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A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

ABSTRACT: Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.

SUBMITTER: Vijayan K

PROVIDER: S-EPMC9481707 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

Vijayan Kamalakannan K Arang Nadia N Wei Ling L Morrison Robert R Geiger Rechel R Parks K Rachael KR Lewis Adam J AJ Mast Fred D FD Douglass Alyse N AN Kain Heather S HS Aitchison John D JD Johnson Jarrod S JS Aderem Alan A Kaushansky Alexis A

Cell chemical biology 20220622 9

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ...[more]

PMID: 35738280

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Project description:IntroductionThe differentiation of B cells into antibody-secreting plasma cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood.MethodsWe have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells.ResultsAmong known genes whose deletion preferentially or mostly affected plasmablast formation were the transcription factors Prdm1 (BLIMP1), Irf4 and Pou2af1 (OBF-1), and the Ern1 gene encoding IRE1a, while deletion of XBP1, the transcriptional master regulator that specifies the expansion of the secretory program in plasma cells, had no effect. Defective plasmablast formation caused by Ern1 deletion could not be rescued by the active, spliced form of XBP1 whose processing is dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1. Moreover, we newly identified several genes involved in NF-kB signaling (Nfkbia), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complex (Hdac1, Mta2, Mbd2) to be required for plasmablast formation. Deletion of ARF4, a small GTPase required for COPI vesicle formation, impaired plasmablast formation and blocked antibody secretion. After Hdac1 deletion plasmablast differentiation was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that govern plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5).DiscussionTaken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation.

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A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

Publications

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

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