ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model.

BACKGROUND: Parasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown. The rodent malaria parasites may be useful to model interactions of parasite species, which lack the var genes with their respective hosts, as other multigene families are shared between the species. The role of the endothelial receptors ICAM-1 and CD36 in cytoadherence and in the development of pathology was investigated in a Plasmodium chabaudi infection in C57BL/6 mice lacking these receptors. The schizont membrane-associated cytoadherence (SMAC) protein of Plasmodium berghei has been shown to exhibit reduced CD36-associated cytoadherence in P. berghei ANKA-infected mice. METHODS: Parasite tissue sequestration and the development of acute stage pathology in P. chabaudi infections of mice lacking CD36 or ICAM-1, their respective wild type controls, and in infections with mutant P. chabaudi parasites lacking the smac gene were compared. Peripheral blood parasitaemia, red blood cell numbers and weight change were monitored throughout the courses of infection. Imaging of bioluminescent parasites in isolated tissues (spleen, lungs, liver, kidney and gut) was used to measure tissue parasite load. RESULTS: This study shows that neither the lack of CD36 nor the deletion of the smac gene from P. chabaudi significantly impacted on acute-stage pathology or parasite sequestration. By contrast, in the absence of ICAM-1, infected animals experience less anaemia and weight loss, reduced parasite accumulation in both spleen and liver and higher peripheral blood parasitaemia during acute stage malaria. The reduction in parasite tissue sequestration in infections of ICAM-1 null mice is maintained after mosquito transmission. CONCLUSIONS: These results indicate that ICAM-1-mediated cytoadherence is important in the P. chabaudi model of malaria and suggest that for rodent malarias, as for P. falciparum, there may be multiple host and parasite molecules involved in sequestration.

Blood-stage immunity to Plasmodium chabaudi malaria following chemoprophylaxis and sporozoite immunization.

Protection against malaria in humans can be achieved by repeated exposure to infected mosquito bites during prophylactic chloroquine treatment (chemoprophylaxis and sporozoites (CPS)). We established a new mouse model of CPS immunization to investigate the stage and strain-specificity of malaria immunity. Immunization with Plasmodium chabaudi by mosquito bite under chloroquine cover does not generate pre-erythrocytic immunity, which is acquired only after immunization with high sporozoite doses. Instead, CPS immunization by bite elicits long-lived protection against blood-stage parasites. Blood-stage immunity is effective against a virulent, genetically distinct strain of P. chabaudi. Importantly, if exposure to blood-stage parasitemia is extended, blood-stage parasites induce cross-stage immunity targeting pre-erythrocytic stages. We therefore show that CPS immunization can induce robust, long-lived heterologous blood-stage immunity, in addition to protection against pre-erythrocytic parasites following high dose sporozoite immunization. Cross-stage immunity elicited by blood-stage parasites may further enhance efficacy of this immunization regimen.

Sequestration and histopathology in Plasmodium chabaudi malaria are influenced by the immune response in an organ-specific manner.

Infection with the malaria parasite, Plasmodium, is associated with a strong inflammatory response and parasite cytoadhesion (sequestration) in several organs. Here, we have carried out a systematic study of sequestration and histopathology during infection of C57Bl/6 mice with Plasmodium chabaudi AS and determined the influence of the immune response. This parasite sequesters predominantly in liver and lung, but not in the brain, kidney or gut. Histopathological changes occur in multiple organs during the acute infection, but are not restricted to the organs where sequestration takes place. Adaptive immunity, and signalling through the IFNγ receptor increased sequestration and histopathology in the liver, but not in the lung, suggesting that there are differences in the adhesion molecules and/or parasite ligands utilized and mechanisms of pathogenesis in these two organs. Exacerbation of pro-inflammatory responses during infection by deletion of the il10 gene resultsin the aggravation of damage to lung and kidney irrespective of the degree of sequestration. The immune response therefore affected both sequestration and histopathology in an organ-specific manner. P. chabaudi AS provides a good model to investigate the influence of the host response on the sequestration and specific organ pathology, which is applicable to human malaria.

Vector transmission regulates immune control of Plasmodium virulence.

Defining mechanisms by which Plasmodium virulence is regulated is central to understanding the pathogenesis of human malaria. Serial blood passage of Plasmodium through rodents, primates or humans increases parasite virulence, suggesting that vector transmission regulates Plasmodium virulence within the mammalian host. In agreement, disease severity can be modified by vector transmission, which is assumed to 'reset' Plasmodium to its original character. However, direct evidence that vector transmission regulates Plasmodium virulence is lacking. Here we use mosquito transmission of serially blood passaged (SBP) Plasmodium chabaudi chabaudi to interrogate regulation of parasite virulence. Analysis of SBP P. c. chabaudi before and after mosquito transmission demonstrates that vector transmission intrinsically modifies the asexual blood-stage parasite, which in turn modifies the elicited mammalian immune response, which in turn attenuates parasite growth and associated pathology. Attenuated parasite virulence associates with modified expression of the pir multi-gene family. Vector transmission of Plasmodium therefore regulates gene expression of probable variant antigens in the erythrocytic cycle, modifies the elicited mammalian immune response, and thus regulates parasite virulence. These results place the mosquito at the centre of our efforts to dissect mechanisms of protective immunity to malaria for the development of an effective vaccine.

Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi.

BACKGROUND: Serial blood passage of Plasmodium increases virulence, whilst mosquito transmission inherently regulates parasite virulence within the mammalian host. It is, therefore, imperative that all aspects of experimental malaria research are studied in the context of the complete Plasmodium life cycle. METHODS: Plasmodium chabaudi chabaudi displays many characteristics associated with human Plasmodium infection of natural mosquito vectors and the mammalian host, and thus provides a unique opportunity to study the pathogenesis of malaria in a single infection setting. An optimized protocol that permits efficient and reproducible vector transmission of P. c. chabaudi via Anopheles stephensi was developed. RESULTS AND CONCLUSIONS: This protocol was utilized for mosquito transmission of genetically distinct P. c. chabaudi isolates, highlighting differential parasite virulence within the mosquito vector and the spectrum of host susceptibility to infection initiated via the natural route, mosquito bite. An apposite experimental system in which to delineate the pathogenesis of malaria is described in detail.

IL-22 Protects Against Liver Pathology and Lethality of an Experimental Blood-Stage Malaria Infection.

The host response following malaria infection depends on a fine balance between levels of pro-inflammatory and anti-inflammatory mediators resulting in the resolution of the infection or immune-mediated pathology. Whilst other components of the innate immune system contribute to the pro-inflammatory milieu, T cells play a major role. For blood-stage malaria, CD4(+) and γδ T cells are major producers of the IFN-γ that controls parasitemia, however, a role for TH17 cells secreting IL-17A and other cytokines, including IL-17F and IL-22 has not yet been investigated in malaria. TH17 cells have been shown to play a role in some protozoan infections, but they also are a source of pro-inflammatory cytokines known to be involved in protection or pathogenicity of infections. In the present study, we have investigated whether IL-17A and IL-22 are induced during a Plasmodium chabaudi infection in mice, and whether these cytokines contribute to either protection or to pathology induced during the infection. Although small numbers of IL-17- and IL-22-producing CD4 T cells are induced in the spleens of infected mice, a more pronounced induction is observed in the liver, where increases in mRNA for IL-17A and, to a lesser extent, IL-22 were observed and CD8(+) T cells, rather than CD4 T cells, are a major source of these cytokines in this organ. Although the lack of IL-17 did not affect the outcome of infection or pathology, lack of IL-22 resulted in 50% mortality within 12 days after infection with significantly greater weight loss at the peak of infection and significant increase in alanine transaminase in the plasma in the acute infection. As parasitemias and temperature were similar in IL-22 KO and wild-type control mice, our observations support the idea that IL-22 but not IL-17 provides protection from the potentially lethal effects of liver damage during a primary P. chabaudi infection.

Transformation of the rodent malaria parasite Plasmodium chabaudi.

The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and immunopathology. This protocol describes an optimized method for the transformation of mature blood-stage P.c. chabaudi and a description of a vector that targets efficient, single crossover integration into the P.c. chabaudi genome. Transformed lines are reproducibly generated and selected within 14-20 d, and show stable long-term protein expression even in the absence of drug selection. This protocol, therefore, provides the scientific community with a robust and reproducible method to generate transformed P.c. chabaudi parasites expressing fluorescent, bioluminescent and model antigens that can be used in vivo to dissect many of the fundamental principles of malaria infection.

Induction of an IL7-R(+)c-Kit(hi) myelolymphoid progenitor critically dependent on IFN-gamma signaling during acute malaria.

Although the relationship between hematopoietic stem cells and progenitor populations has been investigated extensively under steady-state conditions, the dynamic response of the hematopoietic compartment during acute infection is largely unknown. Here we show that after infection of mice with Plasmodium chabaudi, a c-Kit(hi) progenitor subset positive for interleukin 7 receptor-alpha (IL-7Ralpha) emerged that had both lymphoid and myeloid potential in vitro. After being transferred into uninfected alymphoid or malaria-infected hosts, IL-7Ralpha(+)c-Kit(hi) progenitors generated mainly myeloid cells that contributed to the clearance of infected erythrocytes in infected hosts. The generation of these infection-induced progenitors was critically dependent on interferon-gamma (IFN-gamma) signaling in hematopoietic progenitors. Thus, IFN-gamma is a key modulator of hematopoiesis and innate and adaptive immunity during acute malaria infection.

The pir multigene family of Plasmodium: antigenic variation and beyond.

Multigene families are present on the telomeric and sub-telomeric regions of most chromosomes of the malaria parasite, Plasmodium. The largest gene family identified so far is the Plasmodium interspersed repeat (pir) multigene gene family and is shared by Plasmodium vivax, and simian and rodent malaria species. Most pir genes share a similar structure across the different species; a short first exon, long second exon and a third exon encoding a trans-membrane domain, and some pir genes can be assigned to specific sub-families. Although pir genes can be differentially transcribed in different life cycle stages, suggesting different functions, there is no clear link between sub-family and transcription pattern. Some of the pir genes encode proteins expressed on or near the surface of infected erythrocytes, and therefore could be potential targets of the host's immune response, and involved in antigenic variation and immune evasion. Other functions such as signalling, trafficking and adhesion have been also postulated. The presence of pir in rodent models will allow the investigation of this gene family in vivo and thus their potential as vaccines or in other interventions in human P. vivax infections.

Migrating monocytes recruited to the spleen play an important role in control of blood stage malaria.

Host responses controlling blood-stage malaria include both innate and acquired immune effector mechanisms. During Plasmodium chabaudi infection in mice, a population of CD11b(high)Ly6C(+) monocytes are generated in bone marrow, most of which depend on the chemokine receptor CCR2 for migration from bone marrow to the spleen. In the absence of this receptor mice harbor higher parasitemias. Most importantly, splenic CD11b(high)Ly6C(+) cells from P chabaudi-infected wild-type mice significantly reduce acute-stage parasitemia in CCR2(-/-) mice. The CD11b(high)Ly6C(+) cells in this malaria infection display effector functions such as production of inducible nitric oxide synthase and reactive oxygen intermediates, and phagocytose P chabaudi parasites in vitro, and in a proportion of the cells, in vivo in the spleen, suggesting possible mechanisms of parasite killing. In contrast to monocyte-derived dendritic cells, CD11b(high)Ly6C(+) cells isolated from malaria-infected mice express low levels of major histocompatibility complex II and have limited ability to present the P chabaudi antigen, merozoite surface protein-1, to specific T-cell receptor transgenic CD4 T cells and fail to activate these T cells. We propose that these monocytes, which are rapidly produced in the bone marrow as part of the early defense mechanism against invading pathogens, are important for controlling blood-stage malaria parasites.

Rapid changes in transcription profiles of the Plasmodium yoelii yir multigene family in clonal populations: lack of epigenetic memory?

The pir multigene family, found in the genomes of Plasmodium vivax, P. knowlesi and the rodent malaria species, encode variant antigens that could be targets of the immune response. Individual parasites of the rodent malaria Plasmodium yoelii, selected by micromanipulation, transcribe only 1 to 3 different pir (yir) suggesting tight transcriptional control at the level of individual cells. Using microarray and quantitative RT-PCR, we show that despite this very restricted transcription in a single cell, many yir genes are transcribed throughout the intra-erythrocytic asexual cycle. The timing and level of transcription differs between genes, with some being more highly transcribed in ring and trophozoite stages, whereas others are more highly transcribed in schizonts. Infection of immunodeficient mice with single infected erythrocytes results in populations of parasites each with transcriptional profiles different from that of the parent parasite population and from each other. This drift away from the original 'set' of transcribed genes does not appear to follow a preset pattern and "epigenetic memory" of the yir transcribed in the parent parasite can be rapidly lost. Thus, regulation of pir gene transcription may be different from that of the well-characterised multigene family, var, of Plasmodium falciparum.

Transcription and alternative splicing in the yir multigene family of the malaria parasite Plasmodium y. yoelii: identification of motifs suggesting epigenetic and post-transcriptional control of RNA expression.

The Plasmodium interspersed repeat (pir) genes represent the largest multigene family in Plasmodium genomes, and the only one shared between the human pathogen, P. vivax, the simian malaria species P. knowlesi and the rodent malaria species P.y. yoelii, P. berghei and P.c. chabaudi. PIR have been shown to be expressed on the surface of red blood cells and are thought to play a role in antigenic variation. Here we have used a range of bioinformatic and experimental approaches to investigate the existence of gene subsets within P.y. yoelii pir. We have identified five groups of yir genes which could be further distinguished by chromosomal location and different alternative splicing events. Two of the groups were not highly represented among the transcribed pirs in blood stage parasites. Together these data suggest that different pir genes may be active at different stages of the life cycle of P. yoelii and may have different functions. Analysis of the 5' UTR identified a unique highly conserved yir/bir/cir specific promoter motif, which could serve as a general recognition element for yir transcription. However, its presence in front of all yirs makes it unlikely to play a role in regulating differential expression.

Epitope-specific regulation of immunoglobulin class switching in mice immunized with malarial merozoite surface proteins.

Antibodies that bind to Fc receptors and activate complement are implicated in the efficient control of pathogens, but the processes that regulate their induction are still not well understood. To investigate antigen-dependent factors that regulate class switching, we have developed an in vivo model of class switching to immunoglobulin G2b (IgG2b) using the malaria antigen Plasmodium falciparum merozoite surface protein 2 (MSP2). C57BL/6 mice were immunized with recombinant proteins representing discrete domains of MSP2, and a T-cell epitope (C8) was identified within the conserved C terminus of the protein that preferentially induces IgG2b antibodies. The ability of C8 to induce IgG2b is ablated in both homozygous gamma interferon-negative and interleukin 10-negative mice. The IgG2b-inducing properties of C8 override the IgG1-inducing properties of both the fusion protein partner, glutathione S-transferase, and the adjuvant. Furthermore, when attached to other proteins that normally induce IgG1 responses, C8 induces a switch to IgG2b secretion. This is the first description of a defined T-cell epitope that drives specific IgG2b subclass switching, and our data offer proof of the concept that chimeric vaccines incorporating specific T-cell "switch epitopes" might be used to enhance qualitative aspects of the antibody response.

Host immunity modulates transcriptional changes in a multigene family (yir) of rodent malaria.

Variant antigens, encoded by multigene families, and expressed at the surface of erythrocytes infected with the human malaria parasite Plasmodium falciparum and the simian parasite Plasmodium knowlesi, are important in evasion of host immunity. The vir multigene family, encoding a very large number of variant antigens, has been identified in the human parasite Plasmodium vivax and homologues (yir) of this family exist in the rodent parasite Plasmodium yoelii. These genes are part of a superfamily (pir) which are found in Plasmodium species infecting rodents, monkeys and humans (P. yoelii, P. berghei, P. chabaudi, P. knowlesi and P. vivax). Here, we show that YIR proteins are expressed on the surface of erythrocytes infected with late-stage asexual parasites, and that host immunity modulates transcription of yir genes. The surface location and expression pattern of YIR is consistent with a role in antigenic variation. This provides a unique opportunity to study the regulation and expression of the pir superfamily, and its role in both protective immunity and antigenic variation, in an easily accessible animal model system.

Distinct trafficking and localization of STEVOR proteins in three stages of the Plasmodium falciparum life cycle.

The genome of Plasmodium falciparum harbors three extensive multigene families, var, rif, and stevor (for subtelomeric variable open reading frame), located mainly in the subtelomeric regions of the parasite's 14 chromosomes. STEVOR variants are known to be expressed in asexual parasites, but no function has as yet been ascribed to this protein family. We have examined the expression of STEVOR proteins in intraerythrocytic sexual stages, gametocytes, and extracellular sporozoites isolated from infected Anopheles mosquitoes. In gametocytes, stevor transcripts appear transiently early in development but STEVOR proteins persist for several days and are transported out of the parasite, travel through the host cell cytoplasm, and localize to the erythrocyte plasma membrane. In contrast to asexual parasites, gametocytes move STEVOR to the periphery via a trafficking pathway independent of Maurer's clefts. In sporozoites, STEVOR appear dispersed throughout the cytoplasm in vesicle-like structures. The pattern of STEVOR localization we have observed in gametocytes and sporozoites differs significantly from that in asexual parasite stages. STEVOR variants are therefore likely to perform different functions in each stage of the parasites life cycle in which they occur.

Conservation and developmental control of alternative splicing in maebl among malaria parasites.

Genes of malaria parasites and other unicellular organisms have larger exons with fewer and smaller introns than metaozoans. Such differences in gene structure are perceived to extend to simpler mechanisms for transcriptional control and mRNA processing. Instead, we discovered a surprisingly complex level of post-transcriptional mRNA processing in analysis of maebl transcripts in several Plasmodium species. Mechanisms for internal alternative cis-splicing and exon skipping were active in multiple life cycle stages to change exon structure in the deduced coding sequence (CDS). The major alternatively spliced transcript utilized a less favorable acceptor splice site, which shifted codon triplet usage to a different CDS with a hydrophilic C terminus, changing the canonical type I membrane MAEBL product to a predicted soluble isoform. We found that developmental control of the alternative splicing pattern was distinct from the canonical splicing pattern. Western blot analysis indicated that MAEBL expression was better correlated with the appearance of the canonical ORF1 transcript. Together these data reveal that RNA metabolism in unicellular eukaryotes like Plasmodium is more sophisticated than believed and may have a significant role regulating gene expression in Plasmodium.

The Py235 proteins: glimpses into the versatility of a malaria multigene family.

Py235 rhoptry proteins, encoded by a multigene family of the rodent malaria parasite Plasmodium yoelii, combine a functional role in invasion with one of immune evasion, and have homologues in malaria parasites of humans. Investigations of Py235 are summarised and the perspectives for dissecting the molecular and biological mechanisms underlying these crucial phenomena are discussed.

Antibodies against MAEBL ligand domains M1 and M2 inhibit sporozoite development in vitro.

MAEBL is a type 1 membrane protein that is implicated in the merozoite invasion of erythrocytes and sporozoite invasion of mosquito salivary glands. This apical organelle protein is structurally similar to the ebl erythrocyte binding proteins, such as EBA-175, except that the tandem ligand domains of MAEBL are similar to part of the extracellular domain of apical membrane antigen 1 and not the Duffy binding-like domain. Although midgut and salivary gland sporozoites are morphologically similar, salivary gland sporozoites undergo a period of new gene expression after infecting the salivary glands, display distinct phenotypic differences, and are more infectious for the mammalian host. The objectives of this project were to determine the molecular form of MAEBL in the infectious salivary gland sporozoites and whether the ligand has a role in the sporozoite development to exoerythrocytic stages in hepatocytes. We determined that MAEBL is newly expressed in salivary gland sporozoites and in a form distinct from what is present in the midgut sporozoites or present in erythrocytic stages. Both ligand domains (M1 and M2) were expressed as part of a full-length membrane form of MAEBL in the salivary gland sporozoites in contrast to the other stages that retain only the M2 ligand domain as part of the membrane form of the protein. Antisera developed against the cysteine-rich regions of the extracellular portion of MAEBL inhibited sporozoite development to exoerythrocytic forms in vitro. Together these data indicate that MAEBL has a role in this third developmental stage in the life cycle of the malaria parasite. Thus, MAEBL is another target for pre-erythrocytic-stage vaccine development against malaria parasites.

Cerebral edema and cerebral hemorrhages in interleukin-10-deficient mice infected with Plasmodium chabaudi.

During a Plasmodium chabaudi infection in interleukin-10 (IL-10) knockout mice, there is greater parasite sequestration, more severe cerebral edema, and a high frequency of cerebral hemorrhage compared with infection of C57BL/6 mice. Anti-tumor necrosis factor alpha treatment ameliorated both cerebral edema and hemorrhages, suggesting that proinflammatory responses contributed to cerebral complications in infected IL-10(-/-) mice.