Quantitative proteomic analysis of amastigotes from <i>Leishmania (L</i>.<i>) amazonensis</i> LV79 and PH8 strains reveals molecular traits associated with the virulence phenotype

<div><p>Background</p><p>Leishmaniasis is an antropozoonosis caused by <i>Leishmania</i> parasites that affects around 12 million people in 98 different countries. The disease has different clinical forms, which depend mainly on the parasite genetics and on the immunologic status of the host. The promastigote form of the parasite is transmitted by an infected female phlebotomine sand fly, is internalized by phagocytic cells, mainly macrophages, and converts into amastigotes which replicate inside these cells. Macrophages are important cells of the immune system, capable of efficiently killing intracellular pathogens. However, <i>Leishmania</i> can evade these mechanisms due to expression of virulence factors. Different strains of the same <i>Leishmania</i> species may have different infectivity and metastatic phenotypes <i>in vivo</i>, and <i>w</i>e have previously shown that analysis of amastigote proteome can give important information on parasite infectivity. Differential abundance of virulence factors probably accounts for the higher virulence of PH8 strain parasites shown in this work. In order to test this hypothesis, we have quantitatively compared the proteomes of PH8 and LV79 lesion-derived amastigotes using a label-free proteomic approach.</p><p>Methodology/Principal findings</p><p>In the present work, we have compared lesion development by <i>L</i>. <i>(L</i>.<i>) amazonensis</i> PH8 and LV79 strains in mice, showing that they have different virulence <i>in vivo</i>. Viability and numbers of lesion-derived amastigotes were accordingly significantly different. Proteome profiles can discriminate parasites from the two strains and several proteins were differentially expressed.</p><p>Conclusions/Significance</p><p>This work shows that PH8 strain is more virulent in mice, and that lesion-derived parasites from this strain are more viable and more infective <i>in vitro</i>. Amastigote proteome comparison identified GP63 as highly expressed in PH8 strain, and Superoxide Dismutase, Tryparedoxin Peroxidase and Heat Shock Protein 70 as more abundant in LV79 strain. The expression profile of all proteins and of the differential ones precisely classified PH8 and LV79 samples, indicating that the two strains have proteins with different abundances and that proteome profiles correlate with their phenotypes.</p></div

Validation of proteome data by Western blot.

<p>Western blot images and corresponding graphs showing expression of A. GP63, B. CPx in three samples of amastigotes of PH8 and LV79 strains. Statistical analysis by T test, **:p<0.01.</p

Comparison between PH8 and LV79 lesion-derived amastigotes regarding infective characteristics.

<p>A. Estimation of amastigote viability by MTT. Three independent experiments, T test, **:p<0.01. B. Trypsin-like activity of amastigote extracts. One experiment (representative of two) with technical triplicates, T test, ***:p<0.001. C. Density of promastigote cultures, indicating efficiency of conversion of amastigotes to promastigotes after 4 days in culture (one experiment with five technical replicates) T test, ***:p<0.001. D. Lesion development graph and E. the respective area under curve after infection with lesion-derived amastigotes from PH8, LV79 and normalized numbers (using viability percentages) of LV79- named as LV79 normalized. Results from three independent experiments, ANOVA followed by Tukey post test, **:p<0.01, ***:p<0.001 (6 weeks: ** for PH8 x LV79, *** for PH8 x LV79norm, 7 and 8 weeks: *** for PH8 x LV79 and LV79norm). F. Representative image in the last day of infection of BALB/c: I, infected with PH8 amastigotes, II, infected with LV79 amastigotes and III, infected with normalized numbers of LV79 amastigotes.</p

Protein comparison in PH8 and LV79 lesion-derived amastigotes.

<p>A. Venn diagram showing the overlap between all identified proteins in PH8 and LV79. B. Heat map representing log2 fold changes of the quantitative data (green—lowest abundance and red—highest abundance) of the differentially expressed proteins (T test, p <0.05). C. Main component analysis based on all proteins identified in LV79 and/or PH8. D. Heat map representing log2 fold changes of the quantitative data of all proteins identified. The first number (1 or 2) after strain name (1, 2 or 3) indicates infection experiment, the second corresponds to the technical replicate.</p

Lesions in BALB/c mice infected with <i>L</i>. <i>(L</i>.<i>) amazonensis</i> promastigotes from LV79 and PH8 strains for 12 weeks.

<p>HE staining of BALB/c mice footpads infected with PH8 (A) and LV79 (B) with necrosis area (*). Parasite labeling in PH8 (C) and LV79 (D) lesions by IHQ using anti-<i>Leishmania</i> serum and anti-rabbit HRP antibody. In E, higher magnification of IHQ of PH8 lesion, in F, negative control with secondary antibody but no anti-<i>Leishmania</i> serum. A, B, C, D and F 400X magnification, E with 1000x magnification.</p

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

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

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

<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>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