Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes

Abstract

Sequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake of malaria pigment requires an intact DV membrane and does not occur when the pigment is extracted from the organelle. Merozoites are not opsonized and escape phagocytosis in nonimmune serum. Antimalarial Abs mediate some uptake of the parasites, but to an extent that is not sufficient to markedly reduce reinvasion rates. Phagocytosis of DVs induces a vigorous respiratory burst that drives the cells into IntroductionSevere malaria develops as a consequence of capillaric sequestration of parasitized RBCs (pRBCs), 1-3 activation of complement [4][5][6] and coagulation, 7-9 and increases in vascular permeability, which together can lead to microcirculatory disturbances with comatous death as the ultimate outcome. 1,7,10,11 One unresolved puzzle relates to the fact that children with severe malaria frequently suffer from septicemia due to bacteria that otherwise play no major role in this potentially fatal affliction. [12][13][14] Approximately 50% of these infections are caused by nontyphoidal Salmonellae and other Enterobacteriae, indicating a gut origin. Current hypotheses regarding the causes underlying this general state of immune compromise focus mainly on disturbances in the function of macrophages, dendritic cells, and the adaptive immune system. 15 During its developmental cycle in the RBC, the malaria parasite ingests hemoglobin and packages the waste product hemozoin, which is also known as malaria pigment, in an organelle designated the digestive vacuole (DV). 16 Copious numbers of DVs are released into the bloodstream in patients with severe Plasmodium falciparum malaria, yet their biologic properties and fates have been only minimally studied. Hematin is widely regarded to be equivalent to hemozoin, 17,18 and most investigators have focused on the effects of this artificial molecule on the immune system. [19][20][21][22][23][24][25] However, hematin is not packaged within a membrane-bound organelle and any biologic activity deriving from the DV membrane would therefore be missed. In a different study, we have shown that the DV membrane, but not hemozoin itself, is endowed with the unique capacity to dually activate the alternative complement and the intrinsic clotting pathway (P.D., S. Heber, S. Baumeister, K.L., K.R., S.C.B., and S. Bhakdi, unpublished data, September 2011). Overactivation of these ancient enzyme cascades ...

Malarial anemia: digestive vacuole of Plasmodium falciparum mediates complement deposition on bystander cells to provoke hemophagocytosis

“…1b). We have reported that the activating particles become coated with C3 and C5b-9 [14,18]. Now, the striking finding was made that focal deposits of C3 also became detectable on the bystander RBCs (Fig.…”

“…DVs were isolated, enumerated and banked in 50 % glycerol at −20 °C as described [14,18]. Immediately prior to use, they were washed twice in veronal-buffered saline (VBS) (Virion/Serion, Würzburg, Germany) and, unless otherwise stated, employed at a 1:1 ratio with target cells.…”

“…To detect iC3b, Western blots were developed by probing with a 1:2,000 dilution of primary polyclonal rabbit antihuman C3d antibody, followed by 1:2,000 diluted secondary HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz biotechnology). To detect DV proteins, blots were probed with IgG isolated from a pool of high-titered sera containing antibodies against P. falciparum [18] (diluted 1:500) and developed with secondary HRP-conjugated goat anti-human IgG. Bands were visualized by chemiluminescence [14].…”

Recruitment of Factor H as a Novel Complement Evasion Strategy for Blood-Stage Plasmodium falciparum Infection

“…In bloodstage infection, schizont rupture releases merozoites and digestive vacuoles leading to immune system exposure. A recent study has found digestive vacuoles serve as major complement activation targets with MAC forming on their surface (36)(37)(38). Intriguingly, these studies did not observe terminal pathway formation on merozoites, a strong indication that complement evasion strategies are in place to protect this cell type from complement-mediated attack.…”

Digestive vacuole of Plasmodium falciparum released during erythrocyte rupture dually activates complement and coagulation

“…17,18 We reported recently that when parasitized erythrocytes rupture in blood, the DVs are rapidly phagocytosed by polymorphonuclear granulocytes (PMNs). 19 Phagocytosis only occurred in active serum and complement factor C3 was found attached to the surface of the organelle. It followed that the DV might directly activate complement, so experiments were performed to explore this possibility, leading to the novel findings reported herein.…”