Biology subjects

Brooks, C. F.

Publications and source records attributed to Brooks, C. F..

4 recordsLinked to original sources

bioRxiv2025

Spatiotemporal dynamics of signal dependent exocytosis and parasitophorous vacuolar membrane rupture during Plasmodium falciparum egress.

Malaria, caused by intracellular Plasmodium parasites, remains a major global health concern. These parasites reside and replicate within a vacuole in host red blood cells. Egress of daughter parasites out of the vacuolar and host membranes is tightly regulated via a complex mechanism. Prior studies have suggested that a cyclic-GMP driven calcium signaling pathway leads to the signal-dependent exocytosis of egress-specific vesicles that discharge several proteases into the parasitophorous vacuole. However, signal-dependent exocytosis during egress has not yet been observed in live parasitized RBCs. We targeted the exocytosis reporter, superecliptic pHlourin or SEP, to these egress-specific vesicles and utilized time-lapse imaging to observe exocytosis in live P. falciparum parasites. The spatiotemporal relationship between exocytosis and the breakdown of the parasitophorous vacuolar membrane (PVM) as well as parasite egress was also determined using a fluorescent reporter targeted to the PVM. Our data showed that exocytosis is triggered as early as 3 hours prior to merozoite egress. These data suggest that the PVM rupture occurs at a single site and rapidly expands from that initial site of rupture, releasing the merozoites into the RBC. This is followed by RBC membrane rupture and egress of merozoites. Using conditional mutants of the P. falciparum endoplasmic reticulum calcium-binding protein (PfERC), we demonstrate that knockdown of PfERC inhibits signal-dependent exocytosis of egress-specific vesicles. Together, these data demonstrate that signal-dependent exocytosis of egress-specific vesicles starts well before merozoites are formed via cytokinesis, PVM ruptures at a single site, and that PfERC is required for exocytosis of egress-specific vesicles.

cell biology↗

bioRxiv2022

RAPID, TIME-RESOLVED PROXIMITY LABELING BY SBP1 IDENTIFIES A PORIN DOMAIN PROTEIN AT THE MALARIA PARASITE PERIPHERY

The deadly human malaria-causing parasite, Plasmodium falciparum relies on its capacity to completely remodel its host red blood cell (RBC) through the export of hundreds of parasite proteins across several membranes to the RBC. Among these exported proteins are numerous membrane proteins that are inserted into the parasite plasma membrane (PPM) during their transport via the secretory pathway. It is not known how these exported membrane proteins are extracted from the PPM for export. To answer this question, we fused the exported membrane protein skeleton binding protein 1 (SBP1) with the rapid, efficient, and promiscuous biotin ligase known as TurboID (SBP1TbID). Our data show that the SBP1TbID fusion protein was exported efficiently to the host RBC and was able to rapidly biotinylate proteins at the host-parasite interface during its export as well as at its final destination in the host RBC. Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified early (pre-export) interactors and late (post-export) interactors of SBP1TbID. This led to the identification of 24 proteins that were 10-fold or more enriched in the pre-export time point compared to the post-export time point. Among these early interactors were two promising membrane-associated proteins, one of which has a predicted porin domain, that could potentially act as a translocon at the PPM for exported membrane proteins (Plasmodium translocon of exported membrane proteins or PTEM). Both proteins localize to the host-parasite interface during early stages of the intraerythrocytic cycle and conditional knockdown of these candidates show that they play essential roles in the asexual lifecycle of the parasite. Taken together, our data suggest that these two proteins may play a role in extracting membrane proteins from the PPM for export to the host RBC.

cell biology↗

bioRxiv2019

The Clp proteolytic system acts as an essential nexus for Plasmodium plastid biogenesis

The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most of apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrated that the Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9 based system to express catalytically-dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and matured via trans-autocatalysis. The expression of a Clp chaperone (PfClpC) mutant led to the discovery of a functional chaperone-protease interaction essential for plastid function. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

microbiology↗

bioRxiv2018

An ER-resident calcium binding protein modulates egress of malaria parasites from erythrocytes

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be the calcium (Ca2+) signaling hub and required for biogenesis of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum Endoplasmic Reticulum-resident Calcium-binding protein (PfERC), a member of the CREC family. Knockdown of PfERC shows that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythrocytic lifecycle revealed that PfERC is essential for parasite egress but not required for protein trafficking or Ca2+ storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease, SUB1. Using double mutant parasites, we show that PfERC is required for the proteolytic maturation of the essential aspartic protease, Plasmepsin X, which cleaves SUB1. Further, we show that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish the ER-resident CREC family protein, PfERC, as a key early regulator of the egress proteolytic cascade of malaria parasites.

cell biology↗