Author response: The Plasmodium falciparum rhoptry protein RhopH3 plays essential roles in host cell invasion and nutrient uptake

ABSTRACT Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610. IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.

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Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family

mBio ◽

10.1128/mbio.01500-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

J. Stephan Wichers ◽

Judith A. M. Scholz ◽

Jan Strauss ◽

Susanne Witt ◽

Andrés Lill ◽

...

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Plasma Membrane ◽

Host Cell ◽

Cell Invasion ◽

Surface Antigens ◽

Membrane Topology ◽

Host Cell Invasion ◽

Content Type ◽

Variant Surface Antigens

ABSTRACT During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparum stevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface. IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

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Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.6b01109 ◽

2016 ◽

Vol 59 (24) ◽

pp. 10994-11005 ◽

Cited By ~ 7

Author(s):

Swapna Johnson ◽

Raphaël Rahmani ◽

Damien R. Drew ◽

Melanie J. Williams ◽

Mark Wilkinson ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Host Cell ◽

Cell Invasion ◽

Host Cell Invasion

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Critical Glycosylated Residues in Exon Three of Erythrocyte Glycophorin A Engage Plasmodium falciparum EBA-175 and Define Receptor Specificity

mBio ◽

10.1128/mbio.01606-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 25

Author(s):

Nichole D. Salinas ◽

May M. Paing ◽

Niraj H. Tolia

Keyword(s):

Plasmodium Falciparum ◽

Host Cell ◽

Cell Invasion ◽

Expression System ◽

Glycophorin A ◽

High Avidity ◽

Host Cell Invasion ◽

Specific Receptor ◽

Parasite Invasion ◽

Content Type

ABSTRACT Erythrocyte invasion is an essential step in the pathogenesis of malaria. The erythrocyte binding-like (EBL) family of Plasmodium falciparum proteins recognizes glycophorins (Gp) on erythrocytes and plays a critical role in attachment during invasion. However, the molecular basis for specific receptor recognition by each parasite ligand has remained elusive, as is the case with the ligand/receptor pair P. falciparum EBA-175 (PfEBA-175)/GpA. This is due largely to difficulties in producing properly glycosylated and functional receptors. Here, we developed an expression system to produce recombinant glycosylated and functional GpA, as well as mutations and truncations. We identified the essential binding region and determinants for PfEBA-175 engagement, demonstrated that these determinants are required for the inhibition of parasite growth, and identified the glycans important in mediating the PfEBA-175–GpA interaction. The results suggest that PfEBA-175 engages multiple glycans of GpA encoded by exon 3 and that the presentation of glycans is likely required for high-avidity binding. The absence of exon 3 in GpB and GpE due to a splice site mutation confers specific recognition of GpA by PfEBA-175. We speculate that GpB and GpE may have arisen due to selective pressure to lose the PfEBA-175 binding site in GpA. The expression system described here has wider application for examining other EBL members important in parasite invasion, as well as additional pathogens that recognize glycophorins. The ability to define critical binding determinants in receptor-ligand interactions, as well as a system to genetically manipulate glycosylated receptors, opens new avenues for the design of interventions that disrupt parasite invasion. IMPORTANCE Plasmodium falciparum uses distinct ligands that bind host cell receptors for invasion of red blood cells (RBCs) during malaria infection. A key entry pathway involves P. falciparum EBA-175 (PfEBA-175) recognizing glycophorin A (GpA) on RBCs. Despite knowledge of this protein-protein interaction, the complete mechanism for specific receptor engagement is not known. PfEBA-175 recognizes GpA but is unable to engage the related RBC receptor GpB or GpE. Understanding the necessary elements that enable PfEBA-175 to specifically recognize GpA is critical in developing specific and potent inhibitors of PfEBA-175 that disrupt host cell invasion and aid in malaria control. Here, we describe a novel system to produce and manipulate the host receptor GpA. Using this system, we probed the elements in GpA necessary for engagement and thus for host cell invasion. These studies have important implications for understanding how ligands and receptors interact and for the future development of malaria interventions.

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Phosphorylation of Rhoptry Protein RhopH3 Is Critical for Host Cell Invasion by the Malaria Parasite

mBio ◽

10.1128/mbio.00166-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Roseleen Ekka ◽

Ankit Gupta ◽

Sonika Bhatnagar ◽

Pawan Malhotra ◽

Pushkar Sharma

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Blood Cells ◽

Malaria Infection ◽

Malaria Parasite ◽

Host Cell Invasion ◽

Human Malaria Parasite ◽

Content Type ◽

Rhoptry Protein

ABSTRACT Merozoites formed after asexual division of the malaria parasite invade the host red blood cells (RBCs), which is critical for initiating malaria infection. The process of invasion involves specialized organelles like micronemes and rhoptries that discharge key proteins involved in interaction with host RBC receptors. RhopH complex comprises at least three proteins, which include RhopH3. RhopH3 is critical for the process of red blood cell (RBC) invasion as well as intraerythrocytic development of human malaria parasite Plasmodium falciparum. It is phosphorylated at serine 804 (S804) in the parasite; however, it is unclear if phosphorylation regulates its function. To address this, a CRISPR-CAS9-based approach was used to mutate S804 to alanine (A) in P. falciparum. Using this phosphomutant (R3_S804A) of RhopH3, we demonstrate that the phosphorylation of S804 is critical for host RBC invasion by the parasite but not for its intraerythrocytic development. Importantly, the phosphorylation of RhopH3 regulates its localization to the rhoptries and discharge from the parasite, which is critical for RBC invasion. We also identified P. falciparum CDPK1 (PfCDPK1) as a possible candidate kinase for RhopH3-S804 phosphorylation and found that it regulates RhopH3 secretion from the parasite. These findings provide novel insights into the role of phosphorylation in rhoptry release and invasion, which is poorly understood. IMPORTANCE Host cell invasion by the malaria parasite is critical for establishing infection in human host and is dependent on discharge of key ligands from organelles like rhoptry and microneme, and these ligands interact with host RBC receptors. In the present study, we demonstrate that phosphorylation of a key rhoptry protein, RhopH3, is critical for host invasion. Phosphorylation regulates its localization to rhoptries and discharge from the parasite.

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