Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.     

Comparative evolutionary genomics of human malaria parasites

The parasites Plasmodium falciparum and Plasmodium vivax are responsible for the majority of human malaria cases worldwide. Despite many similarities in their biology, they frequently are studied in isolation. With the completion of the P. vivax genome and the generation of an initial P. falciparum genetic diversity map, attempts are being made to infer inter- and intra-species genome evolution. Here, we briefly review our current knowledge of comparative evolutionary genomics of the two species in the light of several presentations at the Molecular Approaches to Malaria 2008 meeting in Lorne, Australia and ask the question: can evolutionary genomics of one species inform the other?     

Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites

 The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the β-pleated sheets and by frequent exchange of fixed sequence motifs in the α-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates. Received: 10 August 1999 / Revised: 11 October 1999     

The immune landscape in tuberculosis reveals populations linked to disease and latency

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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.     

"Fingerprints" of malaria parasites in human genome

The effect of malaria epidemics on human genome alteration is discussed. Global and regional features of human polymorphism are examined through the genetic selection lens implemented by different malaria parasites species over the course of Homo sapiens evolution.     

Rapidly evolving genes in human. I. The glycophorins and their possible role in evading malaria parasites

In an attempt to identify all fast-evolving genes between human and other primates, we found three glycophorins, GPA, GPB, and GPE, to have the highest rate of nonsynonymous substitutions among the 280 genes surveyed. The Ka/Ks ratios are generally greater than 3 for GPA, GPB, and GPE in human, chimpanzee, and gorilla, indicating positive selection. The uniformly high substitution rate across loci can be explained by the frequent sequence exchanges among genes. GPA is the receptor for the binding ligand EBA-175 of the malaria parasite, Plasmodium falciparum. The levels of nonsynonymous divergence and polymorphism of EBA-175 are also the highest in the genome of P. falciparum. We hypothesize that GPA has been evolving rapidly to evade malaria parasites. Both the high rate of nonsynonymous substitutions and the frequent interlocus conversions may be means of evasion. The support for the evasion hypothesis is still indirect, but, unlike other hypotheses, it can be tested specifically and systematically.     

Species concepts and malaria parasites: detecting a cryptic species of Plasmodium.

Species of malaria parasite (phylum Apicomplexa: genus Plasmodium) have traditionally been described using the similarity species concept (based primarily on differences in morphological or life-history characteristics). The biological species concept (reproductive isolation) and phylogenetic species concept (based on monophyly) have not been used before in defining species of Plasmodium. Plasmodium azurophilum, described from Anolis lizards in the eastern Caribbean, is actually a two-species cryptic complex. The parasites were studied from eight islands, from Puerto Rico in the north to Grenada in the south. Morphology of the two species is very similar (differences are indistinguishable to the eye), but one infects only erythrocytes and the other only white blood cells. Molecular data for the cytochrome b gene reveal that the two forms are reproductively isolated; distinct haplotypes are present on each island and are never shared between the erythrocyte-infecting and leucocyte-infecting species. Each forms a monophyletic lineage indicating that they diverged before becoming established in the anoles of the eastern Caribbean. This comparison of the similarity, biological and phylogenetic species concepts for malaria parasites reveals the limited value of using only similarity measures in defining protozoan species.     

Temporal dynamics and diversity of avian malaria parasites in a single host species

1. We have used molecular methods to unravel a remarkable diversity of parasite lineages in a long-term population study of great reed warblers Acrocephalus arundinaceus that was not foreseen from traditional microscopic examination of blood smears. This diversity includes eight Haemoproteus and 10 Plasmodium lineages of which most probably represent good biological species. 2. Contrary to expectation, the relative frequency of parasite lineages seemed not to change over the 17-year study period and we found no effects of the parasites on a male secondary sexual ornament (song repertoire size) and two measures of fitness (adult survival and production of recruited offspring). 3. We discuss whether the absence of fitness consequences of the parasites might relate to the fact that we have studied the host at the breeding sites in Europe, whereas the transmission seems to take place at the wintering sites in Africa, where the na?ve birds encounter the parasites for the first time and the resulting primary infections likely make them sicker than during the chronic phase of the infection. 4. The prevalence of the three most common lineages appeared to fluctuate in parallel with a periodicity of approximately 3-4 years. Theoretical models based on intrinsic interactions between parasite antigen and host immune genes cannot explain such dynamics, suggesting that knowledge of extrinsic parameters such as vector distribution and alternative hosts are required to understand these patterns.     

Immune responses to asexual blood-stages of malaria parasites

The blood stage of the malaria parasite's life cycle is responsible for all the clinical symptoms of malaria. The development of clinical disease is dependent on the interplay of the infecting parasite with the immune status and genetic background of the host. Following repeated exposure to malaria parasites, individuals residing in endemic areas develop immunity. Naturally acquired immunity provides protection against clinical disease, especially severe malaria and death from malaria, although sterilizing immunity is never achieved. Given the absence of antigen processing in erythrocytes, immunity to blood stage malaria parasites is primarily conferred by humoral immune responses. Cellular and innate immune responses play a role in controlling parasite growth but may also contribute to malaria pathology. Here, we analyze the natural humoral immune responses acquired by individuals residing in P. falciparum endemic areas and review their role in providing protection against malaria. In addition, we review the dual potential of cellular and innate immune responses to control parasite multiplication and promote pathology.     

Co-expression analysis reveals a group of genes potentially involved in regulation of plant response to iron-deficiency

Iron (Fe) is an essential element for plant growth and development. Iron deficiency results in abnormal metabolisms from respiration to photosynthesis. Exploration of Fe-deficient responsive genes and their networks is critically important to understand molecular mechanisms leading to the plant adaptation to soil Fe-limitation. Co-expression genes are a cluster of genes that have a similar expression pattern to execute relatively biological functions at a stage of development or under a certain environmental condition. They may share a common regulatory mechanism. In this study, we investigated Fe-starved-related co-expression genes from Arabidopsis. From the biological process GO annotation of TAIR (The Arabidopsis Information Resource), 180 iron-deficient responsive genes were detected. Using ATTED-II database, we generated six gene co-expression networks. Among these, two modules of PYE and IRT1 were successfully constructed. There are 30 co-expression genes that are incorporated in the two modules (12 in PYE-module and 18 in IRT1-module). Sixteen of the co-expression genes were well characterized. The remaining genes (14) are poorly or not functionally identified with iron stress. Validation of the 14 genes using real-time PCR showed differential expression under iron-deficiency. Most of the co-expression genes (23/30) could be validated in pye and fit mutant plants with iron-deficiency. We further identified iron-responsive cis-elements upstream of the co-expression genes and found that 22 out of 30 genes contain the iron-responsive motif IDE1. Furthermore, some auxin and ethylene-responsive elements were detected in the promoters of the co-expression genes. These results suggest that some of the genes can be also involved in iron stress response through the phytohormone-responsive pathways.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

Nima- and Aurora-related kinases of malaria parasites

Completion of the life cycle of malaria parasite requires a succession of developmental stages which vary greatly with respect to proliferation status, implying a tightly regulated control of the parasite's cell cycle, which remains to be understood at the molecular level. Progression of the eukaryotic cell cycle is controlled by members of mitotic kinase of the families CDK (cyclin-dependent kinases), Aurora, Polo and NIMA. Plasmodium parasites possess cyclin-dependent protein kinases and cyclins, which strongly suggests that some of the principles underlying cell cycle control in higher eukaryotes also operate in this organism. However, atypical features of Plasmodium cell cycle organization and important divergences in the composition of the cell cycle machinery suggest the existence of regulatory mechanisms that are at variance with those of higher eukaryotes. This review focuses on several recently described Plasmodium protein kinases related to the NIMA and Aurora kinase families and discusses their functional involvement in parasite's biology. Given their demonstrated essential roles in the erythrocytic asexual cycle and/or sexual stages, these enzymes represent novel potential drug targets for antimalarial intervention aiming at inhibiting parasite replication and/or blocking transmission of the disease. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Analysis of DNA from various species and strains of malaria parasites by restriction endonuclease fingerprinting

1. DNA from various rodent Plasmodium species and strains and from P. falciparum, the human parasite, were analysed by agarose gel electrophoresis following digestion with restriction endonucleases EcoRI, Hind III and Bam Hl. Complex patterns of ethidium-stained bands were obtained, which showed similarity but reproducible differences among the various parasite species (P. chabaudi, P. yoelii, P. berghei and P. falciparum). 2. No differences could be discerned among two cloned strains of P. yoelii (33X, and YM) and among pyrimethamine-resistant (pyrimethamine + chloroquine)-resistant and the drug-sensitive P. chabaudi clone from which the resistant clones were derived. 3. From the known complexity of Plasmodium DNA it could be concluded that the visible bands were derived from repetitive DNA fractions.     

Genome comparison among species of the genus Arthroascus von Arx

Genome comparison in strains of the genus Arthroascus indicates that two species, A. javanensis (CBS 2555, Type) and A. schoenii (CBS 7223, Type), can be recognized.     

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Characterisation of the dihydrofolate reductase-thymidylate synthetase gene from human malaria parasites highly resistant to pyrimethamine

To investigate the genetic basis of drug resistance in human malaria parasites, we have sequenced the entire dihydrofolate reductase thymidylate synthetase DHFR-TS bifunctional gene from the highly pyrimethamine-resistant K1 isolate of Plasmodium falciparum. The protein is predicted to consist of 607 amino acids (aa), (71,685 Da), with an N-terminal methionine encoded by the second start codon of the open reading frame. Compared to the sequence from drug-sensitive parasites, there are two nucleotide changes in the coding region which bring about a substitution of Arg for Cys at aa position 59 and Asn for Thr at aa position 108. Both changes occur in regions of the DHFR domain involved in inhibitor and cofactor binding and are hence strongly implicated in drug resistance. The gene is present as a single copy in both K1 and drug-sensitive FCR3 isolates, and is assigned to chromosome 4. Codon usage follows the pattern observed in that of malarial surface antigen genes, with the exception fo codons corresponding to Val and Pro. The Asn and Lys contents of the predicted protein are exceptionally high, these residues being particularly concentrated in the DHFR and junction domains.