Studies on enzyme variation in the murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi by starch gel electrophoresis.

Electrophoretic variation of the enzymes glucose phosphate isomerase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase (NADP-dependent) has been studied in the African murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi and their subspecies. Horizontal starch gel electrophoresis was used throughout. The number of isolates examined in each subspecies varied from 1 (P. y. nigeriensis) to 24 (P. c. chabaudi). Extensive enzyme variation was found among isolates of most of the subspecies from which more than two such isolates were available for study. It is clear that the phenomenon of enzyme polymorphism is of common occurrence among malaria parasites. With the exception of P. berghei and P. yoelii, of which all isolates share an identical electrophoretic form of lactate dehydrogenase, no enzyme forms are shared between any of the 4 species of murine plasmodia. By contrast, within each species common enzyme forms are shared among each of the subspecies. The subspecies are nevertheless, distinguished from each other by the electrophoretic forms of at least one enzyme. The distribution and reassortment of enzyme variation among isolates of a single subspecies is in accordance with the concept of malaria parasites as sexually reproducing organisms. The study of variation among parasites present in individual wild-caught rodent hosts demonstrates that natural malarial infections usually comprise genetically heterogeneous populations of parasites. Nevertheless, the number of genetically distinct types of parasite of any one species present in a single infected host appears to be small. Generally not more than 2 or 3 clones of parasite of distinct genetic constitution are present in a single infected animal.     

Growth in vitro and metabolism of Plasmodium vinckei chabaudi.

An in vitro system, based on the rocker dilution technic, has been developed that supports intraerythrocytic growth of a rat-adapted strain of Plasmodium vinckei chabaudi from ring to schizont stages; some reinvasion was obtained, although invariably, this was associated with a decrease in parasite numbers. Pertinent features were the very high buffer content of the medium and the low oxygen tension of the gaseous phase. Lactate production, glucose utilization, and 3H-leucine and 3H-adenosine incorporations were investigated for their suitability to monitor parasite growth. Throughout an 18-hr incubation there was a continuous and increasing production of lactate and utilization of glucose, which correlated well with the development of the parasites from ring to schizont stages. During the same period, there was a low but continuous and increasing incorporation of 3H-leucine into parasite protein. However, 3H-adenosine was incorporated only for the 1st hr of incubation, after which time no net incorporation occurred. Parasites grew normally from ring to schizont stages even in the absence of adenosine from the dilution medium.     

Growth in vitro and Metabolism of Plasmodium vinckei chabaudi*

SYNOPSIS. An in vitro system, based on the rocker dilution technic, has been developed that supports intraerythrocytic growth of a rat-adapted strain of Plasmodium vinckei chabaudi from ring to schizont stages; some reinvasion was obtained, although invariably, this was associated with a decrease in parasite numbers. Pertinent features were the very high buffer content of the medium and the low oxygen tension of the gaseous phase. Lactate production, glucose utilization, and ³H-leucine and ³H-adenosine incorporations were investigated for their suitability to monitor parasite growth. Throughout an 18-hr incubation there was a continuous and increasing production of lactate and utilization of glucose, which correlated well with the development of the parasites from ring to schizont stages. During the same period, there was a low but continuous and increasing incorporation of ³H-leucine into parasite protein. However, ³H-adenosine was incorporated only for the 1st hr of incubation, after which time no net incorporation occurred. Parasites grew normally from ring to schizont stages even in the absence of adenosine from the dilution medium.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).     

Interactions between malaria parasites and their mosquito hosts in the midgut

This review examines what is presently known of the molecular interactions between Plasmodium and Anopheles that take place in the latter's midgut upon ingestion of the parasites with an infectious blood meal. In order to become 'established' in the gut and to transform into a sporozoite-producing oocyst, the malaria parasite needs to undergo different developmental steps that are often characterized by the use of selected resources provided by the mosquito vector. Moreover, some of these resources may be used by the parasite in order to overcome the insect host's defence mechanisms. The molecular partners of this interplay are now in the process of being defined and analyzed for both Plasmodium and mosquito and, thus, understood; these will be presented here in some detail.     

Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites

A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31–40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30–39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-γ production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.     

Coevolutionary genetics of Plasmodium malaria parasites and their human hosts

Malaria has been invoked, perhaps more than any other infectious disease, as a force for the selection of human genetic polymorphisms. Evidence for genome-shaping interactions can be found in the geographic and ethnic distributions of the hemoglobins, blood group antigens, thalassemias, red cell membrane molecules, human lymphocyte antigen (HLA) classes, and cytokines. Human immune responses and genetic variations can correspondingly influence the structure and polymorphisms of Plasmodium populations, notably in genes that affect the success and virulence of infection. In Africa, where the burden from Plasmodium falciparum predominates, disease severity and manifestations vary in prevalence among human populations. The evolutionary history and spread of Plasmodium species inform our assessment of malaria as a selective force. Longstanding host-pathogen relationships, as well as recent changes in this dynamic, illustrate the selective pressures human and Plasmodium species place on one another. Investigations of malaria protection determinants and virulence factors that contribute to the complexity of the disease should advance our understanding of malaria pathogenesis.     

Lipid transport proteins in malaria,from Plasmodium parasites to their hosts

Eukaryotic unicellular pathogens from the genus Plasmodium are the etiological agents of malaria, a disease that persists over a wide range of vertebrate species, including humans. During its dynamic lifecycle, survival in the different hosts depends on the parasite's ability to establish a suitable environmental milieu. To achieve this, specific host processes are exploited to support optimal growth, including extensive modifications to the infected host cell. These modifications include the formation of novel membranous structures, which are induced by the parasite. Consequently, to maintain a finely tuned and dynamic lipid environment, the organisation and distribution of lipids to different cell sites likely requires specialised lipid transfer proteins (LTPs). Indeed, several parasite and host-derived LTPs have been identified and shown to be essential at specific stages. Here we describe the roles of LTPs in parasite development and adaptation to its host including how the latest studies are profiting from the improved genetic, lipidomic and imaging toolkits available to study Plasmodium parasites. Lastly, a list of predicted Plasmodium LTPs is provided to encourage research in this field.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gametocyte development of Plasmodium chabaudi in mice and rats: evidence for host induction of gametocytogenesis

Gametocytogenesis in Plasmodium chabaudi was studied in intact and splenectomized mice and rats. Blood forms inoculated into mice entered a new asexual cycle, resulting in high parasitaemias 4 days after inoculation. Blood forms inoculated into intact rats were almost eliminated by day 4 after injection. In splenectomized rats, however, the parasitaemia remained constant over this period. With an immunofluorescence test (IFA), using an antiserum to mouse erythrocytes, it was found that the majority of parasites invaded rat erythrocytes, but were unable to enter a new asexual cycle. It was found that up to 50% of the parasites in splenectomized rats developed into gametocytes. The IFA showed that the proportion of gametocytes to asexual forms was very high in splenectomized animals, irrespective of the origin of the host cells-mouse or rat.     

Midgut specific immune response of vector mosquito Anopheles stephensi to malaria parasite Plasmodium

Innate immune-related polypeptides expression in midgut in the ageing vector mosquito A. stephensi following infection by malaria parasite, Plasmodium yoelii yoelii has been studied. Twenty polypeptides were induced by an infected blood meal during various stages of adult life. A 24 kDa polypeptide was induced generally in most of the stages. Maximum parasite induced polypeptides i.e. 22, 33, 111, 122, 127, 140, 143 and 146 kDa were found in 5 days of post blood feeding (PBF) which coincides with the presence of oocysts on the midgut. However, in addition, three polypeptides in 11 days PBF and 8 polypeptides in 20 days PBF were also induced due to parasite infection in aged mosquitoes. Quantitatively, the amount of soluble proteins in the midgut in oocyst-sporozoite-positive mosquitoes was always less as compared to their normal counterparts. The parasite evidently elicits defined immune responses by inducing specific polypeptides in the midgut of the mosquito.     

Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria

Blood-stage malaria of Plasmodium chabaudi is characterized by its responsiveness to testosterone (T): T suppresses development of protective immunity, whereas once acquired immunity is T-unresponsive. Here, we have analyzed the liver, a T target and lymphoid organ with anti-malaria activity, for its T-responsiveness of gene expression in immune mice. Using Affymetrix microarray technology, in combination with quantitative RT-PCR, we have identified (i) T-unresponsive expression of newly acquired mRNAs encoding diverse sequences of IgG- and IgM-antibodies, (ii) 24 genes whose expression has become T-unresponsive including those encoding the T(H)2 response promoting EHMT2 and the erythrocyte membrane protein band 7.2 STOM, (iii) T-unresponsive expression of mRNAs for the cytokines IL-1β, IL-6, TNFα, and IFNγ, as well as iNOS, which are even not inducible by infection, and (iv) 35 genes retaining their T-responsiveness, which include those encoding the infection-inducible acute phase proteins SAA1, SAA2, and ORM2 as well as those of liver metabolism which encode the T-downregulated female-prevalent enzymes CYP2B9, CYP2B13, CYP3A41, CYP7A1, and SULT2A2 and the T-upregulated male-prevalent enzymes CYP2D9, CYP7B1, UGT2B1, HSD3B2, HSD3B5, respectively. The mRNA of the latter T-metabolizing enzyme is even 5-fold increased by T, suggesting a decrease in the effective T concentrations in the liver of immune mice. Collectively, our data suggest that the liver, which has acquired a selective T-unresponsiveness of gene expression, contributes to the acquired T-unresponsive, antibody-mediated protective immunity to blood-stage malaria of P. chabaudi.     

Selection for high and low virulence in the malaria parasite Plasmodium chabaudi.

What stops parasites becoming ever more virulent? Conventional wisdom and most parasite-centred models of the evolution of virulence suppose that risk of host (and, hence, parasite) death imposes selection against more virulent strains. Here we selected for high and low virulence within each of two clones of the rodent malaria parasite Plasmodium chabaudi on the basis of between-host differences in a surrogate measure of virulence--loss of live weight post-infection. Despite imposing strong selection for low virulence which mimicked 50-75% host mortality, the low virulence lines increased in virulence as much as the high virulence lines. Thus, artificial selection on between-host differences in virulence was unable to counteract natural selection for increased virulence caused by within-host selection processes. The parasite''s asexual replication rate and number of sexual transmission forms also increased in all lines, consistent with evolutionary models explaining high virulence. An upper bound to virulence, though not the asexual replication rate, was apparent, but this bound was not imposed by host mortality. Thus, we found evidence of the factors assumed to drive evolution of increased virulence, but not those thought to counter this selection.     

Immunological relationship of Plasmodium inui with two other quartan malaria parasites, P. malariae and P. brasilianum

A series of monoclonal antibodies was produced against sporozoites of the OS strain of Plasmodium inui, a simian quartan malaria parasite, and used to characterize the circumsporozoite protein of this parasite. The results confirm that the immunodominant epitope of the circumsporozoite protein of P. inui is immunologically distinct from those of 2 other quartan parasites, the human P. malariae and simian P. brasilianum, which are identical.