scholarly journals Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes

2009 ◽  
Vol 42 (2) ◽  
pp. 103-106 ◽  
Author(s):  
Maria Imaculada Muniz-Junqueira ◽  
Carlos Eduardo Tosta
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Circulation ◽  
Defense Mechanisms ◽  
Healthy Adult ◽  
Critical Role ◽  
Immune Serum ◽  
Human Monocytes ◽  
Malaria Parasites

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.

scholarly journals Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration

2006 ◽  
Vol 50 (10) ◽  
pp. 3343-3349 ◽  
Author(s):  
Halima Kaddouri ◽  
Serge Nakache ◽  
Sandrine Houzé ◽  
France Mentré ◽  
Jacques Le Bras
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Lactate Dehydrogenase ◽  
Active Metabolite ◽  
Inhibitory Concentration ◽  
Drug Susceptibility ◽  
Maximum Effect ◽  
Malaria Parasites ◽  
Effect Model

ABSTRACT The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-3H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC50) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (r = 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC50s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

scholarly journals EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

2019 ◽  
Author(s):  
Timothy Nessel ◽  
John M. Beck ◽  
Shima Rayatpisheh ◽  
Yasaman Jami-Alahmadi ◽  
James A. Wohlschlegel ◽  
...  
Keyword(s):  
Genome Editing ◽  
Translational Control ◽  
Protein Identification ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Transferase Activity ◽  
Glutathione S Transferase ◽  
Malaria Parasites ◽  
Vacuole Membrane

AbstractIntraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between parasite and host compartments. The PVM is the site of several essential transport activities but the basis for organization of this membrane system is unknown. We utilized the second-generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of the PVM, identifying known and novel candidate PVM proteins. Among the best represented hits were members of a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated the function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by profound changes in vacuole ultrastructure, including increased separation of the PVM and PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant version of EXP1 lacking a residue important for this activity in vitro still provides substantial rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM organization.ImportanceLike other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. Although the vacuole is the site of several transport activities essential to parasite survival, little is known about its organization. To explore vacuole biology, we adopted recently developed proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which allowed us to query the function of the prototypical vacuole membrane protein EXP1.Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and genome editing in P. falciparum.

scholarly journals Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites

2021 ◽  
Author(s):  
Megan Okada ◽  
Krithika Rajaram ◽  
Russell P. Swift ◽  
Amanda Mixon ◽  
J. Alan Maschek ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Fluidity ◽  
Critical Role ◽  
Vesicular Trafficking ◽  
Malaria Parasites ◽  
Therapeutic Targeting ◽  
Isopentenyl Pyrophosphate ◽  
Cellular Processes ◽  
Metabolic Output ◽  
Long Chain

Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast lumen. PPS knockdown is lethal to parasites, rescued by IPP, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.

scholarly journals Small, Clonally Variant Antigens Expressed on the Surface of the Plasmodium falciparum–Infected Erythrocyte Are Encoded by the rif Gene Family and Are the Target of Human Immune Responses

1999 ◽  
Vol 190 (10) ◽  
pp. 1393-1404 ◽  
Author(s):  
Victor Fernandez ◽  
Marcel Hommel ◽  
Qijun Chen ◽  
Per Hagblom ◽  
Mats Wahlgren
Keyword(s):  
Plasmodium Falciparum ◽  
Immune Responses ◽  
Defense Mechanisms ◽  
Infected Erythrocyte ◽  
Direct Consequence ◽  
Two Dimensional Electrophoresis ◽  
Human Immune ◽  
Dimensional Electrophoresis ◽  
Falciparum Erythrocyte Membrane Protein

Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30–45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P. falciparum. Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5–6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum. Prominent polypeptides of 20, 22, 76–80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro–propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P. falciparum on the surface of the infected erythrocyte.

scholarly journals Fibronectin enhances the opsonic and protective activity of monoclonal and polyclonal antibody against group B streptococci.

1984 ◽  
Vol 159 (6) ◽  
pp. 1618-1628 ◽  
Author(s):  
H R Hill ◽  
A O Shigeoka ◽  
N H Augustine ◽  
D Pritchard ◽  
J L Lundblad ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Defense Mechanisms ◽  
Critical Role ◽  
Major Effect ◽  
Protective Effects ◽  
Group B Streptococci ◽  
Opsonic Activity ◽  
Surface Receptors ◽  
Group B

We have investigated the opsonic and protective effects of fibronectin (FN) against type III group B streptococci. When used by itself, the FN failed to promote actual internalization of group B organisms. The addition of FN to group B streptococci that had been preopsonized in an immunoglobulin preparation modified for intravenous use ( IgIV ) or a type-specific, murine monoclonal antibody of IgG isotype markedly enhanced interaction with human polymorphonuclear leukocytes (PMN). A similar enhanced effect was observed when the FN was combined with type-specific monoclonal antibody preparations of IgM and, surprisingly, IgA isotype. Preincubation experiments indicated that the major effect was upon the PMN rather than directly on the bacteria, but we could not demonstrate an effect of FN on cell surface receptors for the Fc fragment of Ig or C3b using rosetting techniques. In addition to enhancing the in vitro opsonic activity of Ig, the FN significantly increased the protective effect of the polyclonal and monoclonal Ig preparations in an animal model of neonatal group B streptococcal disease. Thus, FN appears to have a critical role in the host defense mechanisms against group B streptococci.

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