Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii.

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

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.

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.

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 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.

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.

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.

Preliminary crystallographic data for Pneumocystis carinii dihydrofolate reductase.

Dihydrofolate reductase from Pneumocystis carinii has been crystallized in a form suitable for high resolution X-ray diffraction studies. Recombinant enzyme that had been refolded following solubilization in guanidinium hydrochloride was crystallized as both a ternary complex with the cofactor NADPH and the inhibitor trimethoprim as well as a binary complex with NADPH. The two types of complex crystallized isomorphously from polyethylene glycol using sitting-drop vapour diffusion. The crystals were of space group P2(1) with unit cell parameters, a = 69.9 A, b = 43.6 A, c = 37.6 A, beta = 117.7 degrees, with one molecule per asymmetric unit. The crystals diffracted to 1.8 A resolution.

Refolding of recombinant Pneumocystis carinii dihydrofolate reductase and characterization of the enzyme.

The isolation of dihydrofolate reductase (DHFR) cDNA sequences from the messenger RNA of Pneumocystis carinii using the polymerase chain reaction is described. The 206-amino acid P. carinii DHFR was expressed to high levels in Escherichia coli inclusion bodies using the T7 promoter expression system. Solubilization of the inclusion bodies in 4 M guanidine hydrochloride and refolding of the recombinant protein in the presence of 0.5% polyethylene glycol 1450 yielded correctly folded DHFR which was purified to homogeneity by methotrexate-Sepharose affinity chromatography. The refolded enzyme was readily crystallized as a ternary complex with NADPH and various inhibitors. The enzyme exhibited a sharp pH optimum with maximum activity at pH 7.0 (turnover number = 6500 min-1). Km values for dihydrofolate (DHF) and NADPH were 2.3 and 3.0 microM, respectively, in 0.1 m imidazole buffer, pH 7. Folate did not act as a substrate. Comparison of the kinetic properties of the refolded enzyme with soluble P. carinii DHFR expressed at low levels in the T7 expression system showed similar pH-activity profiles, Km values for DHF and NADPH, and IC50 values for several known antifolates which were tested as inhibitors of the enzyme.

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.

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.

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.

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.

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.

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.