The origin of malarial parasites in orangutans

Background

Recent findings of Plasmodium in African apes have changed our perspectives on the evolution of malarial parasites in hominids. However, phylogenetic analyses of primate malarias are still missing information from Southeast Asian apes. In this study, we report molecular data for a malaria parasite lineage found in orangutans.

Methodology/Principal Findings

We screened twenty-four blood samples from Pongo pygmaeus (Kalimantan, Indonesia) for Plasmodium parasites by PCR. For all the malaria positive orangutan samples, parasite mitochondrial genomes (mtDNA) and two antigens: merozoite surface protein 1 42 kDa (MSP-1₄₂) and circumsporozoite protein gene (CSP) were amplified, cloned, and sequenced. Fifteen orangutans tested positive and yielded 5 distinct mitochondrial haplotypes not previously found. The haplotypes detected exhibited low genetic divergence among them, indicating that they belong to one species. We report phylogenetic analyses using mitochondrial genomes, MSP-1₄₂ and CSP. We found that the orangutan malaria parasite lineage was part of a monophyletic group that includes all the known non-human primate malaria parasites found in Southeast Asia; specifically, it shares a recent common ancestor with P. inui (a macaque parasite) and P. hylobati (a gibbon parasite) suggesting that this lineage originated as a result of a host switch. The genetic diversity of MSP-1₄₂ in orangutans seems to be under negative selection. This result is similar to previous findings in non-human primate malarias closely related to P. vivax. As has been previously observed in the other Plasmodium species found in non-human primates, the CSP shows high polymorphism in the number of repeats. However, it has clearly distinctive motifs from those previously found in other malarial parasites.

Conclusion

The evidence available from Asian apes indicates that these parasites originated independently from those found in Africa, likely as the result of host switches from other non-human primates.     

Positive selection underlies the species‐specific binding of Plasmodium falciparum RH5 to human basigin

Plasmodium falciparum, the causative agent of the deadliest form of malaria, is a member of the Laverania subgenus, which includes ape‐infecting parasites. P. falciparum is thought to have originated in gorillas, although infection is now restricted to humans. Laverania parasites display remarkable host‐specificity, which is partially mediated by the interaction between parasite ligands and host receptors. We analyse the evolution of BSG (basigin) and GYPA (glycophorin A) in primates/hominins, as well as of their Plasmodium‐encoded ligands, PfRH5 and PfEBA175. We show that, in primates, positive selection targeted two sites in BSG (F27 and H102), both involved in PfRH5 binding. A population genetics–phylogenetics approach detected the strongest selection for the gorilla lineage: one of the positively selected sites (K191) is a major determinant of PfRH5 binding affinity. Analysis of RH5 genes indicated episodic selection on the P. falciparum branch; the positively selected W447 site is known to stabilize the interaction with human basigin. Conversely, we detect no selection in the receptor‐binding region of EBA175 in the P. falciparum lineage. Its host receptor, GYPA, shows evidence of positive selection in all hominid lineages; selected codons include glycosylation sites that modulate PfEBA175 binding affinity. Data herein provide an evolutionary explanation for species‐specific binding of the PfRH5‐BSG ligand–receptor pair and support the hypothesis that positive selection at these genes drove the host shift leading to the emergence of P. falciparum as a human pathogen.     

Selection shapes malaria genomes and drives divergence between pathogens infecting hominids versus rodents

Background  

Malaria kills more people worldwide than all inherited human genetic disorders combined. To characterize how the parasites causing this disease adapt to different host environments, we compared the evolutionary genomics of two distinct groups of malaria pathogens in order to identify critical properties associated with infection of different hosts: those parasites infecting hominids (Plasmodium falciparum and P. reichenowi) versus parasites infecting rodent hosts (P. yoelii yoelii, P. berghei, and P. chabaudi). Adaptation by the parasite to its host is likely highly critical to the evolution of these species.     

Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

Background  

Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites.     

No Evidence for Ape Plasmodium Infections in Humans in Gabon

African great apes are naturally infected by a multitude of Plasmodium species most of them recently discovered, among which several are closely related to human malaria agents. However, it is still unknown whether these animals can serve as source of infections for humans living in their vicinity. To evaluate this possibility, we analysed the nature of Plasmodium infections from a bank of 4281 human blood samples collected in 210 villages of Gabon, Central Africa. Among them, 2255 were detected positive to Plasmodium using molecular methods (Plasmodium Cytochrome b amplification). A high throughput sequencing technology (454 GS-FLX Titanium technology, Roche) was then used to identify the Plasmodium species present within each positive sample. Overall, we identified with confidence only three species infecting humans in Gabon: P. falciparum, P. malariae and P. ovale. None of the species known to infect non-human primates in Central Africa was found. Our study shows that ape Plasmodium parasites of the subgenus Laverania do not constitute a frequent source of infection for humans. It also suggests that some strong host genetic barriers must exist to prevent the cross species transmission of ape Plasmodium in a context of ever increasing contacts between humans and wildlife.     

Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes

Background  

Functional screens based on dsRNA-mediated gene silencing identified several Anopheles gambiae genes that limit Plasmodium berghei infection. However, some of the genes identified in these screens have no effect on the human malaria parasite Plasmodium falciparum; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different Plasmodium species.     

Identification of Plasmodium malariae,a Human Malaria Parasite,in Imported Chimpanzees

It is widely believed that human malaria parasites infect only man as a natural host. However, earlier morphological observations suggest that great apes are likely to be natural reservoirs as well. To identify malaria parasites in great apes, we screened 60 chimpanzees imported into Japan. Using the sequences of small subunit rRNA and the mitochondrial genome, we identified infection of Plasmodium malariae, a human malaria parasite, in two chimpanzees that were imported about thirty years ago. The chimpanzees have been asymptomatic to the present. In Japan, indigenous malaria disappeared more than fifty years ago; and thus, it is most likely inferred that the chimpanzees were infected in Africa, and P. malariae isolates were brought into Japan from Africa with their hosts, suggesting persistence of parasites at low level for thirty years. Such a long term latent infection is a unique feature of P. malariae infection in humans. To our knowledge, this is the first to report P. malariae infection in chimpanzees and a human malaria parasite from nonhuman primates imported to a nonendemic country.     

A fresh look at the origin of Plasmodium falciparum, the most malignant malaria agent

From which host did the most malignant human malaria come: birds, primates, or rodents? When did the transfer occur? Over the last half century, these have been some of the questions up for debate about the origin of Plasmodium falciparum, the most common and deadliest human malaria parasite, which is responsible for at least one million deaths every year. Recent findings bring elements in favor of a transfer from great apes, but are these evidences really solid? What are the grey areas that remain to be clarified? Here, we examine in depth these new elements and discuss how they modify our perception of the origin and evolution of P. falciparum. We also discuss the perspectives these new discoveries open.     

Cladistic relationships of extant and fossil hominoids

There is general agreement that the hominoid primates form a monophyletic group, that the extant great apes and humans form a second clade within that group with the gibbons as the sister group, and that the African apes and humans form a third clade. Although it has recently been proposed that humans and orang utans are sister taxa and also that the great apes form a clade to the exclusion of humans, our analysis, particularly of the molecular evidence, supports the existence of an African ape and human clade. The major problem in hominoid phylogeny at present is the relationships of the species within this clade: morphological data generally support the existence of an African ape clade which is the sister group to humans; some molecular data also support this conclusion, but most molecular evidence indicates the existence of a chimpanzee/human clade. We have cladistically re-analysed the DNA and protein sequence data for which apomorphic character states can be assessed. It is clear that there is a high degree of homoplasy whichever branching pattern is produced, with some characters supporting the existence of a chimpanzee/human clade and others supporting an African ape clade. When the cladistic analyses of morphological and molecular data are combined we believe that the most parsimonious interpretation of the data is that the African apes form a clade which is the sister taxon of the human (i.e., Australopithecus, Homo and Paranthropus) clade.This paper is not intended as a survey of all hominoid fossils but as a study of branching points in hominoid evolution and fossils are included which are relevant to this branching pattern. The analysis of fossil taxa in this study leads us to conclude that Proconsul is the sister taxon to the later Hominoidea. A number of middle Miocene forms such as Dryopithecus, Kenyapithecus, Heliopithecus and Afropithecus are shown to share derived characters with great apes and humans and provide evidence for the divergence of that clade from the gibbon lineage prior to 18 Ma. The position that Sivapithecus represents the sister group of the orang utan clade is supported here and shows that the orang utan lineage had diverged from the African ape and human lineage prior to 11·5 Ma. There is unfortunately no definitive fossil cvidence on branching sequences within the African ape and human clade, although a new specimen from Samburu, Kenya may be related to the gorilla.     

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor,artemisinic acid

Background  

Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.     

On the Diversity of Malaria Parasites in African Apes and the Origin of Plasmodium falciparum from Bonobos

The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites'' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria.     

Investigating zoonotic infection barriers to ape Plasmodium parasites using faecal DNA analysis

African apes are endemically infected with numerous Plasmodium spp. including close relatives of human Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Although these ape parasites are not believed to pose a zoonotic threat, their ability to colonise humans has not been fully explored. In particular, it remains unknown whether ape parasites are able to initiate exo-erythrocytic replication in human hepatocytes following the bite of an infective mosquito. Since animal studies have shown that liver stage infection can result in the excretion of parasite nucleic acids into the bile, we screened faecal samples from 504 rural Cameroonians for Plasmodium DNA. Using pan-Laverania as well as P. malariae- and P. vivax-specific primer sets, we amplified human P. falciparum (n?=?14), P. malariae (n?=?1), and P. ovale wallikeri (n?=?1) mitochondrial sequences from faecal DNA of 15 individuals. However, despite using an intensified PCR screening approach we failed to detect ape Laverania, ape P. vivax or ape P. malariae parasites in these same subjects. One faecal sample from a hunter-gatherer contained a sequence closely related to the porcupine parasite Plasmodium atheruri. Since this same faecal sample also contained porcupine mitochondrial DNA, but a matching blood sample was Plasmodium-negative, it is likely that this hunter-gatherer consumed Plasmodium-infected bushmeat. Faecal Plasmodium detection was not secondary to intestinal bleeding and/or infection with gastrointestinal parasites, but indicative of blood parasitaemia. Quantitative PCR identified 26-fold more parasite DNA in the blood of faecal Plasmodium-positive than faecal Plasmodium-negative individuals (P?=?0.01). However, among blood-positive individuals only 10% - 20% had detectable Plasmodium sequences in their stool. Thus, faecal screening of rural Cameroonians failed to uncover abortive ape Plasmodium infections, but detected infection with human parasites, albeit with reduced sensitivity compared with blood analysis.     

Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes

The uptake and expression of extracellular DNA has been established as a mechanism for horizontal transfer of genes between bacterial species. Such transfer can support acquisition of advantageous elements, including determinants that affect the interactions between infectious organisms and their hosts. Here we show that erythrocyte-stage Plasmodium falciparum malaria parasites spontaneously take up DNA from the host cell cytoplasm into their nuclei. We have exploited this finding to produce levels of reporter expression in P.falciparum that are substantially improved over those obtained by electroporation protocols currently used to transfect malaria parasites. Parasites were transformed to a drug-resistant state when placed into cell culture with erythrocytes containing a plasmid encoding the human dihydrofolate reductase sequence. The findings reported here suggest that the malaria genome may be continually exposed to exogenous DNA from residual nuclear material in host erythrocytes.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

New insights into the evolutionary history of Plasmodium falciparum from mitochondrial genome sequence analyses of Indian isolates

Estimating genetic diversity and inferring the evolutionary history of Plasmodium falciparum could be helpful in understanding origin and spread of virulent and drug‐resistant forms of the malaria pathogen and therefore contribute to malaria control programme. Genetic diversity of the whole mitochondrial (mt) genome of P. falciparum sampled across the major distribution ranges had been reported, but no Indian P. falciparum isolate had been analysed so far, even though India is highly endemic to P. falciparum malaria. We have sequenced the whole mt genome of 44 Indian field isolates and utilized published data set of 96 genome sequences to present global genetic diversity and to revisit the evolutionary history of P. falciparum. Indian P. falciparum presents high genetic diversity with several characteristics of ancestral populations and shares many of the genetic features with African and to some extent Papua New Guinean (PNG) isolates. Similar to African isolates, Indian P. falciparum populations have maintained high effective population size and undergone rapid expansion in the past with oldest time to the most recent common ancestor (TMRCA). Interestingly, one of the four single nucleotide polymorphisms (SNPs) that differentiates P. falciparum from P. falciparum‐like isolates (infecting non‐human primates in Africa) was found to be segregating in five Indian P. falciparum isolates. This SNP was in tight linkage with other two novel SNPs that were found exclusively in these five Indian isolates. The results on the mt genome sequence analyses of Indian isolates on the whole add to the current understanding on the evolutionary history of P. falciparum.     

Cellular immune response to Plasmodium falciparum after pregnancy is related to previous placental infection and parity

Background  

Malaria in pregnancy is characterised by the sequestration of Plasmodium falciparum -infected erythrocytes in placental intervillous spaces. Placental parasites express a specific phenotype, which allows them to cytoadhere to chondroitin sulfate A expressed by syncytiotrophoblasts. Malaria infection during pregnancy allows the acquisition of antibodies against placental parasites, these antibodies are thought to be involved in protection during subsequent pregnancies.     

An AFLP-based genetic linkage map of Plasmodium chabaudi chabaudi

Background

Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.

Methods

The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers (used as chromosomal anchors) were organized into linkage groups using Map Manager software.

Results

614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, Plasmodium falciparum

Conclusion

The P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.