Critical role of Erythrocyte Binding-Like protein of the rodent malaria parasite Plasmodium yoelii to establish an irreversible connection with the erythrocyte during invasion

Abstract

Plasmodium malaria parasites multiply within erythrocytes and possess a repertoire of proteins whose function is to recognize and invade these vertebrate host cells. One such protein involved in erythrocyte invasion is the micronemal protein, Erythrocyte Binding-Like (EBL), which has been studied as a potential target of vaccine development in Plasmodium vivax (PvDBP) and Plasmodium falciparum (EBA-175). In the rodent malaria parasite model Plasmodium yoelii, specific substitutions in the EBL regions responsible for intracellular trafficking (17XL parasite line) or receptor recognition (17X1.1pp. parasite line), paradoxically increase invasion ability and virulence rather than abolish EBL function. Attempts to disrupt the ebl gene locus in the 17XL and 17XNL lines were unsuccessful, suggesting EBL essentiality. To understand the mechanisms behind these potentially conflicting outcomes, we generated 17XL-based transfectants in which ebl expression is suppressed with anhydrotetracycline (ATc) and investigated merozoite behavior during erythrocyte invasion. In the absence of ATc, EBL was secreted to the merozoite surface, whereas following ATc administration parasitemia was negligible in vivo. Merozoites lacking EBL were unable to invade erythrocytes in vitro, indicating that EBL has a critical role for erythrocyte invasion. Quantitative time-lapse imaging revealed that with ATc administration a significant number of merozoites were detached from the erythrocyte after the erythrocyte deformation event and no echinocytosis was observed, indicating that EBL is required for merozoites to establish an irreversible connection with erythrocytes during invasion.

Plasmodium yoelii Erythrocyte-Binding-like Protein Modulates Host Cell Membrane Structure, Immunity, and Disease Severity

“…S1A) (6,8), which may affect PyEBL protein localization. To investigate PyEBL protein expression and localization, we performed an immunofluorescence assay (IFA) using anti-EBL antibodies (8,10) and examined protein localization under a confocal microscope. The C741 PyEBL was expressed as a focused dot in merozoites ( Fig.…”

“…IFA and confocal microscopy analyses showed diffused PyEBL expression in Y741 merozoites and a focused dot in C741 merozoites (mostly two dots in N67 Y-C , or partial reversal), similar to those observed in 17XL and 17XNL parasites (8), suggesting that some Y741 PyEBL protein may traffic to dense granules (DGs) in merozoites. A recent study showed that PyEBL of the 17XL line is translocated to the merozoite surface after parasite egress from the iRBC, despite its intracellular localization to DGs (10). Ring-infected erythrocyte surface antigen (RESA) is another DG protein that is translocated to the iRBC membrane, where it binds to spectrin (47,48).…”

“…Although these results suggest that PyEBL has a direct role in parasite invasion and virulence, it is difficult to explain the observation that abnormal localization of the variant PyEBL can greatly improve the parasite growth rate. Recently, PyEBL was found to be translocated to the merozoite surface as a confined patch in both the 17XL and 17XNL lines of P. yoelii (10), which may help maintain normal invasion of RBCs but cannot explain the enhanced growth rate of 17XL. The activation of other genes playing a role in invasion, such as those encoding Py235 rhoptry proteins, may help explain these paradoxical observations (8,11,12).…”

cAMP-dependent protein kinase regulates secretion of apical membrane antigen 1 (AMA1) in Plasmodium yoelii

“…All PCR products were purified using a gel extraction kit and ligated into pDC2-Cas9-PyU6-hDHFR plasmid using an In-Fusion HD cloning kit, yielding pDC2-Cas9-PyU6-PKAc-iKO-hDHFR/yFCU. Transfection to P. yoelii was performed as described [13]. Drinking water containing 1 mg/mL 5-fluorocytosine (5-FC; Sigma-Aldrich, St. Louis, Missouri, USA) was orally administrated to mice infected with DiCre-expressing P. yoelii parasites to obtain parasites without the drug cassette, followed by cloning of transgenic parasites by limiting dilution [18].…”

“…To utilize reverse genetics to describe the function of essential parasite proteins for erythrocyte invasion, technologies are required to inducibly knock down or knock out target molecules. To this end, we have recently established a tetracycline-repressive transactivator (Tetoff) system in P. yoelii, but this system requires a time-consuming optimization process to modify the 5 ′ untranslated region of the target gene [13]. In the present study we established a dimerisable Cre-recombinase (DiCre)-loxP inducible gene knockout system in P. yoelii by adapting methods developed for P. falciparum and P. berghei [14,15], and assessed the consequence of the disruption of PKAc in the erythrocyte invasion process of P. yoelii.…”