Cloning and sequence of a beta-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy.

This work describes the isolation and characterization of a full-length cDNA clone encoding beta-tubulin from the pathogen Pneumocystis carinii. P. carinii contains a single gene encoding beta-tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the beta-tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. carinii beta-tubulin and those of fungal beta-tubulins resistant to benomyl, a beta-tubulin-binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.

The multifunctional folic acid synthesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase.

We describe the cloning of a multifunctional folic acid synthesis (fas) gene from Pneumocystis carinii. The nucleotide sequence contains an open reading frame interrupted by three introns, that encodes a protein of 740 amino acids with an Mr of 97,278. The predicted Fas protein has homology to two enzyme domains, dihydropteroate synthase and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, both of which are involved in folate synthesis, and at least one other region of unknown function.

Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum.

The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing. The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli. The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites. In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte. This is the first time a P. falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol. Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway.

An exported protein of Plasmodium falciparum is synthesized as an integral membrane protein.

Exp-1 is an antigen of Plasmodium falciparum which is transported from the parasite cell to the membrane of the parasitophorous vacuole and to membranous compartments in the erythrocyte. To investigate how this protein is transported, we studied the synthesis and membrane translocation of exp-1 in a cell-free system. The protein was translocated into canine pancreatic microsomes. Its N-terminal half was thus protected from proteinase K digestion, suggesting that exp-1 is an integral membrane protein with its N-terminus facing the lumen of the microsomes. This conclusion has been confirmed in vivo. In parasitized erythrocytes, exp-1 is membrane-associated and resistant to extraction with alkali, as would be expected for an integral membrane protein. Moreover, using segment-specific monoclonal antibodies, we have shown that here again the N-terminus of exp-1 faces the inside of vesicles, inaccessible to proteases, whereas the C-terminus is degraded. We conclude that exp-1 is an integral membrane protein and infer that it is transported by vesicles from the parasite to a compartment in the host cell cytoplasm.

Structural and antigenic polymorphism of the 35- to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum.

Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.

Expression of alpha and beta tubulin genes during the asexual and sexual blood stages of Plasmodium falciparum.

Malaria parasites switch to sexual development after a period of vegetative growth in the host's erythrocytes. This switch, vital for parasite transmission to mosquitoes, is little understood at the genetic level. Likely candidates for developmental control are the alpha- and beta-tubulin subunits required for microtubule assembly. We report here that the transcription of the alpha- and beta-tubulin genes in Plasmodium falciparum show a radically different pattern of transcription in the sexual and sexual phases of parasite growth. Our studies lead to the conclusion that three transcripts of the beta-tubulin gene differ by sequences in their 5'- or 3'-untranslated regions.

The tubulin genes of the human malaria parasite Plasmodium falciparum, their chromosomal location and sequence analysis of the alpha-tubulin II gene.

We report the isolation and sequencing of genomic clones encompassing the entire alpha-tubulin II gene from the human malaria parasite Plasmodium falciparum. This gene is closely related to, but significant different from the alpha-tubulin I gene that we have described previously. These two genes represent the entire complement of alpha-tubulin sequences in this organism and are expressed in a stage-specific manner. The alpha-II gene is present as a single copy and encodes a tubulin molecule with a predicted length of 450 amino acid residues (49.7 kDa). Like the alpha-I gene, it contains two introns, which are in identical positions to those of alpha-I, but are about one-third smaller. The deduced alpha-II protein is very similar to alpha-tubulin I (94.2% amino acid identity), except for notable differences across residues 40-45. In addition, unlike the great majority of alpha-tubulin genes (including alpha-I), alpha-II does not encode a terminal tyrosine residue. Using pulsed field gel electrophoresis we demonstrate that the two alpha-tubulin genes, together with the single beta-tubulin gene, are unlinked, all residing on different chromosomes. We assign alpha-I to chromosome 9, alpha-II to chromosome 4 and beta-tubulin to chromosome 10.

A rhoptry antigen of Plasmodium falciparum is protective in Saimiri monkeys.

A non-polymorphic antigen associated with the rhoptry organelles of Plasmodium falciparum has been purified by immunoaffinity chromatography. The antigen, RAP-1 (rhoptry associated protein-1), which is defined by monoclonal antibodies which inhibit parasite growth in vitro, is a multi-component antigen consisting of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa. These proteins were electro-eluted from preparative sodium dodecyl sulphate polyacrylamide gels and protected Saimiri sciureus monkeys from a lethal blood-stage infection of P. falciparum malaria. Sera from the protected animals recognized only proteins of the RAP-1 antigen when used to probe a Western blot of total parasite protein extract, confirming that RAP-1 is responsible for eliciting the protective immune response.

Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum.

We have recently demonstrated that a non-polymorphic rhoptry antigen, RAP-1 (rhoptry associated protein-1), which is recognised by human immune serum, can successfully protect Saimiri monkeys from a lethal infection of Plasmodium falciparum malaria. In this report we further characterise the antigen, which consists of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa, and present the antigen's gene sequence. Monoclonal antibody evidence, autocatalytic processing and immunological cross-reactivity suggest that all components of this antigen are derived from the same precursor protein. The antigen is lipophilic, and disulphide bonding plays an important role in its structure. We discuss the structure and function of RAP-1 in the light of its deduced amino acid sequence and consider the relationship of this antigen to other rhoptry antigens of similar subunit size and composition.

[Preliminary study on the surface antigen gene of Plasmodium malariae].

The genomic Hind III lambda gt1149 library from Plasmodium malariae was screened with two types of oligonucleotides, both of which were from the P190 gene of P. falciparum. One, MAD20 type, was made from spanning nucleotides 4488-4562 in the P190 gene of MAD20 strain and another, K1 type, was made from spanning nucleotides 3652-3692 in the P190 of K1 strain. Both were end-labelled with 32P-ATP as probes before hybridization. Two clones were selected. One clone, designated lambda MSA-1, was specifically recognized by the MAD20 type oligo probe; the other, designated lambda MSA-2, by the K1 type oligo probe. lambda MSA-1 and lambda MSA-2 inserts were obtained by Hind III digestion of the two clone phage DNA. The analysis of the two inserts showed that the size of lambda MSA-1 is 5.5 kb whilst that of lambda MSA-2 is 3.5kb. The MSA-1 and MSA-2 inserts recloned into PUC8 were digested with the restriction enzymes Bg1 II, EcoR I, Xba I, Hind II and Pst I. The results showed that the MSA-1 DNA had one Bg1 II site, one EcoR I site, two Xba I sites, one Hind II site and one Pst I site. The MSA-2 DNA had only one Hind II site. The surface antigen gene of P. malariae was little known. This result also showed that there was probably an analogue of P190 on the surface of P. malariae, and they might fall into two types. This study is informative for further investigation on malariae parasites.

Isolation of alpha-tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of alpha-tubulin.

As a step towards identifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire alpha-tubulin gene (designated alpha-tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other alpha-tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the alpha-tubulin I gene itself was found, although a second gene designated alpha-II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The alpha-I and beta-tubulin genes were found to reside on different chromosomes.

Cloning of a beta-tubulin gene from Plasmodium falciparum.

We describe the isolation and characterization of a gene for beta-tubulin from the malaria parasite, Plasmodium falciparum. This organism appears to contain a single gene encoding beta-tubulin. A single transcript from this gene can be detected in the total RNA of the parasite's asexual blood stages. The complete sequence for the gene has been elucidated. It has two introns, one of which has a position identical to that of a related parasite, Toxoplasma gondii. The gene shows the usual preference for codons with A or T in the third position. The predicted amino acid sequence is compared with that of T. gondii and the human host. Further comparisons between these and fungal sequences of beta-tubulins resistant to benomyl, a drug binding this protein, highlight differences that could be exploited in the development of parasite-specific antitubulin drugs.

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA.

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.

Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum.

Merozoites of the malaria parasite Plasmodium falciparum carry surface proteins processed from a precursor termed p190 or p195. Polymorphism has been reported in this protein. Since the protein is a candidate for a malaria vaccine, it is important to understand the nature of this polymorphism. We have determined the complete nucleotide sequence of the p190 gene from the MAD20 strain (a Papua New Guinea isolate). Comparisons of the gene with that from other strains of P. falciparum allowed us to study the genetic basis of the antigen's polymorphism. The gene consists of sequences distributed in variable blocks, which are separated by conserved or semi-conserved sequences. Variable sequences occur both in regions that code for tripeptide repeats and in regions with no apparent repeats. Interestingly, according to the present data, variable sequences are not widely polymorphic but fall into two distinct types. We argue that the p190 protein is encoded by dimorphic alleles capable of limited genetic exchange and present evidence at the nucleotide level documenting intragenic recombination in Plasmodium.

Characterization and complete nucleotide sequence of a 5.8S ribosomal RNA gene from Plasmodium falciparum.

The 5.8S and 5S rRNA components from the FCR-3/The Gambia strain of Plasmodium falciparum have been identified and the complete nucleotide sequence of a 5.8S ribosomal RNA gene determined. Unlike the 5S rRNA species, the 5.8S is a single homogeneous population of molecules of 157 nucleotides. Comparison of its nucleotide sequence with previously reported 5.8S rRNA sequences indicates that it is homologous to these molecules, but distantly related to them. The sequence of the 5.8S rRNA coding region from the pfrib-2 recombinant of the HG13 Gambian isolate of P. falciparum is identical.

Gametocytogenesis and ribosomal rRNA gene organisation in the rodent malarias Plasmodium chabaudi and Plasmodium berghei.

A cloned Plasmodium berghei (ANKA) isolate was syringe passaged repeatedly to generate a line that was non-infective to Anopheles stephensi. Ribosomal gene organisation of this non-infective line was then compared to its infective ancestor. DNA was also prepared from asexual parasites and gametocytes of P. chabaudi and the arrangement of the rRNA genes of this species was studied. Although macrogametocytes have many more ribosomes than microgametocytes, this increase does not appear to stem from an amplification of the rRNA genes, as no differences either in the quantity or the arrangement of the rDNA could be detected. Furthermore, the loss of infectivity of the P. berghei gametocytes does not seem to be due to a reduction or rearrangement of sequences coding for the rRNA genes. P. chabaudi and P. berghei DNA failed to show any homology to a repetitive DNA sequence cloned from P. falciparum. We conclude that this probe, PFH8rep20, is specific for P. falciparum.

Processing, polymorphism, and biological significance of P190, a major surface antigen of the erythrocytic forms of Plasmodium falciparum.

A detailed analysis of P190, a major surface associated protein of Plasmodium falciparum erythrocytic stages has been undertaken. We have demonstrated that this protein is recognised by two monoclonal antibodies, one of which recognises a constant feature (2.2) and one a variable feature (7.3). Cell free protein synthesis demonstrates that the variable 7.3 epitope is encoded in the structural gene for P190. The 7.3 epitope is only present on late trophozoites and schizonts whilst the 2.2 epitope is present on all erythrocytic stages. Labelling of synchronised cultures demonstrates that P190 is made only from 30 h onwards, (i.e. by trophozoites and schizonts). By pulse chase analysis we show that P190 undergoes processing and is lost at release/re-invasion, correlating with a lack of 7.3 immunofluorescence reactivity on rings. Sera from Nigeria recognise P190 from a Thai isolate of malaria. They also react with purified P190 in a micro-ELISA assay. A model for the role of P190 in re-invasion is presented, and the possible clinical significance of this protein is discussed.