A crucial role for the C‐terminal domain of exported protein 1 during the mosquito and hepatic stages of the<i>Plasmodium berghei</i>life cycle

Wolanin, Kamil; Fontinha, Diana; Sanches-Vaz, Margarida; Nyboer, Britta; Heiß, Kirsten; Mueller, Ann‐Kristin; Prudêncio, Miguel

doi:10.1111/cmi.13088

Cited by 6 publications

(5 citation statements)

References 33 publications

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“…Alternatively, the mNG fusion could interfere with interactions that enable EXP1 to organise into oligomeric arrays (Spielmann et al, ) as the C ‐terminus of P. berghei SEP/ETRAMP family members have been shown to be important for oligomer formation (Currà et al, ). However, the entire C ‐terminal domain of P. berghei EXP1 has recently been shown to be dispensable during the blood stage (although it is important in both the mosquito vector and in the hepatocyte where truncated EXP1 fails to properly traffic to the PVM, illustrating distinct P. berghei EXP1 functional roles and trafficking constraints between parasite life stages; Wolanin et al, ). The inability to similarly truncate the EXP1 N ‐terminus in P. berghei implies its essential blood stage function occurs within the PV lumen (Wolanin et al, ).…”

Section: Discussionmentioning

confidence: 99%

“…However, the entire C ‐terminal domain of P. berghei EXP1 has recently been shown to be dispensable during the blood stage (although it is important in both the mosquito vector and in the hepatocyte where truncated EXP1 fails to properly traffic to the PVM, illustrating distinct P. berghei EXP1 functional roles and trafficking constraints between parasite life stages; Wolanin et al, ). The inability to similarly truncate the EXP1 N ‐terminus in P. berghei implies its essential blood stage function occurs within the PV lumen (Wolanin et al, ).…”

Section: Discussionmentioning

confidence: 99%

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

Nessel

Beck

Rayatpisheh

et al. 2020

Cellular Microbiology

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Nessel

Beck

Rayatpisheh

et al. 2020

Cellular Microbiology

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“…Mello-Vieira et al (2020) show that EXP2 is required for liver invasion and maturation and for establishing blood stage infection in a mouse model, which corroborates with our findings of its importance in maturation ( Mello-Vieira et al, 2020 ). Furthermore, the EXP1 C-terminal region exposed to the host cytoplasm interacts with host Apolipoprotein A (ApoH) which is pivotal for successful liver-stage development in Pb , although it is unclear whether it performs the same role in Pf ( Sá et al, 2017 ; Mesén-Ramírez et al, 2019 ; Wolanin et al, 2019 ). EXP1 is continuously trafficking to the PVM throughout the first days of the liver stage ( Sá et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Mid-Liver Stage Arrest of Plasmodium falciparum Schizonts in Primary Porcine Hepatocytes

Boor

Gemert

Hanssen

et al. 2022

Front. Cell. Infect. Microbiol.

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During co-evolution Plasmodium parasites and vertebrates went through a process of selection resulting in defined and preferred parasite-host combinations. As such, Plasmodium falciparum (Pf) sporozoites can infect human hepatocytes while seemingly incompatible with host cellular machinery of other species. The compatibility between parasite invasion ligands and their respective human hepatocyte receptors plays a key role in Pf host selectivity. However, it is unclear whether the ability of Pf sporozoites to mature in cross-species infection also plays a role in host tropism. Here we used fresh hepatocytes isolated from porcine livers to study permissiveness to Pf sporozoite invasion and development. We monitored intra-hepatic development via immunofluorescence using anti-HSP70, MSP1, EXP1, and EXP2 antibodies. Our data shows that Pf sporozoites can invade non-human hepatocytes and undergo partial maturation with a significant decrease in schizont numbers between day three and day five. A possible explanation is that Pf sporozoites fail to form a parasitophorous vacuolar membrane (PVM) during invasion. Indeed, the observed aberrant EXP1 and EXP2 staining supports the presence of an atypical PVM. Functions of the PVM include the transport of nutrients, export of waste, and offering a protective barrier against intracellular host effectors. Therefore, an atypical PVM likely results in deficiencies that may detrimentally impact parasite development at multiple levels. In summary, despite successful invasion of porcine hepatocytes, Pf development arrests at mid-stage, possibly due to an inability to mobilize critical nutrients across the PVM. These findings underscore the potential of a porcine liver model for understanding the importance of host factors required for Pf mid-liver stage development.

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“…The mNG fusion may interfere with interactions that enable EXP1 to organize into oligomeric arrays (22) as the C-terminus of P. berghei SEP/ETRAMP family members have been shown to be important for oligomer formation (19). However, the entire C-terminal domain of P. berghei EXP1 has recently been shown to be dispensable during the blood stage (although it is important in both the mosquito vector and in the hepatocyte where truncated EXP1 fails to properly traffic to the PVM, illustrating distinct EXP1 functional roles and trafficking constraints between parasite life stages) (59). The inability to similarly truncate the EXP1 N-terminus in P. berghei implies its essential blood stage function occurs within the PV lumen (59).…”

Section: Discussionmentioning

confidence: 99%

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

Nessel

Beck

Rayatpisheh³

et al. 2019

Preprint

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word count: 250 22 23 24 Importance word count: 149 25 26 Abstract 27Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) 28 that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between 29 parasite and host compartments. The PVM is the site of several essential transport activities but 30 the basis for organization of this membrane system is unknown. We utilized the second-31 generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of 32 the PVM, identifying known and novel candidate PVM proteins. Among the best represented 33 hits were members of a group of single-pass integral membrane proteins that constitute a major 34 component of the PVM proteome but whose function remains unclear. We investigated the 35 function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 36 genome editing system to install the TetR-DOZI-aptamers system for conditional translational 37 control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by 38 profound changes in vacuole ultrastructure, including increased separation of the PVM and 39 PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While 40 previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant 41 version of EXP1 lacking a residue important for this activity in vitro still provides substantial 42 rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium 43 translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the 44 translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal 45 a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM 46 organization. 47 48 Importance 49 Like other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a 50 membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. 51Although the vacuole is the site of several transport activities essential to parasite survival, little 52 is known about its organization. To explore vacuole biology, we adopted recently developed 53 proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which 54 allowed us to query the function of the prototypical vacuole membrane protein EXP1. 55Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot 56 fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in 57 maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new 58 insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-59 rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and 60 genome editing in P. falciparum. 61 62 65principal barrier between the parasite and its host cell (1). Durin...

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A crucial role for the C‐terminal domain of exported protein 1 during the mosquito and hepatic stages of thePlasmodium bergheilife cycle

Cited by 6 publications

References 33 publications

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

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

Mid-Liver Stage Arrest of Plasmodium falciparum Schizonts in Primary Porcine Hepatocytes

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

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