Date Published: March 7, 2019
Publisher: Public Library of Science
Author(s): Valeria Messina, Stefano Loizzo, Sara Travaglione, Lucia Bertuccini, Maria Condello, Fabiana Superti, Marco Guidotti, Pietro Alano, Francesco Silvestrini, Carla Fiorentini, Alister G. Craig.
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.
Plasmodium falciparum malaria is a leading cause of ill health, neuro-disability and death in tropical countries . Every year, there are over 500 million clinical cases, with one percent of symptomatic infections that may become complicated and develop into severe malaria. Severe falciparum malaria encompasses a broad range of disease manifestations, including cerebral malaria (CM) . Although the CM mechanisms leading to death are still debated , CM pathology starts from sequestration of infected and non-infected red blood cells in the venules and capillaries of the brain, a process called ‘cytoadherence’ , with the consequent formation of microvascular obstruction that can lead to hypoxia and inadequate tissue perfusion . 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 . It has been hypothesized that pRBC adhesion to endothelial cells directly triggers the Rho signaling activation in the host cells . 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, thus playing a pivotal signaling role in cytoadherence . The Rho GTPases are also 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].