Biology subjects

Docampo, R.

Publications and source records attributed to Docampo, R..

5 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↗

bioRxiv2021

Poly(A)-binding protein is an ataxin-2 chaperone that emulsifies biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs) which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar that dictates the behavior of a few key scaffold proteins that make up these phases but how the partitioning of hundreds of other SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, a SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14aa sequence that acts as an ataxin-2 condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal a new molecular function for ancient poly(A)-binding proteins as emulsifiers of biomolecular condensate proteins. These findings may inspire novel approaches to therapeutically target aberrant phases in disease.

cell biology↗

bioRxiv2020

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress in Leishmania major

Lathosterol oxidase (LSO) catalyzes the formation of C5-C6 double bond in the synthesis of various types of sterols in mammals, fungi, plants and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C5-C6 desaturation, we generated a LSO-null mutant line (lso-) in Leishmania major, the causative agent for cutaneous leishmaniasis. Lso- parasites lacked the ergostane-based sterols commonly found in wild type L. major and instead accumulated equivalent sterol species without the C5-C6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso- mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alteration in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C5-C6 double bond is vital for the structure of sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCESterols are essential membrane components in eukaryotes and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. Lathosterol oxidase (aka sterol C5-desaturase) is required for the formation of C5-C6 double bond in the sterol core structure in mammals, fungi, protozoans, plants and algae. Functions of this C5-C6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that the C5-C6 double bond is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.

microbiology↗

bioRxiv2020

Protein Targets of Inositol Pyrophosphate (5-IP7) in the parasite Trypanosoma cruzi

Diphosphoinositol-5-pentakisphosphate (5-PP-P5), also known as inositol heptakisphosphate (5-IP7), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a post-translational modification involving a {beta}-phosphoryl transfer. In this study, utilizing an immobilized 5-IP7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP7-regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. The highest number of protein targets was detected in the absence of metal ions, with an overrepresentation of proteins involved in purine metabolism, protein phosphorylation, nucleosome assembly, cell redox homeostasis, parasite-surface components, and exocytosis, and only a 37% overlap between the two proliferative stages. In the presence of magnesium, there was a lower enrichment with the isolation of about 30 proteins with overrepresented candidates involved in tRNA amino acylation, phosphate homeostasis, lipid and glucose catabolism, and cell division, and 15% overlap between proliferative stages. To validate the pull-down results, we endogenously tagged four protein candidates and immunopurified them. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP7-mediated phosphorylation events at a polyserine tract (Ser578-580). We also identified a novel SPX domain-containing phosphoribosyl transferase [EC 2.7.6.1] herein termed as TcPRPS4. Our data revealed new functional roles of 5-IP7 in this divergent eukaryote, and provided potential new targets for chemotherapy.

microbiology↗