Project PXD013136

PRIDE Assigned Tags:
Biological Dataset Biomedical Dataset

Summary

Title

Quantitative proteomic map of the trypanosomatid Strigomonas culicis: the biological contribution of its endosymbiotic bacterium

Description

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, that is highly integrated into the protozoa metabolism: it furnishes essential compounds and divides in synchrony with the nuclear host. The protozoa, conversely, can be rid of the endosymbiont, producing a cured cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of a organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid metabolism, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacterium in monoxenic trypanosomatid biology, particularly because these organisms have a polycistronic open reading frame organization and post-transcriptional gene regulation.

Sample Processing Protocol

S. culicis wild type (WT) and aposymbiotic (Apo) strains, COLPROT041 and COLPROT034, respectively, were provided by Fiocruz Protozoa Collection (http://colprot.fiocruz.br) and grown at 28 °C in liver infusion and tryptose medium (LIT) supplemented with 20% heat-inactivated fetal bovine serum (Sigma) and 0.1% hemin. To define the growth phases for each strain, three aliquots from the total cellular culture (100 mL) were collected and quantified in a Neubauer chamber. This experiment was performed in biological triplicate. For each strain (WT or Apo), three independent biological replicates were processed at different times of growth (24 h, 56 h, and 80 h). Parasites (1 x 108 cells) were initially washed three times by successive cycles of phosphate buffered saline (pH 7.4) addition, centrifugation at 3,000 g for 10 min, and supernatant disposal. Washed parasites were incubated with 100 µL of 0.25% (w/v) Rapigest SF surfactant (Waters Corporation, MA, USA) in 50 mM ammonium bicarbonate. Subsequently, 5 freeze-thaw cycles with liquid nitrogen were performed, followed by 5 min in boiling water, 5 min on ice, and centrifugation at 14,000 g for 10 min. The supernatants containing the protein lysates were collected and quantified by the bicinchoninic acid protein assay (Merck, Damstadt, GE). Fifty micrograms per sample were further processed by the addition of dithiotreitol to a final 20 mM concentration and heated for 30 min at 60 C, followed by cooling to room temperature, addition of iodoacetamide (67 mM final concentration), and incubation for 15 min (in the dark) at room temperature. Porcine trypsin (V511, Promega Corporation, Madison, USA) was added at 1:50 (m/m) enzyme to substrate ratio and incubation proceeded for 16 h at 37 C, followed by 45 min at 56 C. Reaction was stopped by addition of trifluoroacetic acid to a 0.5% (v/v) final concentration and incubation for 45 min at 37 C. Samples were centrifuged at 16.000 g for 10 min and supernatants were subjected to reversed-phase desalting with Poros R2 matrix homemade tip-columns, followed by dryness to completion. Desalted tryptic digests, prepared as described in the previous step, were each resuspended in 30 µL of 1% (v/v) formic acid. Each sample was analyzed in technical triplicate on an EASY-nLC-System (Proxeon Biosystems, West Palm Beach, USA) hyphenated to an LTQ-Orbitrap XL mass spectrometer via a nanoscale LC interface (Thermo, USA).

Data Processing Protocol

The RAW data of the technical triplicate for each biological replicate were analyzed in the computer environment PatternLab for Proteomics (version 4.0, http://patternlabforproteomics.org). Peptide-spectrum matching (PSM) was done using the Comet search engine (version 2016.01) against a database containing: a) gi numbered protein sequences from Strigomonas culicis and Candidatus Kinetoplastibacterium blastocrithidii; b) reversed decoy entries for each protein sequence; c) 127 common protein contaminants sequences – total database number of entries of 19,452. For the Comet search, used parameters were: fully tryptic and semi-tryptic peptide candidates with masses between 550 and 5,500 Da; peptide sequences with up to two missed cleavages; 20 ppm for precursor mass and bins of 1.0005 m/z for MS/MS; methionine oxidation, and asparagine and glutamine deamidations as variable modifications, and carbamidomethyl cysteine as fixed modification. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino, 2013 doi: 10.1093/nar/gks126) with the dataset identifier . The validity of the PSM was assessed using the Search Engine Processor (SEPro). For each PSM, the XCorr, DeltaCN, and Spectral Count score values were used to generate a Bayesian discriminator. The spectra were filtered in 3 levels: first, 3% of decoys were accepted; secondly, the spectra were grouped according to their respective identified peptide sequences, converging to a classification that accepted only 2% of decoy peptides and 1% of decoy proteins (for a peptide to be considered identified, a minimum sequence length of 6 amino acid residues was required) and, finally, by applying post-processing filters, only identifications with < 10 ppm error and proteins with at least one peptide spectrum match with XCorr > 2.5 or two or more peptide spectrum matches with XCorr > 2.0 were accepted. The quantitative comparison was done using the TFold module, and only proteins for which the comparison reached a Benjamini–Hochberg q-value of 0.05 and presented a minimum of 8 spectra in one replicate per condition were reported.

Contact

Richard Hemmi Valente, Fundação Oswaldo Cruz
Richard Hemmi Valente, Laboratory of Toxinology, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil ( lab head )

Submission Date

19/03/2019

Publication Date

08/11/2019

Publication

Publication pending