A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization duringPlasmodiumliver infection

Abstract

SummaryPrior to initiating symptomatic malaria, Plasmodium parasites infect and develop within hepatocytes. We performed a forward genetic, genome-wide CRISPR-Cas9 screen to identify host regulators of Plasmodium liver infection. Single guide RNAs targeting genes involved in vesicle trafficking, cytoskeleton organization and lipid biogenesis altered Plasmodium liver development. We observed a redistribution of Golgi-derived vesicles and fragmented Golgi stacks with the parasitophorous vacuolar membrane (PVM). The host microtubule network and non-centrosomal microtubule organizing centers (ncMTOC) also re-localized following infection, closely associating with the parasite. Knocking out the centrosomal MTOC protein CENPJ exasperated the re-localization of MTOCs to the parasite and increased infection, suggesting that the parasite relies on ncMTOC assembly. Thus, we have uncovered a mechanism by which parasites sequester host material for survival and development. Our data provide a wealth of yet untested hypotheses about the elusive biology of the liver stage parasite and serves as a foundation for future investigation.

Forward Genetics in Apicomplexa Biology: The Host Side of the Story

“…Hence, it may be helpful for the field to compile existent information from all FGS reports to aid interpretation. For example, in the recent pre-print, Vijayan and colleagues noted that while there was little overlap at an individual gene level between RNAi and CRISPR screens for gene candidates regulating sporozoite infection by P. berghei , there was significant overall enrichment for shared host pathways that the targeted genes belonged to ( Vijayan et al., 2021 ). We have attempted to compile this data in hopes of facilitating a more rapid lookup of suggested candidates ( Supplemental Table 1 ) and firmly proven genes determined from these FGS approaches ( Table 1 ).…”

“…A preprint uploaded in bioRxiv used a genome-wide CRISPR/Cas9 library containing 123,642 sgRNA to target 19,031 protein-coding genes and 1,864 microRNAs. The screen reported the identification of a host regulator of microtubule organizing centers (MTOCs), CENPJ (centromere protein J), that was required for restricting liver stage development of P. yoelli ( Vijayan et al., 2021 ). In the absence of CENPJ enhanced microtubule association with the sporozoite vacuole was observed.…”

Elucidating Spatially-Resolved Changes in Host Signaling During Plasmodium Liver-Stage Infection

“…Plasmodium parasites traverse multiple hepatocytes before invading one ( Mota et al., 2001 ; Coppi et al., 2007 ; Loubens et al., 2021 ) and preferentially invade both particular liver zones ( Yang et al., 2021 ) and hepatocytes with specific phenotypes, such as high ploidy and particular surface receptor compositions ( Austin et al., 2014 ; Kaushansky et al., 2015a ). In addition to selecting particular hepatocytes for invasion, parasites modify the host cell throughout their development within the liver, including cell size ( Balasubramanian et al., 2019 ), microtubule and organelle organization ( Vijayan et al., 2020 ), and signaling cascades ( Kaushansky et al., 2013a ; Glennon et al., 2019 ).…”