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PhyloQuant approach provides insights into Trypanosoma cruzi evolution using a systems-wide mass spectrometry-based quantitative protein profile.

ABSTRACT: The etiological agent of Chagas disease, Trypanosoma cruzi, is a complex of seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution requires understanding the parasite origin and population structure. In this study, we introduce the PhyloQuant approach to infer the evolutionary relationships between organisms based on differential mass spectrometry-based quantitative features. In particular, large scale quantitative bottom-up proteomics features (MS1, iBAQ and LFQ) were analyzed using maximum parsimony, showing a correlation between T. cruzi DTUs and closely related trypanosomes' protein expression and sequence-based clustering. Character mapping enabled the identification of synapomorphies, herein the proteins and their respective expression profiles that differentiate T. cruzi DTUs and trypanosome species. The distance matrices based on phylogenetics and PhyloQuant clustering showed statistically significant correlation highlighting the complementarity between the two strategies. Moreover, PhyloQuant allows the identification of differentially regulated and strain/DTU/species-specific proteins, and has potential application in the identification of specific biomarkers and candidate therapeutic targets.

SUBMITTER: Mule SN

PROVIDER: S-EPMC7952728 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types.

Project description:?-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (?-D-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gHmdC), for the accurate detection of ?-D-glucosyl-5-hydroxymethyl-2'-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2'-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.?

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PhyloQuant approach provides insights into Trypanosoma cruzi evolution using a systems-wide mass spectrometry-based quantitative protein profile.

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