Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption

Abstract

Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens.DOI: http://dx.doi.org/10.7554/eLife.20621.001

Membrane Lipid Screen to Identify Molecular Targets of Biomolecules

“…Lipid strip probed with PfCelTOS, followed by primary antibody to CelTOS and a secondary antibody conjugated with peroxidase for visualization. PfCelTOS specifically bound to phosphatidic acid (Jimah et al ., 2016). B. Lipid strip probed with primary antibody against His-tag and a secondary antibody conjugated with peroxidase for visualization.…”

“…Examples of lipid-specific pore-formation include Plasmodium CelTOS that depends on phosphatidic acid for pore formation, and the cholesterol dependent cytolysins (Jimah et al ., 2016; Lukoyanova et al ., 2016). CelTOS (cell traversal protein for ookinetes and sporozoites) is a malaria parasite protein that disrupts host cell membranes by pore formation to enable the exit of parasites from invaded host cells during cell traversal (Kariu et al ., 2006; Jimah et al ., 2016). Cholesterol dependent cytolysins are a large class of pore-forming proteins, including virulence factors of gram positive bacteria such as pneumolysin and listeriolysin (Lukoyanova et al ., 2016).…”

“…Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al ., 2016). This protocol has a companion protocol: ‘Liposome disruption assay to examine lytic properties of biomolecules’ which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al , 2017).…”

Liposome Disruption Assay to Examine Lytic Properties of Biomolecules

“…The application of this protocol revealed that CelTOS ( ce ll traversal protein for o okinetes and s porozoites) disrupts membranes containing phosphatidic acid. This insight suggests that CelTOS is secreted by malaria parasites within invaded host cells to disrupt the host cell membrane and enable the exit of parasites (Jimah et al , 2016). Finally, this assay can be readily applied to investigate the inhibition of CelTOS-mediated liposome disruption by small molecules, antibodies or peptides.…”

“…Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al , 2000; Jimah et al , 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al , 2017.…”