Coenzyme A and the Malaria Parasite Plasmodium lophurae

The extracellular development of Plasmodium lophurae in vitro was favored by the addition, to the erythrocyte-extract medium, of a coenzyme A (CoA) preparation of about 75% purity. The effect of CoA was the same regardless of the concentration of free pantothenate in the medium, indicating that the parasites require the complete coenzyme rather than its pantothenic acid moiety. Erythrocyte extracts were found to contain enzymes which hydrolyzed added CoA at a rate such that 8 units per ml. of coenzyme was only slightly destroyed after 3 hours'incubation but almost completely destroyed after 18 hours'incubation at 40°C. The CoA content of erythrocytes from chickens or ducks heavily infected with P. lophurae was about twice as high as that of erythrocytes from uninfected birds. The increased CoA was associated with the parasites, an observation suggesting that malaria parasites can accumulate this essential growth factor which they cannot synthesize. The CoA concentration in the livers of infected chickens was approximately 40% lower than that in the livers of control chickens. The livers of ducks on the 6th day of infection had a slightly lower CoA concentration than those of uninfected ducks. This depletion in CoA, together with the depletion in biotin previously demonstrated in P. lophurae infection, may play a role in the pathology of this infection.     

Parasite richness/sampling effort/host range: the fancy three-piece jigsaw puzzle

In this article, Jean-Fran?ois Guégan and Clive Kennedy propose an alternative explanation for the confounding effects of host geographical range and sampling effort on parasite species richness using pathway analysis procedure. They suggest that much of the species richness revealed by sampling effort is also a reflection of host range. Thus, the total contribution of host range logically incorporates a contribution from sampling effort. The implications of indirect effects of host range on richness estimates have not previously been discussed, and the authors here attempt to redress the balance. The contribution of host range to richness, as derived from control of sampling effort on richness estimates, therefore, is a mathematical expression that does not take into account the cause-and-effect nature of things.     

Parasite prevalence and the size of host populations: an experimental test

Although important in epidemiological theory, the relationship between the size of host populations and the prevalence of parasites has not been investigated empirically. Commonly used models suggest no relationship, but this prediction is sensitive to assumptions about parasite transmission. In laboratory populations, I manipulated the size of Tribolium castaneum flour beetle populations and measured the prevalence and distribution of a parasitic mite, Acarophenax tribolii. I found that parasite prevalence did not vary for a wide range of host population sizes. However, prevalence was lower in populations with less than 40 hosts. This effect cannot be attributed to changes in host population density because host density was held constant among treatments. The reduction in prevalence of small populations below a threshold that I observed is predicted by the extinction debt model, but it is not expected from models of host-parasite interactions that assume density-dependent transmission. The distribution of parasites, measured using Lloyd's patchiness index, was not affected by host population size. The mean crowding of parasites, however, was negatively related with host density. Finally, the prevalence of parasites in large populations did not differ from that found in sets of smaller patches as long as the smaller populations in aggregate were equivalent in size to the large population.     

Preferential Invasion by Plasmodium Merozoites and the Self-Regulation of Parasite Burden

The preferential invasion of particular red blood cell (RBC) age classes may offer a mechanism by which certain species of Plasmodia regulate their population growth. Asexual reproduction of the parasite within RBCs exponentially increases the number of circulating parasites; limiting this explosion in parasite density may be key to providing sufficient time for the parasite to reproduce, and for the host to develop a specific immune response. It is critical that the role of preferential invasion in infection is properly understood to model the within-host dynamics of different Plasmodia species. We develop a simulation model to show that limiting the range of RBC age classes available for invasion is a credible mechanism for restricting parasite density, one which is equally as important as the maximum parasite replication rate and the duration of the erythrocytic cycle. Different species of Plasmodia that regularly infect humans exhibit different preferences for RBC invasion, with all species except P. falciparum appearing to exhibit a combination of characteristics which are able to self-regulate parasite density.     

Parasite interactions in natural populations: insights from longitudinal data

The physiological and immunological state of an animal can be influenced by current infections and infection history. Consequently, both ongoing and previous infections can affect host susceptibility to another parasite, the biology of the subsequent infection (e.g. infection length) and the impact of infection on host morbidity (pathology). In natural populations, most animals will be infected by a succession of different parasites throughout the course of their lives, with probably frequent concomitant infections. The relative timing of different infections experienced by a host (i.e. the sequence of infection events), and the effects on factors such as host susceptibility and host survival, can only be derived from longitudinal data on individual hosts. Here we review some of the evidence for the impact of co-infection on host susceptibility, infection biology and pathology focusing on insights obtained from both longitudinal studies in humans and experiments that explicitly consider the sequence of infection. We then consider the challenges posed by longitudinal infection data collected from natural populations of animals. We illustrate their usefulness using our data of microparasite infections associated with field vole (Microtus agrestis) populations to examine impacts on susceptibility and infection length. Our primary aim is to describe an analytical approach that can be used on such data to identify interactions among the parasites. The preliminary analyses presented here indicate both synergistic and antagonistic interactions between microparasites within this community and emphasise that such interactions could have significant impacts on host-parasite fitness and dynamics.     

A New Saurian Malaria Parasite Plasmodium clelandi sp. n. from Ceylon*

Plasmodium clelandi sp. n. is described from Varanus cepedianus of Mannar District, Ceylon. The earliest asexual stages were seen as a chromatin dot. Trophozoites have a vacuole with chromatin material spread around its periphery. The trophozoites divide and mature to form the schizont. The maximum number of merozoites observed in a schizont was 8. Pigment was scanty and sometimes absent. The gametocytes had a characteristic elongate form and tend to encircle the host cell nucleus.     

A New Saurian Malarial Parasite Plasmodium balli from Panama

SYNOPSIS. Plasmodium balli sp. nov. is described from Anolis lionotus and A. poecilopus of central Panama.
Large, elongate gametocytes and segmenters containing up to 100 merozoites are produced by P. balli. Proerythrocytes and normoblasts are more commonly parasitized than erythrocytes. Pigment is uncommon, but when present consists of a minute dot. Hypertrophy, distortion and lysis of host cell nuclei may result from parasitization of immature blood cells by gametocytes, while enucleated host cells are common.     

Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process.     

Phagotrophy and Two New Structures in the Malaria Parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO₄ with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

A Malaria Parasite, Plasmodium aurulentum sp. nov. from the Neotropical Forest Gecko Thecadactylus rapicaudus

SYNOPSIS. Plasmodium aurulentum sp. nov. from the neotropical forest gecko Thecadactylus rapicaudus in Panama is characterized by oval or round to lenticular gametocytes, 6–22 nuclei in crudely fan-shaped schizonts, and light golden pigment masses. A prominent, pinkish red-staining mass, present in older schizonts, disappears by the time schizonts reach maturation.     

Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO''s lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.     

NADP-Specific Isocitrate Dehydrogenase from the Simian Malaria Parasite Plasmodium knowlesi: Partial Purification and Characterization

ABSTRACT. Cell-free schizonts of Plasmodium knowlesi , a simian malaria parasite, possess significant isocitrate dehydrogenase (IDH) activity, about 90% of which is contributed by the NADP-specific enzyme that is localized in the cytosolic fraction. The enzyme has been partially purified by affinity chromatography using Blue sepharose CL-6B. Although unstable in nature, it is stabilized by citrate and glycerol. Kinetic studies with dl -isocitrate and NADP yielded hyperbolic curves with Michaelis constants of 0.210 and 0.038 mM, respectively. Manganous or magnesium ions are essential for activity. The enzyme is thermosensitive, shows maximum activity at pH 8.0, and has a molecular mass of about 48.5 kDa. It is strongly inhibited by thiolblocking agents but protected against them by thiol-providing agents. Cupric and argentic ions also have a marked inhibitory effect on its activity. The enzyme is significantly inhibited by chloroquine and oxytetracycline in vitro, but to a lesser degree by tetracycline.     

Purine Uptake and Utilization by the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae cannot carry out extensive de novo purine biosynthesis, and depends upon the host erythrocyte for a supply of preformed purines. Exogenous purines taken up by the parasitized erythrocyte may constitute a major source of preformed purines for parasite nucleotide biosynthesis. The uptake of exogenous radioactive purine compounds and their incorporation into nucleic acids by duck erythrocytes parasitized with P. lophurae, uninfected erythrocytes, and erythrocyte-free parasites were studied. P. lophurae was found to have a remarkable ability, both intracellularly and extracellularly, to take up and utilize certain exogenous purines such as adenosine, inosine, and hypoxanthine. Incorporation studies indicated that this species has a functional purine salvage pathway by which inosine, hypoxanthine, and adenosine can be converted to both adenine and guanine nucleotides.