A new blood stage antigen of Plasmodium falciparum transported to the erythrocyte surface

On screening a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum against the 41-kDa protein band, which confers protective immunity to monkeys, two strongly reacting clones were isolated. One of the clones codes for parts of the P. falciparum 41-kDa aldolase, while the other (41-2) codes for parts of an unknown antigen; this was analyzed further. The 41-2 insert was used to identify two genomic fragments which carry the entire gene. The 41-2 gene codes for 184 amino acids specifying a 29-kDa schizont protein as estimated by Western blot analysis. The protein contains no repetitive sequences, and is characterized by a signal sequence and by two additional hydrophobic segments which could function as membrane anchor sequences. Computer analysis of the protein sequence predicted a dominant epitope in the N-terminal part which was confirmed by immunoreactivity data. The 41-2 protein could be localized in the schizont membrane, associated with membranous structures in the erythrocytic cytoplasm and with the erythrocyte membrane.     

Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.     

Analysis of Plasmodium falciparum PfEMP-1/var genes suggests that recombination rearranges constrained sequences.

The var genes of Plasmodium falciparum encode a family of parasite erythrocyte surface antigens, the PfEMP-1 proteins, which function as adhesion ligands for host endothelial and erythrocyte receptors. PfEMP-1 is extremely polymorphic although the extent of this variation in naturally transmitted parasite populations is unclear. We have identified 56 different sequences from the Duffy binding-like (DBL-1) domain of var genes amplified from six different P. falciparum clones isolated from patient infections in a Sudanese village in October-November 1989. These clones have been compared with 25 PfEMP-1 sequences expressed from different var gene loci by the 3D7A clone and 48 PfEMP-1 sequences from different isolates in endemic areas such as Kenya, Brazil, Gambia, Vietnam and Vanuatu to analyse diversity in clonal, local and 'global' P. falciparum populations. Evidence that certain conserved sequences recur in clones from one Sudanese village and in isolates from all over the world suggests that var gene diversity is the result of recombinational reshuffling of a subset of conserved, presumably ancestral sequences. Recurrence of particular var sequence blocks thus leads to 'overlaps' in the PfEMP-1 sequence repertoire of different P. falciparum clones.     

Structure and expression of the knob-associated histidine-rich protein of Plasmodium falciparum

cDNA clones encoding 473 amino acids of the knob-associated histidine-rich protein (PfHRPI) of Plasmodium falciparum clone FCR-3/A2 (Gambia) have been isolated and sequenced. Although a short region close to the amino terminus of the predicted sequence contains three blocks of six, seven or nine consecutive histidine residues, the most abundant amino acid is lysine. The predicted sequence contains a putative amino-terminal signal sequence and two potential asparagine glycosylation sites. A 1284 bp Sau3A cDNA fragment was expressed in Escherichia coli as a fusion protein that was recognized by an anti-PfHRPI monoclonal antibody. Pulsed field gradient electrophoresis indicated that the PfHRPI gene is located on chromosome 2. The PfHRPI gene was present, apparently intact, in knobless parasites derived from one uncloned P. falciparum isolate (St. Lucia). In a knobless derivative of another uncloned isolate (Malayan Camp) and in a cloned knobless line (FCR-3/D4) of a third isolate (Gambian), that part of the gene covered by the cDNA clone has been deleted. Loss of PfHRPI expression may therefore arise via several different mechanisms of gene alteration.     

Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope.

A genomic library of Pseudomonas aeruginosa DNA was screened with a monoclonal antibody (MAb 2528) specific for the P. aeruginosa 60-kDa heat shock protein. A positive clone, pAS-1, was isolated. The gene coding for P. aeruginosa chaperonin (hsp60) was localized to a 2-kb EcoRI fragment subcloned in pAS-2. A sequence analysis of pAS-2 and parts of pAS-1 identified two open reading frames that encoded proteins with calculated molecular masses of 10 and 57 kDa. In amino acid sequence comparison studies the sequences of these proteins, which were designated GroES and GroEL, exhibited up to 78% homology with known prokaryotic sequences of 10- and 60-kDa heat shock proteins (hsp10 and hsp60). In order to map the epitope recognized by MAb 2528, a series of GroEL nested carboxy-terminal deletion clones were tested with MAb 2528. We identified the clone with the shortest insertion that was still recognized by MAb 2528 and the clone with the largest insertion that was not recognized by MAb 2528. The 3' ends of the insertions were determined by sequencing and were found to delimit a region that encoded 25 amino acid residues. Synthetic oligonucleotides that coded for peptides possibly resembling the epitope within this region were ligated into expression vector pGEX-3X, and fusion proteins expressed by these clones were tested for reactivity with MAb 2528. By using this method we determined that the decapeptide QADIEARVLQ (positions 339 to 348 on GroEL) was responsible for the binding of P. aeruginosa-specific MAb 2528.     

Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis.

A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).     

Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies

A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts.     

Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum.

Actin is an ubiquitous and highly conserved microfilament protein which is hypothesized to play a mechanical, force-generating role in the unusual gliding motility of sporozoan zoites and in their active penetration of host cells. We have identified and isolated an actin gene from a Cryptosporidium parvum genomic DNA library using a chicken beta-actin cDNA as an hybridization probe. The nucleotide sequences of two overlapping recombinant clones were identical and the amino acid sequence deduced from the single open reading frame was 85 % identical to the P. falciparum actin I and human gamma-actin proteins. The predicted 42 106-Da Cryptosporidium actin contains 376 amino acids and is encoded by a single-copy gene which contains no introns. The nucleic acid coding sequence is 72% biased to the use of A or T in the third position of codons. Chromosome-sized DNA released from intact C. parvum oocysts was resolved by OFAGE into 5 discrete ethidium bromide-staining DNAs ranging in size from 900 to 1400 kb; the cloned C. parvum actin gene hybridized to a single chromosomal DNA of approximately 1200 kb.     

Chromosomal localization and cDNA sequence of human BTEB,a GC box binding protein

Human BTEB cDNA clones have been isolated, sequenced, and the corresponding gene has been assigned to human chromosome 9, region q13, by fluorescent in situ hybridization and DNA blot analysis using DNAs from hybrid cell clones containing a single human chromosome. The cDNA clone encodes a polypeptide of 244 amino acids whose sequence shows a high sequence similarity with the rat BTEB (98% amino acid identity).     

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.     

Evidence for segmental gene conversion between a cognate hsp 70 gene and the temperature-sensitively transcribed hsp70 genes of Trypanosoma brucei

The protozoan parasite Trypanosoma brucei expresses several heat shock proteins of 70 kDa (hsp70). We show that, from 5' to 3', a diverged cognate hsp70 gene (gene 1) is separated by about 6 kb of DNA from a cluster of five identical hsp70 genes (genes 2-6). The hsp70 cognate gene has a predicted open reading frame of 676 amino-acids. The steady-state mRNA levels of gene 1 are unaffected by temperature shifts up to 42 degrees C. Hsps of diverse organisms share several fully conserved amino acid domains in the N-terminal region of the hsp70 proteins. These conserved amino acid domains are also observed in the T. brucei hsp70 genes 1-6. However they are, in contrast to the heat shock genes of other eukaryotes, encoded by nucleotide sequence blocks that are identical in all six hsp70 genes. These conserved domains, located in the 5' coding region, range in size from several to hundreds of nucleotides and are separated by highly diverged nucleotide sequences. The nucleotide sequence conservation between hsp70 gene 1 and hsp70 genes 2-6 indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino acid sequence conservation.     

Identification of a common Plasmodium epitope (CPE) recognised by a pan-specific inhibitory monoclonal antibody.

A Plasmodium falciparum genomic expression library was screened with a monoclonal antibody produced from mice infected with Plasmodium yoelii. Eleven unique clones were isolated all of which contained the sequence NKND, IKND or KKND. This sequence was confirmed as the epitope of M26-32 by testing a series of overlapping peptides and the allowable substitutions determined by testing the binding of M26-32 to peptides containing all possible single amino acid replacements of NKND. Potential epitopes of M26-32 occur in many plasmodial proteins and this is consistent with the large number of proteins recognised in these parasites by Western blotting. Since this monoclonal antibody shows marked in vitro inhibition of P. falciparum growth, these data suggest that an anti-malarial vaccine may be produced by targeting such common plasmodial epitopes without necessarily identifying the corresponding antigens.     

The primary structure of Plasmodium falciparum DNA polymerase delta is similar to drug sensitive delta-like viral DNA polymerases.

We report the isolation and sequencing of genomic DNA clones that encode the 1094-amino acid catalytic subunit of DNA polymerase delta from the human malaria parasite Plasmodium falciparum. Protein sequence comparison to other DNA polymerases revealed the presence of six highly conserved regions found in alpha-like DNA polymerases from different prokaryotic, viral, and eukaryotic sources. Five additional regions of amino acid sequence similarity that are only conserved in delta and delta-like DNA polymerases, so far, were present in P. falciparum DNA polymerase delta. P. falciparum DNA polymerase delta was highly similar to both Saccharomyces cerevisiae DNA polymerase delta (DNA polymerase III; CDC2) and Epstein-Barr virus DNA polymerase at the amino acid sequence, and the predicted protein secondary structure levels. The gene that encodes DNA polymerase delta resides as a single copy on chromosome 10, and is expressed as a 4.5-kb mRNA during the trophozoite and schizont stages when parasite chromosomal DNA synthesis is active.     

Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.     

Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein.

From a Plasmodium falciparum cDNA expression library derived from mRNA of the asexual blood stages, we isolated and sequenced five different cDNA clones whose predicted protein products were unusually rich in asparagine (Asn). Two of the clones, R5 and G5, contain tandem imperfectly repeated sequences based on Asn-Asn-Thr (NNT) and Asn-Asn-Met (NNM) respectively. The other three, E4, C5 and R13, as well as G5, contain stretches of polyasparagine varying in length from 2 to 26 residues. Results of DNA blotting experiments with the individual cDNA sequences as probes suggest that each of the five clones corresponds to a different P. falciparum gene. The fragments of P. falciparum proteins expressed by the cDNA clones shared cross-reactive antigenic determinants which were present on multiple P. falciparum proteins. In immunoblotting experiments, owl monkey antibodies selected for binding to the polypeptide expressed by clone E4, C5 or G5 reacted with the expressed proteins from all 5 clones, and with at least 10 proteins from schizont infected erythrocytes. The cross-reactive epitopes could be modeled by two Asn-rich peptide structures: (1) (NNT)8, whose sequence was based on the R5 repeat; and (2) (NPNA)6, whose sequence was based on the Asn-rich repeat of the P. falciparum circumsporozoite protein (CSP). Antibodies that bound to each peptide were selected from sera of immune monkeys that had never been exposed to sporozoites. The selected antibodies bound all 5 expressed proteins in immunoblotting assays and also bound to several proteins from parasitized erythrocytes. Such cross reactivity between the CSP repeating unit and several blood-stage antigens has not been previously reported.     

Isolation and characterization of a cysteine proteinase gene of Plasmodium falciparum.

We have previously identified a 28-kDa cysteine proteinase of Plasmodium falciparum trophozoites that appears to be an essential malarial hemoglobinase and a potential target for antimalarial chemotherapy. The trophozoite cysteine proteinase (TCP) shares a number of biochemical properties with the lysosomal cysteine proteinase cathepsin L. To isolate the gene encoding TCP, we synthesized degenerate oligonucleotides based on two amino acid sequences of cathepsin L that are well conserved among papain-family cysteine proteinases, and used the oligonucleotides to prime the polymerase chain reaction (PCR) with P. falciparum genomic DNA. A 549-bp DNA fragment was amplified by PCR. This fragment was used as a hybridization probe to screen a lambda gt11 library of P. falciparum genomic DNA and isolate a 1.8-kb genomic clone (C1.8) that encoded an intact malarial cysteine proteinase gene. The sequence of C1.8 predicted a 67-kDa protein containing a typical signal sequence, a large pro sequence, and a 26.8-kDa mature proteinase with 37% amino acid identity to cathepsin L. Antisera directed against a peptide encoded by C1.8 recognized a 28-kDa trophozoite protein on immunoblots. In a Northern analysis, C1.8 hybridized predominantly with RNA from rings, the life-cycle stage immediately preceding the trophozoite stage. Taken together, these results strongly suggest that the P. falciparum cysteine proteinase gene we have isolated and characterized encodes TCP.     

Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR

Using broad-spectrum primers, we have amplified, cloned, and sequenced a 620-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp65) from the heart valve of a patient with Whipple's endocarditis. The deduced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific seminested PCR. Seventeen patients shown to be positive by 16S ribosomal DNA (rDNA) PCR and/or internal transcribed spacer (ITS) PCR were also positive by hsp65 PCR. All 33 control specimens from patients without Whipple's disease and negative for "T. whippelii" by both seminested 16S rDNA and ITS PCR remained negative. All amplicons digested with XhoI revealed an identical restriction pattern. Eight of the 17 hsp65 amplicons representing all three previously described ITS types were sequenced. Three of the amplicons showed slight differences, but none of the mutations detected affected the amino acid sequence of the corresponding protein. We conclude that the hsp65 gene is a suitable target for the specific detection of "T. whippelii." Its product represents a putative antigen for a future serodiagnostic assay.