Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase

Kreutzfeld, Oriana; Grützke, Josephine; Ingmundson, Alyssa; Müller, Katja; Matuschewski, Kai

doi:10.3389/fgene.2021.742153

Cited by 6 publications

(12 citation statements)

References 63 publications

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“…As controls, we also immunoprecipitated mCherry from erythrocytes infected with other P. berghei lines expressing mCherry or mCherry fusion proteins (Fig 1A). In these control samples, mCherry was localized in the cytoplasm of the parasite (labeled in Fig 1A as mCherry) [28], the cytoplasm of the host erythrocyte (PEXEL-mCherry) [29], or in the parasitophorous vacuole membrane (EXP2-mCherry) [30]. In the EXP2-mCherry line, full length EXP2 is tagged with mCherry and expressed from the endogenous promoter.…”

Section: Identification Of Plasmodium Proteins Co-precipitating With ...mentioning

confidence: 99%

“…Immunoprecipitations from cell lysates were performed with the RFP-Trap-A kit (Cromotek) according to manufacturer's instructions using equal numbers of parasites per line, which varied from 7x10 8 to 7x10 9 across experimental replicates. The P. berghei lines used included a line expressing IBIS1-mCherry [15], a line expressing mCherry from the HSP70 promoter [28], a line in which mCherry is fused to the predicted PEXEL motif of the circumsporozoite protein and expressed from the IBIS1 promoter [29], and a line expressing EXP2-mCherry [30].…”

Section: Immunoprecipitation Of Ibis1-mcherrymentioning

confidence: 99%

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A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

et al. 2022

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Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues.

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Section: Identification Of Plasmodium Proteins Co-precipitating With ...mentioning

confidence: 99%

Section: Immunoprecipitation Of Ibis1-mcherrymentioning

confidence: 99%

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

et al. 2022

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show abstract

“…As controls, we also immunoprecipitated mCherry from erythrocytes infected with other P. berghei lines expressing mCherry or mCherry fusion proteins (Figure 1A). In these control samples, mCherry was localized in the cytoplasm of the parasite (labeled in figure 1a as mCherry) [28], the cytoplasm of the host erythrocyte (PEXEL-mCherry) [29], or in the parasitophorous vacuole membrane (EXP2-mCherry) [30]. In the EXP2-mCherry line, full length EXP2 is tagged with mCherry and expressed from the endogenous promoter.…”

Section: Identification Of Plasmodium Proteins Co-precipitating With ...mentioning

confidence: 99%

Section: Immunoprecipitation Of Ibis1-mcherrymentioning

confidence: 99%

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

Gabelich¹,

Grützke

Kirscht³

et al. 2022

Preprint

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Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further co-localized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues.

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“…Intriguingly, while EXP2 and PTEX150 are also expressed during hepatocyte infection, HSP101 has not been detected until late liver stage development when it is loaded into the dense granules of forming merozoites to be deployed upon red blood cell (RBC) invasion (15-17). Furthermore, blood-stage export reporters are not translocated beyond the liver-stage PVM, even when HSP101 is ectopically expressed at this stage, indicating a distinct export mechanism (15, 18, 19). Inactivation of exp2 expression in sporozoites using the FLP/FRT stage-specific conditional knockdown system in P. berghei demonstrated a critical role for EXP2 in transition from the mosquito host to the vertebrate blood-stage (15).…”

Section: Introductionmentioning

confidence: 97%

The PTEX pore component EXP2 is important for intrahepatic development during the Plasmodium liver stage

Hussain

Linera-Gonzalez

Beck

et al. 2022

Preprint

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During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To transcend this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are indicated by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3′-UTR enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Post-invasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid to late liver stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding with merosome detachment that marks the culmination of liver stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal EXP2 is important for intrahepatic parasite development, indicating PTEX components also function at the hepatocyte-parasite interface.

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Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase

Cited by 6 publications

References 63 publications

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

The PTEX pore component EXP2 is important for intrahepatic development during the Plasmodium liver stage

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