EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S-transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of citie

Identification of novel factors important for the organization and function of the Plasmodium falciparum parasitophorous vacuole

Malaria is a global disease that impact hundreds of millions of people a year with deadly consequences. There is hope for global initiatives to combat and eradicate this disease, but a deeper understanding of the causative parasite, Plasmodium falciparum, is required in order to develop the needed therapeutics. This apicomplexan parasite develops in a complex, dual-host lifecycle involving transition between humans and an invertebrate mosquito vector. Human pathological symptoms arise exclusively from the asexual blood stage in which the parasite invades and grows within host red blood cells, a process that requires substantial host cell remodeling. During intraerythrocytic development, the parasite is enclosed by a parasitophorous vacuole membrane generated during invasion. Survival within this vacuolar barrier necessitates several transport and trafficking processes, including nutrient uptake, waste efflux, effector protein translocation, and endocytosis of host cytosol. Effector protein export out of the vacuole is mediated by the Plasmodium Translocon of EXported Proteins (PTEX) which consists of the AAA+ chaperone HSP101, the adaptor protein PTEX150, and a membrane pore formed by EXP2. The EXP2 pore also serves a second role as a vacuolar nutrient channel, but little is known about this unique dual functionality is controlled. Here, we performed proximity labeling using multiple PTEX components as bait to identify candidate protein co-factors for the EXP2 nutrient channel. Top hits enriched by EXP2 proximity labeling included the vacuole membrane protein EXP1. We characterize EXP1 with a TetR-DOZI-aptamers knockdown system, revealing it performs an essential role in intraerythrocytic development and that a previously hypothesized glutathione S-transferase activity cannot account for this essential function. Notably, depletion in EXP1 caused major changes to vacuole ultrastructure and the distribution of EXP2 but does not impact protein export, consistent with a specific role in supporting the nutrient channel. To further characterize EXP2 nutrient channel function, we attempted to develop a nutrient import sensor based on the promiscuous biotin ligase TurboID targeted to the vacuole lumen. We quantified biotinylation as a metric for biotin import through the nutrient channel following dimerizable Cre recombinase (diCre)-mediated conditional knockout of EXP2, HSP101, or PTEX150. Finally, we adapted a split-TurboID system to detect protein compartmentalization and topology in the complex membrane systems of the parasite-infected erythrocyte. We demonstrate the functionality of this system by generating an N_TurboID fragment fusion to the endogenous C-terminus of either EXP1 or EXP2 in parasites expressing the C_TurboID fragment trafficked to multiple parasite compartments. Notably, expression of an exported C_TurboID fragment in EXP1-N_TurboID parasites enables proximity labeling experiments at the EXP1 C-terminus exposed on the vacuolar surface for the first time. The establishment of these tools provides novel strategies for proximity labeling of parasite compartments and new approaches for studying the trafficking and topology of parasite proteins. Taken together, our work provides new insights into the biology of the parasite vacuole and the interactions of the EXP2 nutrient channel which may help in the fight against this deadly parasite