Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610.IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.
Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.
A monoclonal antibody, 9.1C3, was used to investigate the mechanism of natural killer (NK) cell-mediated lysis. In addition to blocking NK cell function, the antibody blocked antibody-dependent cellular cytotoxicity against the K562 target cell at the effector cell level. The stage at which 9.1C3 antibody inhibited cytolysis was established with a Ca++ pulse technique, whereby it was shown that the antibody inhibited killing at a discrete step after the Ca++-dependent programming for lysis. The 9.1C3 antigen appeared to be associated with the T200 glycoprotein complex. Thus the 66 and 77 Kd proteins detected by 9.1C3 were also precipitated with a monoclonal antibody to T200, and in sequential immunoprecipitations, 9.1C3 antibody removed these bands from immunoprecipitates with antibody to T200. Also, in co-modulation studies, it was found that antibody to T200 co-capped the 9.1C3 antigen but that capping with 9.1C3 antibody did not induce co-modulation of the T200 antigen. Expression of the 9.1C3 and T200 antigens on different cell types, however, was not identical, and the 9.1C3 antibody did not immunoprecipitate high m.w. proteins in the region of 200 Kd. Functionally, in NK cell killing studies, the antibody to T200 used alone did not block but was synergistic with the 9.1C3 antibody. The differential effect of the enzymes pronase and trypsin on the cell surface expression of the 9.1C3 and T200 antigens reflected the ability of these enzymes to inhibit NK cell killing. These data suggest that the 9.1C3 antigen participates in a late event in the cytolytic pathway.
The structures involved in the recognition of melanoma cells by nonspecific cytotoxic T lymphocytes (CTL) activated in mixed lymphocyte culture were investigated with monoclonal antibodies (MAb) which blocked this anomalous killer (AK) function. Of over 2000 MAb raised against melanoma cells, only three inhibited killing; one of these, an IgMk termed Leo Me13, was investigated in detail. In antibody-binding studies using a large range of cultured tumor cells, it was shown that Leo Me13 was relatively specific for melanoma cells. Of more importance, Leo Me13 inhibited conjugate formation between AK cells and melanoma target cells by 60 to 80% and caused an eight- to 10-fold reduction in killing. The MAb did not immunoprecipitate protein from melanoma cells surface-labeled with 125I, and thin-layer chromatography followed by immunoblotting of the separated glycolipids from melanoma cells indicated that the epitope was on acidic glycolipids migrating between GM1 and GD1a; moreover, treatment of melanoma cells with neuraminidase resulted in complete loss of binding of Leo Me13 but not of other anti-melanoma antibodies which did not inhibit AK cell-mediated lysis. Other melanoma-reactive MAb of the same isotype as Leo Me13 did not block killing of melanoma cells, but one documented antibody, R24, an IgG3 with specificity for the ganglioside GD3, was found to inhibit this function. These data suggest that the AK cells recognize and bind to melanoma cells by a secondary "lectin-type" receptor for a carbohydrate moiety.
Despite the key role that antibodies play in protection, the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria, we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3, both of which contribute to the impaired GC polarization observed in the absence of T-bet, resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics, thereby promoting the differentiation of B cells with increased affinity for antigen.
The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.
ABSTRACT Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate whose function has not been unequivocally defined. Partial complementation of function can be achieved by exchanging the AMA1 of P. falciparum (PfAMA1) with that of P. chabaudi (PcAMA1). In this study, parasites expressing chimeric AMA1 proteins were created to identify domains of PfAMA1 critical in erythrocyte invasion and which are important immune targets. We report that specific chimeric AMA1 proteins containing domains I to III from PfAMA1 and PcAMA1 were able to complement PfAMA1 function in erythrocyte invasion. We demonstrate that domain III does not contain dominant epitope targets of antibodies raised against Escherichia coli expressed and refolded PfAMA1 ectodomain. Furthermore, we generated a parasite line in which the N-terminal pro region of PfAMA1 does not undergo proteolytic cleavage and show that its removal is necessary for PfAMA1 function.
ABSTRACT The 235-kDa family of rhoptry proteins in Plasmodium yoelii and the two reticulocyte binding proteins of P. vivax comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in P. falciparum ( PfRH3 ) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5′ end of the gene.
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