Abstract
Plasmodium falciparum severe malaria causes more than 400,000 deaths every year. One feature of P. falciparum-parasitized erythrocytes (pRBC) leading to cerebral malaria (CM), the most dangerous form of severe malaria, is cytoadherence to endothelium and blockage of the brain microvasculature. Preventing ligand-receptor interactions involved in this process could inhibit pRBC sequestration and insurgence of severe disease whilst reversing existing cytoadherence could be a saving life adjunct therapy. Increasing evidence indicate the endothelial Rho signaling as a crucial player in malaria parasite cytoadherence. Therefore, we have used the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli protein able to modulate the activity of Cdc42, Rac, and Rho, three subfamilies of the Rho GTPases family, to study interactions between infected erythrocytes and cerebral endothelium in co-culture models. The main results are that CNF1 not only prevents cytoadherence but, more importantly, induces the detachment of pRBCs from endothelia monolayers. We first observed that CNF1 does affect neither parasite growth, nor the morphology and concentration of knobs that characterize the parasitized erythrocyte surface, as viewed by scanning electron microscopy. On the other hand, flow cytometry experiments show that cytoadherence reversion induced by CNF1 occurs in parallel with a decreased ICAM-1 receptor expression on the cell surface, suggesting the involvement of a toxin-promoted endocytic activity in such a response. Furthermore, since the endothelial barrier functionality is compromised by P. falciparum, we conducted a permeability assay on endothelial cells, revealing the CNF1 capacity to restore the brain endothelial barrier integrity. Then, using pull-down assays and inhibitory studies, we demonstrated, for the first time, that CNF1 is able not only to prevent but also to cause the parasite detachment by simultaneously activating Rho, Rac and Cdc42 in endothelial cells. All in all our findings indicate that CNF1 may represent a potential novel therapeutic strategy for preventing neurological complications of CM.
Highlights
Plasmodium falciparum malaria is a leading cause of ill health, neuro-disability and death in tropical countries [1]
Cytoadherence needs the formation on the surface of parasitized red blood cells of protrusions named knobs, which bind to several endothelial adhesion molecules variably expressed in different organs, forming a physical ‘engagement’ of pRBCs with endothelial cells [6]
We first investigated whether cytotoxic necrotizing factor 1 (CNF1) inhibited the adhesion of two different parasite lines: the asexual stage parasites of the P. falciparum clone ItG, a reference clone in in vitro studies on parasite cytoadhesion, and a recently adapted isolate, AQ104, of African origin, able to adhere with different affinity to ICAM-1 and cluster of differentiation 36 (CD36) receptors as previously reported [36]
Summary
Plasmodium falciparum malaria is a leading cause of ill health, neuro-disability and death in tropical countries [1]. Cytoadherence needs the formation on the surface of parasitized red blood cells (pRBCs) of protrusions named knobs, which bind to several endothelial adhesion molecules variably expressed in different organs, forming a physical ‘engagement’ of pRBCs with endothelial cells [6]. It has been hypothesized that pRBC adhesion to endothelial cells directly triggers the Rho signaling activation in the host cells [7]. This is supported by the fact that members of the Rho family of small GTPases, which are activated and inactivated by binding to GTP and GDP, respectively, are the first intermediates of the intracellular signaling mediating the engagement of various receptors, including ICAM-1, VCAM-1 and selectins, playing a pivotal signaling role in cytoadherence [8]. The Rho GTPases are involved, albeit indirectly as effectors, in the pathways governed by the Endothelial cell protein C receptor (EPCR), an additional important actor in this scenario [9,10]
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