Biology subjects

Ramon-Zamorano, G.

Publications and source records attributed to Ramon-Zamorano, G..

4 recordsLinked to original sources

bioRxiv2025

Protein landscape of the chromatin states in the malaria parasite Plasmodium falciparum

Epigenetic regulation is essential for development and adaptation across eukaryotes. However, a comprehensive overview of the molecular framework of chromatin-mediated regulation, particularly in non-model organisms, is lacking. Here, we present a systematic proteomic characterization of the chromatin states in P. falciparum, an ancient human pathogen with unique genome composition and epigenetic blueprint. We adapted and systematically employed three proximity-labelling approaches to provide a high-confidence and comprehensive proteome of heterochromatin, euchromatin and (peri)centromeric chromatin comprised of 214 proteins, including both expected and new chromatin components. Characterization of 20 proteins validated our approach and i) uncovered a protein influencing parasite transmission, ii) defined complexes relevant for histone variant exchange and chromatin-RNA interactions and iii) provided evidence for the so far believed to be absent spindle assembly checkpoint and the corresponding Bub1-like kinase. This study hence offers a reference proteome of the chromatin states and a resource to uncover novel chromatin biology.

cell biology↗

bioRxiv2024

CUT&Tag and DiBioCUT&Tag enable investigation of the AT-rich and dynamic epigenome of Plasmodium falciparum from low input samples.

Phenotypic variation between malaria parasites is one of the major contributors to the pathogens success and is regulated by differences in heterochromatin-mediated gene silencing. Currently, the heterochromatin landscape is mostly profiled utilising chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). However this technique has drawbacks regarding AT-content-related artifacts and requires substantial material and time investment, severely limiting profiling of scarce sample types (e.g. field isolates). In order to facilitate assessments of epigenetic states in low-input samples, we adopted the epigenetic profiling technique Cleavage Under Targets and Tagmentation (CUT&Tag) to Plasmodium falciparum parasites. Performing the reaction with 100,000 or even only 10,000 nuclei yielded reproducible results coherent with bulk-ChIP-seq data while using significantly less material. We also optimised sample preparation, permitting the use of crude saponin lysates, which decreases sample loss due to inefficient nuclei isolation and increases versatility of the protocol. Finally, we developed DiBioCUT&Tag, a novel way of utilising dimerisation-induced recruitment of biotin ligases for signal amplification prior to anti-biotin CUT&Tag, which we successfully deployed to profile both heterochromatin occupancy and a dynamically chromatin-associated protein (BDP5). Methods described here hence provide substantially improved means for epigenetic profiling of (transiently) chromatin-associated proteins from low-input samples in the malaria parasite and beyond.

genomics↗

bioRxiv2024

A system for functional studies of the major virulence factor of malaria parasites

PfEMP1 is a variable antigen displayed on erythrocytes infected with the malaria parasite Plasmodium falciparum. PfEMP1 mediates binding of the infected cell to the endothelium of blood vessels, a cause of severe malaria. Each parasite encodes [~]60 different PfEMP1 variants but only one is expressed at a time. Switching between variants underlies immune evasion in the host and variant-specific severity of disease. PfEMP1 is difficult to study due to expression heterogeneity between parasites which also renders genetic modification approaches ineffective. Here, we used selection linked integration (SLI) to generate parasites all expressing the same PfEMP1 variant and genome edit the expressed locus. Moving this system from the reference strain 3D7 to IT4 resulted in PfEMP1 expressor parasites with effective receptor binding capacities. We also introduce a second version of SLI (SLI2) to introduce additional genome edits. Using these systems, we study PfEMP1 trafficking, generate cell lines binding to the most common endothelial receptors, survey the protein environment from functional PfEMP1 in the host cell and identify new proteins needed for PfEMP1 mediated sequestration. These findings show the usefulness of the system to study the key virulence factor of malaria parasites.

microbiology↗

bioRxiv2022

Gene-by-gene screen of the unknown proteins encoded on P. falciparum chromosome 3

Taxa-specific proteins are key determinants defining the biology of all organisms and represent prime drug targets in pathogens. However, lacking comparability with proteins in other lineages makes them particularly difficult to study. In malaria parasites this is exacerbated by technical limitations. Here, we analysed the cellular location, essentiality, function and, in selected cases, interactome of all unknown non-secretory proteins encoded on an entire P. falciparum chromosome. The nucleus was the most common localisation, indicating it is a hotspot of parasite-specific biology. More in-depth functional studies with four proteins revealed essential roles in DNA replication and mitosis. The novel mitosis proteins defined a possible orphan complex and a highly diverged complex needed for the spindle-kinetochore connection. Structure-function comparisons indicated that the taxa-specific proteins evolved by different mechanisms. This work demonstrates the feasibility of gene-by-gene screens to elucidate the biology of malaria parasites and reveal critical parasite-specific processes of interest as drug targets.

cell biology↗