Papers from the COST Action 857 Workshop V Biodiversity and population genetics of the Apicomplexa (Congress Centre of the Bambino Gesu Hospital, Rome, November 8-9, 2004).
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Animals↗
Biomedical subjects
P Alano
Publications and source records attributed to P Alano.
Molecular approaches to monitor parasite genetic complexity in the transmission of Plasmodium falciparum malaria.
Determination of the number of parasite clones present in a malaria infection is usually based on Polymerase Chain Reaction (PCR) amplification of Plasmodium polymorphic genomic sequences from peripheral blood. This method however does not provide information on the developmental stages of the parasites detected, or on the potential trasmissibility of the detected genotypes to the Anopheles vector. Reverse Transcriptase-PCR assays on P. falciparum mRNAs produced specifically in sexual stages have been developed in the past few years in order to detect and genotype circulating gametocytes, the parasite transmission stages, and are discussed in this review. Assays based on P. falciparum gamete-specific gene pfs25 and gametocyte-specific polymorphic gene pfg377 can detect presence of subpatent gametocytes in infected blood, can identify the pfg377 allele(s) specifically carried by the sexual stages, and detect coexistance of gametocytes of different genotypes. These assay have been used for the first time in field studies in a region of Sudan where malaria is seasonal, and they characterised parasite clonality and pattern of gametocyte production in the subpatent parasitaemias observed in the long malaria-free season. The method of specifically detecting and genotyping gametocytes in natural infections is proving to be a useful tool in investigating parasite transmission dynamics in field studies. This approach can be further improved by developing a multilocus RT-PCR assay which includes additional polymorphic gametocyte-specific transcripts. Candidate genes can be identified from the available data on the P. falciparum genome sequence and from recent analyses of parasite stage-specific transcriptomes.
Alleles↗
Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.
We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.
Amino Acid Sequence↗
Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum.
The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes. orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.
Animals↗
Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.
A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.
Alleles↗
Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum.
Commitment to the production of female and male gametocytes was studied in the NF54 line of the human malaria parasite Plasmodium falciparum. The development of sibling parasites derived from individual schizonts was followed, and 2 antisera against the female gametocyte-specific protein Pfg377 and the male gametocyte-specific protein alpha-tubulin II were used to determine the sex of sibling gametocytes. The experiment showed that individual cohorts of sibling gametocytes were stained in a mutually exclusive fashion by only one or the other antiserum, indicating that individual schizonts committed to yield sexual parasite progeny produce gametocytes of the same sex. This work suggests that in P. falciparum commitment to sexual differentiation occurs prior to schizont maturation, at the same moment when the sex of the resulting gametocytes is determined.
Animals↗
An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum. Identification of a MAPK signature.
The cDNA encoding Pfmap-2, an enzyme of the human malaria parasite Plasmodium falciparum, was cloned, sequenced, and expressed in Escherichia coli. The open reading frame carried by the Pfmap-2 cDNA encodes a 508-amino acid polypeptide of 59.2 kDa with maximal homology to mitogen-activated protein kinases (MAPKs) from various organisms. The purified recombinant enzyme displayed functional characteristics of MAPKs such as (i) ability to undergo autophosphorylation, (ii) ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) regulation of kinase activity by a MAPK-specific phosphatase, and (iv) ability to be activated by component(s) present in cell extracts. Mutational analysis of the recombinant protein allowed the identification of residues that are important for enzymatic activity. Northern blot analysis and immunofluorescence assays indicated that Pfmap-2 is expressed specifically in gametocytes, the form that is responsible for transmission of the parasite to the mosquito vector. Gametocyte extracts activated recombinant Pfmap-2 more efficiently than extracts from asexual parasites, which is consistent with this stage specificity. Despite its overall high level of homology to MAPKs, Pfmap-2 presents the peculiarity of not possessing the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family; instead, it has a threonine-serine-histidine at the same location. This atypical feature formed the basis for a detailed analysis of the primary structure of MAPKs, allowing us to define an operational MAPK signature, which is shared by Pfmap-2. The fact that no MAPK from vertebrates diverge in the activation motif suggests that the fine mechanisms of Pfmap-2 regulation may offer an opportunity for antimalarial drug targeting.
Amino Acid Motifs↗
Chromosome mapping in Cryptosporidium parvum and establishment of a long-range restriction map for chromosome VI.
We used contour-clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvum and establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvum was found to consist of partially co-migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvum chromosomes or isolated chromosome VI using gene-specific probes and an oligonucleotide specific for C. parvum telomeres allowed the development of a long-range restriction map of chromosome VI.
Animals↗
The production of the osmiophilic body protein Pfg377 is associated with stage of maturation and sex in Plasmodium falciparum gametocytes.
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Animals↗
Genome plasticity and sexual differentiation in Plasmodium.
Spontaneous subtelomeric deletions of Plasmodium chromosomes have been observed both in natural infections and in laboratory maintained parasites. In the latter case, functions dispensable for asexual parasite multiplication and encoded at the extremities of the chromosomes are easily lost. In particular, spontaneous subtelomeric deletions have been characterised which affect gametocytogenesis both in Plasmodium berghei maintained in laboratory animals and in Plasmodium falciparum propagated in in vitro cultures. In order to identify these genetic determinants, and, potentially, other genes located subtelomerically, we designed a transfection system able to induce and select for controlled, site-specific subtelomeric deletions.
Animals↗
Structure and polymorphism of the upstream region of the pfg27/25 gene, transcriptionally regulated in gametocytogenesis of Plasmodium falciparum.
Transcription of the early gametocyte-specific gene pfg27/25, and genomic structure and polymorphism of its upstream region were studied in the human malaria parasite Plasmodium falciparum. The upstream genomic sequence of the pfg27/25 gene is characterised by a repetitive region that contains five direct and one inverted repeats of a unit constituted by a perfectly conserved sequence flanked by a poly-dT and a poly-d(AT) tract. Sequences further upstream from the repetitive region are polymorphic in distantly related parasite lines. Nuclear 'run off' experiments indicated that transcription of pfg27/25 is developmentally regulated. Transcription of the gene, undetectable in asexual parasites, is activated at the onset of gametocytogenesis. Te gene is transcribed in a 2.5 kb mature mRNA for the first 2-3 days of sexual differentiation, while transcription is down-regulated in more mature gametocytes.
Animals↗
Plasmodium falciparum: parasites defective in early stages of gametocytogenesis.
Some molecular characteristics of Plasmodium falciparum lines which do not produce gametocytes are described. Parasites carrying a subtelomerically deleted chromosome 9 cannot form even the earliest forms of gametocytes, detectable with antibodies against the gametocyte-specific antigen Pfg27. In a parasite culture of clone HB3, in which both intact and deleted forms of chromosome 9 are present, full-length chromosome 9 molecules are retained mainly in gametocytes. These data suggest that the subtelomeric portion of chromosome 9 is required at an early stage of gametocytogenesis. Parasite subclones derived from gametocyte producing clone 3D7, which completely lost ability to produce gametocytes, are also described. Unlike the previous gametocyteless lines, these parasites stably maintain a full-length chromosome 9 and the ability to cytoadhere to C32 melanoma cells after prolonged asexual propagation. Their defect in sexual development is therefore genetically and functionally distinct from that of parasites carrying a deleted chromosome 9. Gametocyteless subclones derived from 3D7 do not produce any Pfg27 mRNA, while this gene is anomalously expressed in asexual stage parasites of two lines of a different genetic background, 1776sel8 and C10, one able and the other unable to produce gametocytes.
Animals↗
COS cell expression cloning of Pfg377, a Plasmodium falciparum gametocyte antigen associated with osmiophilic bodies.
We report the deduced protein sequence and preliminary characterization of Pfg377, a novel sexual stage antigen of Plasmodium falciparum. An initial cDNA clone (Pfg377-1) encoding the N-terminal 755 amino acids of Pfg377 was isolated by transfecting a 3D7 gametocyte cDNA library into COS7 cells and selecting using a pool of anti-Pfs230 monoclonal antibodies. The protein encoded by Pfg377-1 included an N-terminal hydrophobic signal sequence, but no apparent transmembrane anchor. Instead, the particular cDNA clone selected was fused in-frame at its 3' end with the coding sequence for the human decay acceleration factor membrane anchor, which had been deliberately placed downstream of the vector polylinker in order to attach potential fusion proteins onto the COS cell surface. Northern blots probed with the Pfg377-1 cDNA demonstrated cross-hybridization to a single approximately 9.5-kb transcript, which was present only in sexual stages, and not in a sexual stages. DNA hybridization was used to obtain a series of overlapping genomic clones which collectively yielded the complete DNA sequence for Pfg377. There are no introns within the gene, which contains a 9360-bp open reading frame and encodes a 377-kDa protein. The Pfg377 protein is highly hydrophilic, and has an essentially non-repetitive structure, with only four very limited regions of tandem repeats. The Pfg377 gene resides on chromosome 12, and immunoelectron microscopy with two different anti-Pfg377 polyclonal antisera raised against two separate recombinant sub-fragments of the protein both indicated that the antigen is located in electron-dense organelles of the gametocytes--the osmiophilic bodies--which are proposed to play a role in parasite emergence from the erythrocyte during gametocyte maturation in the Anopheles mosquito midgut. Although it was selected with anti-Pfs230 antibodies, comparison of the sub-cellular locations and protein sequences of Pfg377 and Pfs2 show them to be completely distinct antigens. We hypothesize that Pfg377-1 was initially isolated because it expresses an epitope which is recognized by (i.e., cross-reacts with) one of the anti-Pfs230 monoclonal antibodies used to select the original transfected COS cells.
Amino Acid Sequence↗
Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control.
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Amino Acid Sequence↗
Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control.
On the basis of conserved sequences characteristic of the Ran/TC4 subfamily of the GTPase superfamily, a fragment of the gene encoding a Plasmodium falciparum Ran/TC4 homologue was amplified in the polymerase chain reaction. The fragment was used to screen a cDNA library to obtain clones which allowed determination of the complete gene sequence. The gene, designated pfran (Plasmodium falciparum ras-like nuclear protein), has around 70% amino acid identity with previously characterised Ran/TC4 proteins. Like other malarial mRNAs, the pfran mRNA contains a long (at least 679 bp) 5' untranslated region. Southern blotting experiments show that pfran is a single copy gene located on chromosome 11. RNA hybridisation experiments indicate that pfran mRNA is abundant in late trophozoite and schizont stages, but present at very low levels in gametocytes and early asexual stages.
Amino Acid Sequence↗
The gene encoding DNA polymerase alpha from Plasmodium falciparum.
The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.
Amino Acid Sequence↗
The culture and preparation of gametocytes of Plasmodium falciparum for immunochemical, molecular, and mosquito infectivity studies.
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Aedes↗
Chromosomal polymorphism and sexual differentiation in Plasmodium.
The correlation observed in several instances between the loss of ability to produce gametocytes and chromosomal rearrangements, prompted us to investigate in further detail the molecular bases of chromosomal polymorphism in Plasmodium. Generation of polymorphic karyotypes in Plasmodium involves important rearrangements, mostly occurring in subtelomeric position. Detailed analysis on the organisation of these regions have been carried out on the rodent malaria P. berghei and the human malaria P. falciparum. A 2.3kb sequence, tandemly organised in long clusters is shared by many P. berghei chromosomal ends. Variations in the copy number of this "module" account for most of the observed polymorphisms. In a P. falciparum cloned line (3D7) a common region spanning at least 40 kb, is present. It does not contain any repetitive structure other than the rep20 cluster, that appears to be completely contained within the common region. Notwithstanding the structural differences, human and rodent Plasmodia share the common feature of possessing long subtelomeric regions showing, thus, a homology between the different chromosomes.
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