotinamide adenine dinucleotide<b>; VIII:</b> Nicotinamide adenine dinucleotide phosphate<b>; IX:</b> Tryptophan; <b>X</b>: L-Formylkynurenine<b>; XI:</b> L-Kynurenine<b>; XII:</b> 3-Hydroxy-L-kynurenine<b>; XIII:</b> 3-Hydroxyanthranilate<b>; XIV:</b> 2-Amino-3-carboxymuconate semialdehyde<b>; XV:</b> Nicotinic acid; <b>XVI:</b> Nicotinamide. <b>Enzymes - </b><b>1.4.3.16:</b> L-aspartate oxidase<b>; 1.4.1.21:</b> aspartate dehydrogenase<b>; 2.5.1.72:</b> quinolinate synthase<b>; 2.4.2.19:</b> nicotinate-nucleotide diphosphorylase<b>; 2.7.7.18:; 6.3.5.1:</b> NAD+ synthase<b>; 2.7.1.23:</b> NAD+ kinase<b>; 1.13.11.11:</b> tryptophan 2,3-dioxygenase<b>; 3.5.1.9:</b> arylformamidase<b>; 1.14.13.9:</b> kynurenine 3-monooxygenase<b>; 3.7.1.3</b> kynureninase<b>; 1.13.11.6:</b> 3-hydroxyanthranilate 3,4-dioxygenase; <b>2.4.2.11</b>: nicotinate phosphoribosyltransferase (recently transferred to EC6.3.4.21); <b>3.5.1.19</b>: nicotinamidase.</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

Maximum likelihood phylogenetic tree of UbiC (EC:4.1.3.40).

<p><b>A –</b>overall tree, colored according to taxonomic affiliation of each taxon, as per the legend on the left; distance bar only applies to panel A. B – details of the region of the tree where the Trypanosomatidae are placed. Values on nodes represent bootstrap support (only 50 or greater shown). Panel B is meant to only represent the branching patterns and do not portray estimated distances between sequences.</p

Overview of the biosynthetic pathways of essential vitamins and cofactors in trypanosomatids.

<p>Dashed arrows: metabolite import/exchange; dotted arrows: reaction present in only some of the organisms analyzed; solid arrows: other reactions (circles on the top of the arrows indicate number of steps and fulfilled circles indicate presence of enzyme); arrows surrounded by a gray box: enzymes possibly acquired through horizontal transfer from Bacteria to trypanosomatids (see main text). <b>A -</b> Contribution of SHTs and TPEs based on the analysis of gene content in the genomes of <i>A. deanei</i>, <i>A. desouzai</i>, <i>S. culicis</i>, <i>S. oncopelti</i>, <i>S. galati</i> and respective endosymbionts. <b>B -</b> Biochemical capability of trypanosomatids without symbionts based on the analysis of genomic data of <i>H. muscarum</i>, <i>C. acanthocephali</i> and <i>L. major</i>.</p