The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism

Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC₅₀ ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.     

Selective accumulation of a novel antimalarial rhodacyanine derivative, SSJ-127, in an organelle of Plasmodium berghei

SSJ-127, a novel antimalarial rhodacyanine derivative, has shown potent antimalarial activity against chloroquine-resistant Plasmodium strains in vitro and subcutaneous administration of SSJ-127 results in a complete cure of a mouse malaria model. SSJ-127 was detected by fluorescence microscopy in the mouse malaria parasites Plasmodium berghei after exposure of infected red blood cells to the compound in vitro and in vivo. Selective accumulation of SSJ-127 in an organelle is observed in all blood stages of live malaria parasites. The organelle is clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways.     

Characterisation of carbonic anhydrase in Plasmodium falciparum

Here we report the existence, purification and characterisation of carbonic anhydrase in Plasmodium falciparum. The infected red cells contained carbonic anhydrase approximately 2 times higher than those of normal red cells. The three developmental forms of the asexual stages, ring, trophozoite and schizont were isolated from their host red cells and found to have stage-dependent activity of the carbonic anhydrase. The enzyme was purified to homogeneity from the crude extract of P. falciparum using multiple steps of fast liquid chromatographic techniques. It had a Mr of 32 kDa and was active in a monomeric form. The human red cell enzyme was also purified for comparison with the parasite enzyme. The parasite enzyme activity was sensitive to well-known sulfonamide-based inhibitors of both bacterial and mammalian enzymes, sulfanilamide and acetazolamide. The kinetic properties and the amino terminal sequences of the purified enzymes from the parasite and host red cell were found to be different, indicating that the purified protein most likely exhibited the P. falciparum carbonic anhydrase activity. In addition, the enzyme inhibitors had antimalarial effect against in vitro growth of P. falciparum. Moreover, the vital contribution of the carbonic anhydrase to the parasite survival makes the enzyme an attractive target for therapeutic evaluation.     

The Homeostasis of Plasmodium falciparum-Infected Red Blood Cells

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15–32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before ~48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.     

Pyridoxine kinase in Plasmodium lophurae and duckling erythrocytes.

Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent Km for pyridoxine of the parasite enzyme was 6.6 times 10(-5) M whereas the host red cell enzyme Km was 1.9 times 10(-6) M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent Ki of 1.5 times 10(-6) and 8.6 times 10(-6) M, respectively. These results suggest that the vitamin B6 metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.     

Plasmodium falciparum exported protein PFE60 influences Maurer’s clefts architecture and virulence complex composition

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer’s clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore – unlike all other described resident MC membrane proteins – PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.     

Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum.

The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing. The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli. The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites. In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte. This is the first time a P. falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol. Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway.     

Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite

Selected aspects of the metabolism of Plasmodium falciparum are reviewed, but conclusions based on the study of other species of plasmodia are intentionally not included since these may not be applicable. The parasites increase glucose consumption 50-100 fold as compared to uninfected red cells; most of the glucose is metabolized to lactic acid. The parasite contains a complete set of glycolytic enzymes. Some enzymes such a hexokinase, enolase and pyruvate kinase are vastly increased over corresponding levels in uninfected red cells. However, the pathway for synthesizing 2,3-diphosphoglycerate (2,3-DPG) is absent. Parasitized red cells show a decline in the concentration of 2,3-DPG which may function as an inhibitor for certain essential enzyme pathways. Pentose shunt activity is increased in absolute terms, but as a percent of total glucose consumption, there is a decrease during parasite infection of the red cell. The parasite contains a gene for G6PD and can produce a small quantity of parasite-encoded enzyme. It is not clear if the production of this enzyme can be up-regulated in G6PG deficient host red cells. The NADPH normally produced by the pentose shunt can be obtained from other parasite pathways (such as glutamate dehydrogenase). NADPH may subserve additional needs in the infected red cell such as driving diribonucleotide reductase activity--a rate limiting enzyme in DNA synthesis. The role of NADPH in protecting the parasite-red cell system against oxidative stress (via glutathione reduction) remains controversial. Parasitized red cells contain about 10 times more NAD(H) than uninfected red cells, but the NADP(H) content is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite.

Multiplication of the human malaria parasite Plasmodium falciparum within red blood cells is an energy-dependent process and glucose consumption increases dramatically in infected red blood cells (IRBC) versus normal red blood cells (NRBC). The major pathway for glucose metabolism in P. falciparum IRBC is anaerobic glycolysis. Phosphoglycerate kinase (PGK) is one of the key enzymes of this pathway as it generates ATP. We found that the PGK specific activity in P. falciparum IRBC is seven times higher than that in NRBC. The parasitic origin of the increase in PGK activity is confirmed by isoelectric focusing. Indeed, two P. falciparum isoenzymes with neutral isoelectric points were detected. P. falciparum PGK in purified form has a molecular mass of 48 kDa. Antiserum raised against purified P. falciparum PGK specifically recognizes the 48-kDa protein band in P. falciparum and also reacts with P. berghei and P. yoelii IRBC lysates but does not cross-react with PGK associated with NRBC.     

DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be “sub-microscopic” blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.     

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) – Isoleucine (I) – Lysine (K) – Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite’s ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Histidine-rich protein in rodent malaria

Mouse red blood cells (RBCs) infected with the malaria parasite Plasmodium yoelii nigeriensis were shown to synthesize a histidine-rich protein (His-RP) in vitro. The existence of this protein was demonstrated by comparing fluorograms of infected red blood cells (IRBCs) labelled with either [14C]histidine or [14C]leucine. The molecular weight of this His-RP was estimated to be 43,500, which compares well with the values reported for the avian parasite P. lophurae (45,000) and for the human parasite P. falciparum (42,000). This result supports the idea that such a protein may play an important role in the biology of all plasmodium species.     

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.     

Structure and non-essential function of glycerol kinase in Plasmodium falciparum blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito.     

Cytoadherence, pathogenesis and the infected red cell surface in Plasmodium falciparum.

The particular virulence of Plasmodium falciparum compared with the other malaria species which naturally infect humans is thought to be due to the way in which the parasite modifies the surface of the infected red cell. Approximately 16 hours into the asexual cycle, parasite encoded proteins appear on the red cell surface which mediate adherence to a variety of host tissues. Binding of infected red cells to vascular endothelium, a process which occurs in all infections, is thought to be an important factor in the pathogenesis of severe disease where concentration of organisms in particular organs such as the brain occurs. Binding to uninfected red cells to form erythrocyte rosettes, a property of some isolates, is linked to disease severity. Here we summarise the data on the molecular basis of these interactions on both the host and parasite surfaces and review the evidence for the involvement of particular receptors in specific disease syndromes. Finally we discuss the relevance of these data to the development of new treatments for malaria.     

Human malaria parasite adenosine deaminase. Characterization in host enzyme-deficient erythrocyte culture

Human malaria infected erythrocytes show a dramatic increase in adenosine deaminase activity in vitro. Using recently developed culture techniques, adenosine deaminase-deficient human erythrocytes were infected in vitro with the major human pathogen Plasmodium falciparum. Adenosine deaminase activity was undetectable in the uninfected host red cells, but increased by 2-fold over normal levels in these cells with an 8% parasitemia. The enzyme in these cells appeared unique in that its activity was markedly elevated over that of other parasite purine enzymes, was not cross-reactive with antibody against human erythrocyte adenosine deaminase, and though inhibited competitively by deoxycoformycin was relatively insensitive to erythro-9-(2-hydroxy-3-nonyl) adenine. The use of adenosine deaminase-deficient erythrocytes for the in vitro cultivation of Plasmodium provides a unique system for the study of parasite enzyme and allows further insight into the purine metabolism of the intraerythrocytic malaria parasite.     

Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell.We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia.For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.     

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.     

Inhibition of Ca2+/calmodulin-dependent protein kinase blocks morphological differentiation of plasmodium gallinaceum zygotes to ookinetes

Once ingested by mosquitoes, malaria parasites undergo complex cellular changes. These include zygote formation, transformation of zygote to ookinete, and differentiation from ookinete to oocyst. Within the oocyst, the parasite multiplies into numerous sporozoites. Modulators of intracellular calcium homeostasis, MAPTAM, and TMB-8 blocked ookinete development as did the calmodulin (CaM) antagonists W-7 and calmidazolium. Ca(2+)/CaM-dependent protein kinase inhibitor KN-93 also blocked zygote elongation, while its ineffective analog KN-92 did not have such effect. In vitro both zygote and ookinete extracts efficiently phosphorylated autocamtide-2, a classic CaM kinase substrate, which could be blocked by calmodulin antagonists W-7 and calmidazolium and CaM kinase inhibitor KN-93. These results demonstrated the presence of calmodulin-dependent CaM kinase activity in the parasite. KN-93-treated parasites, however, expressed the ookinete-specific enzyme chitinase and the ookinete surface antigen Pgs28 normally, suggesting that the morphologically untransformed parasites are biochemically mature ookinetes. In mosquitoes, KN-93-treated parasites did not develop as oocysts, while KN-92-treated parasites produced similar numbers of oocysts as controls. These data suggested that in Plasmodium gallinaceum morphological development of zygote to ookinete, but not its biochemical maturation, relies on Ca(2+)/CaM-dependent protein kinase activity and demonstrated that the morphological differentiation is essential for the further development of the parasite in infected blood-fed mosquitoes.