Biology subjects

Pezza, A.

Publications and source records attributed to Pezza, A..

3 recordsLinked to original sources

bioRxiv2025

TcBDF6 deficiency compromises intracellular amastigotes development and infectivity in Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, relies on complex gene regulatory mechanisms to adapt to its diverse host environments. In recent years, it has been established that epigenetics plays an essential role in these mechanisms via the regulation of chromatin structure. Bromodomain-containing factors (BDFs), known for recognizing acetylated lysines on histones, have emerged as key factors in chromatin remodeling complexes. Among the eight predicted BDFs in T. cruzi, BDF6 is part of a TINTIN-like complex with MRGx and MRGBP, homologous to components of the NuA4/TIP60 chromatin remodeling complex. We generated knockout (KO) parasites for bdf6 gene using CRISPR/Cas9 gene editing. BDF6-deficient epimastigotes exhibit normal morphology but decreased size and growth and the resulting metacyclic trypomastigotes displayed drastically reduced infectivity. Strikingly, once inside host cells, BDF6-deficient parasites differentiated into amastigotes but failed to replicate. This intracellular arrest was reversed by episomal complementation of BDF6. Consistently, BDF6-KO parasites also exhibited impaired infectivity in mice, a defect that was also rescued in the add back parasite strains. Our findings highlight BDF6 as a critical regulator of intracellular parasite development, operating in stages beyond epimastigotes where epigenetic plasticity is essential for host adaptation. This striking stage-specific phenotype of BDF6 KO underscores its functional importance and highlights the relevance of epigenetic regulators along T. cruzis life cycle.

microbiology↗

bioRxiv2022

Essential bromodomain TcBDF2 as a drug target against Chagas disease

Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein-containing a bromodomain that was recently described. Using the Tet regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing we were able to demonstrate the essentiality of TcBDF2 in T cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results, point to TcBDF2 as a druggable target against T. cruzi.

cell biology↗

bioRxiv2020

Alpha-tubulin acetylation in Trypanosoma cruzi: a dynamic instability of microtubules is required for replication and cell cycle progression

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of -tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been catalogued in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase that delivers this modification was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates -tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated -tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the acetylated isoform, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that -tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

cell biology↗