Ruud N.H. Konings

Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

Isolation and Functional Characterization of Two Distinct Sexual-Stage-Specific Promoters of the Human Malaria Parasite Plasmodium falciparum

ABSTRACT Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of thepfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activatespfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

Floating stereospecific assignment revisited: Application to an 18 kDa protein and comparison with J-coupling data

We report a floating chirality procedure to treat nonstereospecifically assigned methylene orisopropyl groups in the calculation of protein structures from NMR data using restrainedmolecular dynamics and simulated annealing. The protocol makes use of two strategies toinduce the proper conformation of the prochiral centres: explicit atom ‘swapping’ followingan evaluation of the NOE energy term, and atom ‘floating’ by reducing the angle andimproper force constants that enforce a defined chirality at the prochiral centre. The individualcontributions of both approaches have been investigated. In addition, the effects of accuracyand precision of the interproton distance restraints were studied. The model system employedis the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophagePf3. Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-couplingdata. We conclude that floating stereospecific assignment is a reliable tool in protein structurecalculation. Its use is beneficial because it allows the distance restraints to be extracteddirectly from the measured peak volumes without the need for averaging or addingpseudoatom corrections. As a result, the calculated structures are of a quality almostcomparable to that obtained with stereospecific assignments. As floating chirality furthermoreis the only approach treating prochiral centres that ensures a consistent assignment of the twoproton frequencies in a single structure, it seems to be preferable over using pseudoatoms or(R-6) averaging.

Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe: Comparison with the genome of the F-specific filamentous phages M13, fd and f1

The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff). The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome. The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx. 55%) and that their genomes contain ten homologous genes, the order of which is identical. Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules. The extent of homology between the genes of IKe and Ff differs significantly from one gene to another. Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis. During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities. Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.

Developmentally regulated expression of pfs16, a marker for sexual differentiation of the human malaria parasite Plasmodium falciparum.

Sexual differentiation is essential for the transmission of Plasmodium to mosquitoes and therefore, for the spread of malaria. The molecular mechanisms underlying sexual differentiation are poorly understood but may be elucidated by a detailed study of the regulation of expression of sexual stage specific genes. In the present work we describe the differential expression of the gene encoding the sexual stage specific protein, Pfs16. We have conducted a comparative analysis of pfs16 promoter activity, RNA levels and the rate of de novo protein synthesis during development of Plasmodium falciparum. Furthermore, we have determined the pattern of expression of pfs16 transcripts at the single cell level by in situ hybridisation. We show that the expression of pfs16 is induced immediately following the invasion of a red blood cell in sexually committed ring stage parasites and continues throughout gametocytogenesis and in macrogametes. The expression of pfs16 is regulated at the level of transcription initiation and modulated by a post-transcriptional process. These results demonstrate that the expression of the pfs16 gene is the earliest event in the sexual differentiation process of P. falciparum described to date.

NMR studies of lantibiotics: assignment of the 1H-NMR spectrum of nisin and identification of interresidual contacts

Nisin is a 34 residue long antimicrobial polypeptide, which contains unusual α,β‐unsaturated amino acids as well as lanthionines. By making use of 2D‐NMR methods, the complete 1H‐NMR spectrum of the polypeptide could be assigned. The NMR data indicate that the molecule adopts a well‐defined three‐dimensional structure.

Assembly and expression of a synthetic gene encoding the antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum in yeast.

Pfs48/45 is an important transmission-blocking vaccine candidate antigen of the human malaria parasite Plasmodium falciparum. This study was aimed at synthesis of recombinant Pfs48/45 containing conformation-constrained epitopes of the native antigen in yeast. Since in the yeast Saccharomyces cerevisiae induction of gene-expression led to prematurely terminated transcripts, an entirely synthetic gene of higher GC content was assembled. Replacement of the AT rich natural gene by the synthetic gene relieved the observed premature transcription termination. Nevertheless, recombinant protein expression could not be detected. In contrast, in the yeast Pichia pastoris low levels of recombinant Pfs48/45 were produced upon induction of synthetic gene expression. The recombinant protein was shown to be disulphide-bridge constrained, but was not recognised by transmission-blocking antibodies and did not induce transmission-blocking sera in mice.

A 13C double-filtered NOESY with strongly reduced artefacts and improved sensitivity

SummaryA 1H NOESY experiment with two 13C half-filters is described which has, compared to previously reported versions, an enhanced overall sensitivity and strongly reduced intramolecular cross peaks in any part of the spectrum edited for intermolecular NOEs. By adding a shaped 13C pulse to the half-filter which selectively inverts the aromatic resonances, the filter can be tuned separately and simultaneously for the aliphatic and aromatic regions. Contrary to recently proposed schemes, no magnetization is destroyed, so that full sensitivity is retained for symmetric systems such as homodimers. Furthermore, by replacing the rectangular 180° 13C pulses by high-power hyperbolic secant pulses for inversion of the complete 13C spectral range, offset effects (which are another source of signal loss and artefacts) are eliminated. The spectra edited for intermolecular NOEs clearly demonstrate that residual artefacts are considerably smaller than in the original version of the experiment.

NMR and circular dichroism studies of the lantibiotic nisin in non-aqueous environments

The lantibiotic, nisin, which is known to interact with membranes of certain Gram‐positive bacteria, was studied in three model systems which mimic a membrane‐like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non‐aqueous environments, at 40°C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non‐aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N‐terminus.