The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 A resolution.

BACKGROUND: The fungal pathogen Pneumocystis carinii causes a pneumonia which is an opportunistic infection of AIDS patients. Current therapy includes the dihydrofolate reductase (DHFR) inhibitor trimethoprim which is selective but only a relatively weak inhibitor of the enzyme for P. carinii. Determination of the three-dimensional structure of the enzyme should form the basis for design of more potent and selective therapeutic agents for treatment of the disease. RESULTS: The structure of P. carinii DHFR in complex with reduced nicotinamide adenine dinucleotide phosphate and trimethoprim has accordingly been solved by X-ray crystallography. The structure of the ternary complex has been refined at 1.86 A resolution (R = 0.181). A similar ternary complex with piritrexim (which is a tighter binding, but less selective inhibitor) has also been solved, as has the binary complex holoenzyme, both at 2.5 A resolution. CONCLUSIONS: These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

Two domains with amino-acid sequence similarity are required for dihydroneopterin aldolase function in the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii.

The folic acid synthesized gene (fas) of Pneumocystis carinii (Pc) codes for a multifunctional enzyme (Fas) known to catalyse three consecutive steps leading to the production of dihydropteroate in the de novo folate synthesis pathway. Previously, we predicted that a domain, designated FasB (amino acids (aa) 161-280), of the 740-aa multifunctional protein contains the first of the three enzyme activities in the pathway, namely dihydroneopterin aldolase (DHNA), since it shares 23% aa identity with the DHNA of Streptococcus pneumoniae (Sp). We now extend these findings to show that a second domain, FasA (aa 39-160), whose function was previously unknown, shares 27% sequence identity with the adjacent FasB domain, indicative of functional similarity. FasA is also 18% identical with the DHNA from Sp. Recombinant baculoviruses were constructed which directed the production of either FasA, FasB or FasAB polypeptide species in cultured Spodoptera frugiperda (SF9) insect cells. No DHNA activity is associated with either fasA or fasB when produced as single domains in the insect-baculovirus system. However, DHNA activity was detected in SF9 extracts containing the overproduced FasAB polypeptide. The results of aa sequence alignments and expression studies suggest that FasA and FasB may be two subunits of the DHNA enzyme moiety within the multifunctional Fas protein of Pc. An alternative interpretation of the results is also discussed.

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.

Identity and tissue localization of free and conjugated ecdysteroids in adults of Dirofilaria immitis and Ascaris suum.

Adult males and females of the dog heartworm, Dirofilaria immitis, and of the swine parasite, Ascaris suum, were extracted, the free and polar conjugated ecdysteroid fractions separated and the latter hydrolysed enzymically. The ecdysteroids released by hydrolysis of the conjugates and the free hormones were analysed by radioimmunoassay, high-performance liquid chromatography on reversed phase and adsorption columns monitoring fractions by radioimmunoassay, and by gas-liquid chromatography/mass spectrometry (selected ion monitoring). In both species, males and females contained free and polar conjugated ecdysteroids, with evidence for the presence primarily of ecdysone and 20-hydroxyecdysone together with smaller amounts of 20,26-dihydroxyecdysone. Males and females of both species were then dissected into body fluid, reproductive system, gut and remaining body wall compartments, the ecdysteroids extracted, fractionated and analysed by radioimmunoassay and high-performance liquid chromatography monitoring fractions by radioimmunoassay. The results for both sexes in the two species were similar and indicated that ecdysteroids were not detectable in body fluids and that free ecdysteroids occurred in the reproductive system and the body wall, whereas polar conjugated ecdysteroids were detected in the reproductive system and the gut; a minor portion of the free ecdysteroids in A. suum was also apparently present in the gut. Further localization of the ecdysteroids in the body wall of A. suum females suggested that negligible immunoreactivity was associated with the circumpharyngeal nerve ring. The possible significance of the results is discussed.

Isolation and molecular characterization of the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase gene from Toxoplasma gondii.

Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis. The clinical treatment of choice is the synergistic combination of antifolate agents, pyrimethamine and sulphadiazine, of which the latter targets the parasite's dihydropteroate synthase (DHPS) activity. Here, we describe the isolation of the gene encoding this activity in T. gondii. The nucleotide sequence contains an open reading frame interrupted by five introns, which encodes a protein of 664 amino acids with an M(r) of 72991. Sequence analysis revealed that, in addition to DHPS, the predicted protein contains a second enzyme function, hydroxymethyldihydropterin pyrophosphokinase (PPPK). This enzyme immediately precedes DHPS in the folate biosynthetic pathway. The bifunctional arrangement of the T. gondii pppk-dhps gene is the same as that observed in the related protozoan parasite, Plasmodium falciparum, and confirms previous biochemical data that these activities were inseparable. Recently, specific mutations within conserved motifs of the DHPS gene of P. falciparum have been identified which give rise to sulphonamide drug resistance. Analysis of seven clinical isolates of T. gondii did not reveal any similar mutations in this limited sample of organisms that had been subjected to drug pressure.

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.

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.

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.

Functional cloning of the dihydropteroate synthase gene of Staphylococcus haemolyticus.

A 1,7-kilobase fragment of the Staphylococcus haemolyticus chromosome containing the dihydropteroate synthase gene has been cloned by complementation in a temperature-sensitive mutant of Escherichia coli. The gene, designated folP, predicts a gene product of 29613 Da which shares significant amino acid sequence identity with other known bacterial dihydropteroate synthases. Analysis of the DNA sequence upstream and downstream of folP identified two further, incomplete open reading frames, one of which shows predicted amino acid sequence similarity to a second bacterial folic acid synthesis enzyme, dihydroneopterin aldolase.

Development of Dirofilaria immitis third stage larvae (Nematoda: Filarioidea) in micropore chambers implanted into surrogate hosts.

Groups of 100 third stage larvae of Dirofilaria immitis recovered from Aedes aegypti were loaded into 250 microliters capacity micropore chambers (0.3 micron pore size) and implanted into the peritoneal cavity of mice, jirds, cotton rats and ferrets. In all hosts 74-87% of larvae moulted by 74 hours, with less than 5% mortality. The fourth stage worms recovered at 74 hours were cultured in vitro in L-15 (Leibovitz) medium plus 20% foetal bovine serum with a dog sarcoma feeder cell line. After 96 hours cultivation, the larvae were comparable in size to those recovered from the dog 7 days post-infection (Orihel 1961). Chambers were also recovered from mice at intervals up to day 8. Larval survival at the longer times was approximately 90% and growth was similar to that reported for larvae in the dog. Incubation of third and fourth stage larvae in chambers with Trypan blue demonstrated that the gut is non-functional until the completion of the third moult.

A simple method for the identification of compounds which inhibit tubulin polymerization in filarial worms.

The incubation in vitro of excised ovaries of Dirofilaria immits in medium containing mebendazole between 10(-5) and 10(-8) M for four or six hours results in the accumulation of up to 20% of oogonial cells in arrested mitotic metaphase. In aceto-orcein-stained squashes of the tissue, cells possess condensed chromosomes but no detectable spindle microtubules. Similar results were obtained with colchicine, but the lowest effective concentration of this drug was 10(-7) M. This procedure affords a simple and rapid method for detecting compounds capable of inhibiting tubulin polymerization in filarial worms.

Egg production in Brugia pahangi (Nematoda: Filarioidea).

Oogenesis in Brugia pahangi has been studied by means of the aceto-orcein chromosomal squash technique and light-microscope autoradiography. The use of colchicine has demonstrated a 2-3 mm terminal germinative zone within the ovary, in which continuous and rapid mitotic division of germ cells occurs. In 80% of the gonads, oocytes within a 1-2 mm length of the ovary proximal to the germinative zone were at the prophase of meiosis I. Primary oocytes with markedly less condensed chromatin, apparently interphase cells, were observed in the corresponding region of the ovary in the remaining 20% of material examined. A cyclical or phased development of primary oocytes is suggested. Autoradiographic studies, concerned with the incorporation of [5-3H]uridine into germ cells of B. pahangi in vitro, further suggest that the onset of meiotic prophase is associated with the initiation of high RNA synthetic activity. Following meiotic prophase, oocytes complete meiosis I before entering a period of growth during which the chromatin material is decondensed. Recondensation of chromosomes prior to meiosis II is only observed after fertilization within the seminal receptacle. On completion of meiosis II, with the extrusion of a polar body, the haploid chromosome complement of the female unites with that of the male, re-establishing the diploid number of the zygote (2n = 10).

Gametogenesis and fertilization in Dirofilaria immitis (Nematoda: Filarioidea).

Gametogenesis in Dirofilaria immitis has been studied principally by means of the aceto-orcein chromosomal squash technique, but with additional ultrastructural observations. A terminal germinative zone, in which a continuous and rapid division of germ cells occurs, has been identified in the gonoduct of both male and female worms. Approximately 20% of cells within these germinative zones were in arrested mitotic division following the incubation in vitro of excised gonads in 0.01% colchicine for 4 h. All primary spermatocytes within a 1-2 cm length of the testis proximal to the germinative zone were at the prophase of the 1st meiotic division. In the corresponding region of the ovary, the primary oocytes were similarly at the prophase of the 1st meiotic division in 75% of female worms examined but in the remaining 25% all primary oocytes possessed markedly less condensed, probably interphase nuclei. A possible hormonal control of the cyclical development of primary oocytes, but not primary spermatocytes in D. immitis is suggested. In most of the remaining length of the gonoducts beyond this region of cells at meiotic prophase, the chromatin material of both primary spermatocytes and oocytes is decondensed. Recondensation of chromosomes in the spermatocytes is observed just prior to entry into the seminal vesicle, where meiosis I is completed and meiosis II takes place. In the primary oocyte, completion of meiosis only occurs after fertilization within the seminal receptacle by an entire male gamete. Following the 2 meiotic divisions in the oocyte and subsequent extrusion of the 2 polar bodies, the haploid chromosome complement of the female unites with that of the male, re-establishing the diploid number of the zygote (2n = 10). Male chromosomes within the oocyte remain visible throughout late oogenesis and fusion occurs without the formation of pronuclei.

The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase.

The nucleotide sequence of a folic acid synthesis (fas) gene from Pneumocystis carinii contains an open reading frame (ORF) that predicts a protein of 740 amino acids with an M(r) of 83,979. A recombinant baculovirus was constructed which directed expression of the predicted Fas740 polypeptide in cultured Spodoptera frugiperda (SF9) insect cells. The overexpressed 'full-length' protein migrated anomalously in sodium dodecyl sulfate/polyacrylamide gels, with an apparent molecular mass of 71.5 kDa. An abundant 69-kDa species was also recognized by polyclonal sera specific for the Fas protein in immunoblotting analyses. Dihydroneopterin aldolase, dihydropterin pyrophosphokinase and dihydropteroate synthase activities were readily detected in SF9 extracts in which the 71.5/69-kDa immunoreactive species were overproduced, demonstrating that three enzyme functions involved in catalysing three sequential steps of the folate biosynthetic pathway are encoded by a single gene in P. carinii. Importantly, the polyclonal sera recognize a single 69-kDa species in P. carinii extracts suggesting that the three activities are indeed properties of a single polypeptide, although the nature of the suggested post-translational modification is unknown. Location of the individual enzyme domains with the Fas polypeptide based upon amino acid sequence similarity to their bacterial counterparts is discussed. Furthermore, expression of various truncated fas gene constructs demonstrates that the complete fas ORF, including the N-terminus of the predicted polypeptide (FasA domain) whose enzyme function is unknown, must be expressed for maximum dihydroneopterin aldolase (FasB domain) and dihydropteroate synthase (FasD domain) activities. This suggests interactions between the domains within the larger polypeptide to stabilize the functions of these two enzymes. The FasC domain, which contains 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity, is able to fold and function independently of the other domains. The requirement by mammalian cells for preformed folates, and the absence of dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydropteroate synthase from these tissues opens up the possibility of designing highly selective drugs which inhibit these unique targets.

The hydroxymethyldihydropterin pyrophosphokinase domain of the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii expressed as an independent enzyme in Escherichia coli: refolding and characterization of the recombinant enzyme.

The folic acid synthesis (Fas) protein of Pneumocystis carinii is a multifunctional enzyme containing dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (PPPK), and dihydropteroate synthase activities. Isolation of the stretch of fas cDNA shown by amino acid similarity to the bacterial counterparts to code for PPPK activity (fasC domain) is described. FasC was expressed to high levels in Escherichia coli inclusion bodies using an inducible tac promoter expression system. Solubilization of the inclusion bodies in 6 M guanidine hydrochloride and refolding of the recombinant protein yielded enzymatically active PPPK which was purified to homogeneity by anion-exchange and gel-filtration chromatography. Sequence analysis showed that the first 13 amino acids of the purified protein were in agreement with those predicted from the DNA sequence and, furthermore, that the amino-terminal methionine had been removed. The enzyme is active in the monomeric form, exhibiting maximum activity at around pH 8.0. Isoelectric focusing gave a pI of 9.1. The Km value for 6-hydroxymethyl-7,8-dihydropterin was 3.6 microM in 50 mM Tris buffer, pH 8.2. The production of independently folded, active P. carinii PPPK will allow detailed biochemical and structural studies, increasing our understanding of this enzyme domain.

Neurosecretory-like material in 3rd- and 4th-stage Dirofilaria immitis larvae (Nematoda: Filarioidea).

Phase-interference microscopic examination of the infective, post-infective 3rd-stage and 4th-stage larvae of Dirofilaria immitis has identified a single cell body in each of the paired lateral amphidial nerves which undergoes characteristic morphological change during the development from 3rd- to 4th-stage larvae. Acetaldehyde-fuchsin staining of worm sections revealed fuchsinophilic material in the precise location of the amphidial nerve-cell bodies observed by phase-interference microscopy. This material was found in 70% of infective larvae recovered from mosquitoes and in 100% of larvae recovered from micropore chambers 24 h after implantation into BALB/C mice. In pre-moult larvae recovered at 42 h (Experiment 1) and at 48 h (Experiment 2) fuchsinophilic material was demonstrable, but no staining was observed in those larvae in which separation of the 3rd- and 4th-stage cuticles had occurred. No such material was observed in 4th-stage larvae recovered from chambers after 74 h, and in these larvae the amphidial nerve-cell bodies were not discernible. These cytological observations are consistent with a cycle of elaboration and release of neurosecretion associated with moulting in D. immitis.

Chromosomes of six species of Onchocerca (Nematoda: Filarioidea).

Examination of ovaries and testes from adult Onchocerca ochengi, O. gutturosa, O. armillata and O. lienalis revealed five pairs of chromosomes, but in contrast O. volvulus and O. gibsoni had only four pairs.

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.

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.