Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi

Abstract

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.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity

“…As in other eukaryotic cells, nuclear architecture is dynamic in trypanosomes. Chromatin shows differences through the parasites life cycles and also through cell cycle in proliferating stages regarding its position inside the nucleus, condensation state and protein composition 3,36,37 . These changes thus modify DNA accessibility to protein complexes involved in nuclear functions like transcription and replication.…”

Extracellular Vesicles From Sporothrix brasiliensis Are an Important Virulence Factor That Induce an Increase in Fungal Burden in Experimental Sporotrichosis

“…Peptides were vacuum-dried and cleaned using Zip Tip micro-C18 (Millipore Merck©) before subsequent MS analysis. LC-MS/MS was performed as previously described ( Leandro de Jesus et al, 2017 ). Briefly, peptides were resuspended in 0.1% formic acid and injected to a nano HPLC (NanoLC-1DPlus, Proxeon) coupled to an in-house made pre-column (5 cm × 10 μm) filled with a 10 μm C18 Jupiter ® resins (Phenomenex©).…”

SUMO polymeric chains are involved in nuclear foci formation and chromatin organization in Trypanosoma brucei procyclic forms

“…Micrococcal assay was adapted from [ 51 ] and [ 52 ]. Parasites (5x10 7 ) were washed in a buffer containing 1 mM L-glutamate, 250 mM sucrose, 2.5 mM CaCl 2 and 1 mM PMSF, and then lysed with the same buffer but also containing 1% Triton X-100.…”