Biomedical subjects

J E Donelson

Publications and source records attributed to J E Donelson.

At least 37 records · Page 2Linked to original sources

PubMed1999

Induction of specific cell-mediated immunity in mice by oral immunization with Salmonella expressing Onchocerca volvulus glutathione S-transferase.

Cellular and humoral immune responses of mice to Onchocerca volvulus glutathione S-transferase (OvGST) presented via in vivo expression in attenuated Salmonella typhimurium were examined and compared with the same antigen administered by subcutaneous injection with Freund's adjuvant. After infection with recombinant S. typhimurium, maximal numbers of bacteria were recovered from the mesenteric lymph nodes and spleens during the second week postinfection. By weeks 3-4, bacteria were absent from these tissues. Splenocytes from mice infected with S. typhimurium expressing OvGST showed significant and specific proliferative responses to OvGST, whereas the non-recombinant S. typhimurium controls and those which received the antigen by subcutaneous injection with Freund's adjuvant did not. Mice infected with recombinant S. typhimurium had elevated IFN-gamma levels over non-recombinant S. typhimurium and placebo controls. but IL-4 and IL-5 levels were low and did not differ significantly between these groups. Antibody responses to OvGST antigen expressed by a recombinant Salmonella vaccine or delivered in a purified form with Freund's adjuvant were moderate to high. These data suggest that Salmonella can be used as a vaccine delivery vector that induces specific cellular and humoral immune responses to Onchocerca volvulus antigens. This is the first report to describe the successful application of a filarial antigen in a live-vector delivery system as well as the first recombinant based filarial vaccine to elicit a cellular immune response similar to that described for putative immune endemics.

Administration, Oral↗

PubMed1999

A novel protein targeting domain directs proteins to the anterior cytoplasmic face of the flagellar pocket in African trypanosomes.

The flagellar pocket of African trypanosomes is a critical sorting station for protein and membrane trafficking, and is considered to be an Achilles' heel of this deadly pathogen. Although several proteins, including receptors for host-derived growth factors, are targeted specifically to the flagellar pocket, the signals responsible for this restricted subcellular localization are entirely unknown. Using T lymphocyte triggering factor-green fluorescent protein (TLTF(1)-GFP) fusion proteins, we demonstrate that an internal 144 amino acid domain of TLTF from Trypanosoma brucei is sufficient for directing GFP to the cytoplasmic side of the anterior flagellar pocket. Immuno-gold electron microscopy reveals that the TLTF-GFP fusion protein is located in an electron dense structure that immediately abuts the anterior flagellar pocket membrane. The amino acid sequence of the TLTF targeting domain does not resemble previously characterized protein trafficking signals, and random mutagenesis reveals that flagellar pocket targeting is conferred by a structural motif, rather than a short, contiguous array of amino acids. The aberrant sorting of two mutant proteins into the flagellum, and the targeting of a related human protein to the plus end of the trypanosome's cytoskeletal microtubules, lead us to suggest that flagellar pocket targeting involves interactions with the trypanosome cytoskeleton. The finding that TLTF-GFP is restricted to the anterior, cytoplasmic face of the flagellar pocket membrane, suggests that there is structural heterogeneity in the membrane of this organelle.

Amino Acid Sequence↗

PubMed1998

Generation of expressed sequence tags as physical landmarks in the genome of Trypanosoma brucei.

Previous molecular genetic studies on the African trypanosome have focused on only a few genes and gene products, the majority of which are concerned with surface antigenic variation; consequently, an insignificant number of the genes of this organism have been characterized to date. In order to: (1) identify new genes and analyze their expression profile, (2) generate expressed sequence tags (ESTs) for derivation of a physical map of the trypanosome genome, and (3) make available the partial sequence information and the corresponding clones for general biomedical research on the parasite, we have performed single-pass sequencing of random, directionally cloned cDNAs from a bloodstream form Trypanosoma brucei rhodesiense library. Analysis of 2128 such ESTs sequenced so far in this study showed significant similarities [BLASTX P(n)-value < 10(-4), and a match > 10 amino acid residues] with proteins whose genes have been described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants. A number of the ESTs encode homologues of proteins involved in various functions including signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity. Although some of these genes may have been expected to be present in the African trypanosomes, the majority of them had not previously been described in these organisms. A large proportion, 768 individual ESTs (36%, representing 385 different transcripts), had a significant homology with genes described in organisms other than the African trypanosomes; however, 15% of the ESTs were from genes already described in trypanosomes. Among the ESTs analysed were 462 distinct known genes, only 77 of which have been described in T. brucei. Approximately 52% of the ESTs did not show any significant homology with the sequences in any of the public domain databases.

Animals↗

PubMed1998

Regulatory sequences and a novel gene in the msp (GP63) gene cluster of Leishmania chagasi.

The surface protease GP63 of Leishmania chagasi is encoded by a cluster of more than 18 tandem major surface protease (msp) genes belonging to three classes (mspL, mspS, mspC). mspL and mspS transcripts are differentially expressed during parasite growth. RNAs from mspS genes predominate during stationary phase, the time when parasite virulence and GP63 expression are maximal. We hypothesized that the unique regions downstream of mspS genes contain signals important for gene expression. The 2.8 kb region between tandem mspS genes was found to contain an 882 bp open reading frame designated mag. Copies of mag were found downstream of all mspS genes in the cluster. mag hybridized faintly to bands on Northern blots and a fully processed mag cDNA was identified in a promastigote cDNA library, providing evidence that mag genes are expressed at low levels. Similar to mspS RNAs, the abundance of mag RNAs was greater in stationary phase than logarithmic phase organisms, although mag RNAs were less abundant than mspS RNAs throughout growth. Northern blots and enzyme assays of promastigotes containing plasmid constructs in which the beta-galactosidase gene was followed by sequences between mspS coding regions, either with or without mag and its downstream sequences, suggest these regions have several regulatory effects accounting for the growth-associated changes in mspS expression.

3' Untranslated Regions↗

PubMed1998

Multiple mechanisms of immune evasion by African trypanosomes.

During infection of a mammalian host, African trypanosomes are in constant contact with the host's immune system. These protozoan parasites are infamous for their ability to evade the immune responses by periodically switching their major variant surface glycoprotein (VSG), a phenomenon called antigenic variation. Antigenic variation, however, is likely to be only one of several mechanisms enabling these organisms to thrive in the face of the immune defenses. The ability to grow in high levels of interferon-gamma (IFN-gamma) and to avoid complement-mediated destruction may also facilitate the parasite's survival. In this review we summarize (i) the activation of trypanosome genes for three different VSGs during antigenic variation, (ii) the secretion of a trypanosome protein that induces host CD8 T cells to produce IFN-gamma, and (iii) the evidence for trypanosome protein similar to a surface protease of Leishmania that plays a role in resistance to complement-mediated lysis.

Amino Acid Sequence↗

PubMed1997

African trypanosomes have differentially expressed genes encoding homologues of the Leishmania GP63 surface protease.

The genomes of various Leishmania parasites contain tandemly arrayed genes encoding an abundant 63-kDa surface glycoprotein called GP63. Leishmania GP63s are metalloproteases that play an important role in the invasion and survival of the parasites within the macrophage, and their presence on the Leishmania surface has been correlated with resistance to complement-mediated lysis. Here we report the identification of GP63-like genes in African trypanosomes. The predicted trypanosome and Leishmania GP63s share a metalloprotease catalytic site motif of HEXXH as well as 19 cysteines and 10 prolines, implying a conservation of enzymatic activity and secondary/tertiary structure. The trypanosome GP63 genes are transcribed equally in procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a 50-fold higher steady state level in bloodstream trypanosomes, where the ratio of mRNAs for GP63 and variant surface glycoprotein is about 1:150. Transcription of the GP63 genes is sensitive to alpha-amanitin, indicating that they are transcribed by a different polymerase than the variant surface glycoprotein genes. These results lead to a reconsideration of the potential functions of GP63, inasmuch as African trypanosomes are not known to interact with macrophages and do not have an intracellular stage during their life cycle.

Amino Acid Sequence↗

PubMed1997

Co-duplication of a variant surface glycoprotein gene and its promoter to an expression site in African trypanosomes.

Activation of the metacyclic variant antigen type 7 (MVAT7) variant surface glycoprotein (VSG) gene in bloodstream Trypanosoma brucei rhodesiense involves a duplicative transposition of the gene. The DNA transposition unit extends from a site approximately 3.0 kilobases upstream of the VSG gene through the coding region and includes a 73-base pair sequence that possesses promoter activity in transient transfections. This MVAT7 promoter has 80% identity to a previously characterized promoter for the MVAT4 VSG gene. Nuclear run-on assays demonstrate that the MVAT7 promoter is active in MVAT7 bloodstream organisms and that its transcript is synthesized by an RNA polymerase resistant to alpha-amanitin, consistent with previously published reports regarding VSG gene transcription. The transcription start site was identified by primer extension studies and a modified rapid amplification of cDNA ends protocol. Selective mutational analysis of the MVAT7 promoter showed that two conserved trinucleotide regions are important for full promoter function. This study demonstrates that the MVAT7 VSG gene is co-duplicated with its promoter and transcribed into a monocistronic precursor RNA.

Animals↗

PubMed1997

The gene for a T lymphocyte triggering factor from African trypanosomes.

An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

Amino Acid Sequence↗

PubMed1997

Glycoprotein 46 mRNA abundance is post-transcriptionally regulated during development of Leishmania chagasi promastigotes to an infectious form.

GP46 is an abundant glycoprotein of 46 kDa on the surface of the promastigote form of most Leishmania species. We show that the steady state level of GP46 mRNA increases >>30-fold as Leishmania chagasi promastigotes develop in vitro from a less infectious form during logarithmic growth to a highly infectious form in the stationary phase of cultivation. Nuclear run-on experiments demonstrate that this increase in GP46 mRNA abundance is regulated post-transcriptionally. Plasmids containing the 3'-untranslated regions (UTRs) and downstream intergenic regions (IRs) of two different GP46 genes fused immediately downstream of the beta-galactosidase coding region were transfected into L. chagasi, and beta-galactosidase activity and mRNA levels were examined. The presence of the 3'-UTR + IR of one GP46 gene (gp46A) resulted in a steady increase in beta-galactosidase activity and mRNA level as the transfected promastigotes developed from logarithmic to stationary phase. This differential effect parallels that of the 3'-UTRs + IRs of a family of genes for an unrelated Leishmania surface glycoprotein, GP63. Thus, post-transcriptional regulation of the genes for two different surface glycoproteins of Leishmania occurs via a similar mechanism.

Amino Acid Sequence↗

PubMed1997

A survey of the Trypanosoma brucei rhodesiense genome using shotgun sequencing.

A comparison of the efficiency of sequencing random genomic DNA fragments versus random cDNAs for the discovery of new genes in African trypanosomes was undertaken. Trypanosome DNA was sheared to a 1.5-2.5 kb size distribution, cloned into a plasmid and the sequences at both ends of 183 cloned fragments determined. Sequences of both kinetoplast and nuclear DNA were identified. New coding regions were discovered for a variety of proteins, including cell division proteins, an RNA-binding protein and a homologue of the Leishmania surface protease GP63. In some cases, each end of a fragment was found to contain a different gene, demonstrating the proximity of those genes and suggesting that the density of genes in the African trypanosome genome is quite high. Repetitive sequence elements found included telomeric hexamer repeats, 76 bp repeats associated with VSG gene expression sites, 177 bp satellite repeats in minichromosomes and the Ingi transposon-like elements. In contrast to cDNA sequencing, no ribosomal protein genes were detected. For the sake of comparison, the sequences of 190 expressed sequence tags (ESTs) were also determined, and a similar number of new trypanosomal homologues were found including homologues of another putative surface protein and a human leucine-rich repeat-containing protein. We conclude from this analysis and our previous work that sequencing random DNA fragments in African trypanosomes is as efficient for gene discovery as is sequencing random cDNA clones.

Animals↗

PubMed1996

Differential expression of the expression site-associated gene I family in African trypanosomes.

A minimum of 20 different mRNA species encoding related members of the expression site-associated gene I (ESAG-I) family occur in metacyclic variant antigen type 4 bloodstream trypanosomes. None of these ESAG-I mRNAs are derived from the metacyclic variant antigen type 4 variant surface glycoprotein (VSG) gene expression site, and some appear to come from pseudogenes. The ESAG-Is are transcribed in both procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a detectable steady state level only in bloodstream trypanosomes. At least five different groups of 3'-untranslated regions (3'-UTRs) are represented among these ESAG-I mRNAs, suggesting that the 3'-UTR does not contribute to their differential expression. Some ESAG-I mRNAs completely lack a 3'-UTR or have only a single nucleotide as a 3'-UTR. Transcription of the ESAG-Is is sensitive to alpha-amanitin, indicating that they are transcribed by a different RNA polymerase than the VSG genes. These results collectively demonstrate that ESAG-I's are a heterogeneous population that can be expressed independently of VSG genes, but like the VSG genes, their mRNAs are present in the bloodstream stage of the parasite and not in the procyclic stage.

Amanitins↗

PubMed1996

Leishmania chagasi: a gene encoding a protein kinase with a catalytic domain structurally related to MAP kinase kinase.

The gene for a serine/threonine protein kinase was isolated from Leishmania chagasi. The deduced amino acid sequence encoded by this Leishmania protein kinase (LPK-1) gene possesses all of the 11 conserved subdomains found in most other serine/threonine protein kinase catalytic domains. Sequence alignment with other known protein kinases demonstrates that the 42-kDa LPK-1 is a member of the mitogen-activated protein kinase kinase family involved in the signal-transduction pathways in yeast and mammalian cells. The single copy gene for LPK-1 specifies a 3.1-kb mRNA that is expressed in both logarithmic and stationary phase promastigotes with a twofold higher steady-state level in the infective stationary phase promastigotes.

Amino Acid Sequence↗

PubMed1996

5' sequences essential for trans-splicing of msp (gp63) RNAs in Leishmania chagasi.

The 5' sequences essential for spliced leader (SL) addition to RNA encoding the abundant Leishmania surface protease gp63 were determined. A DNA segment found upstream of all Leishmania chagasi gp63 genes was cloned in front of a luciferase gene, and luciferase activity was measured in transiently transfected L. chagasi promastigotes. Two hundred and twenty bp of the upstream region was needed for optimal luciferase activity. Deletions and point mutations were placed in this segment to determine which nucleotides participate in the splicing events. A region containing 87% pyrimidines located between 31 and 69 bp upstream of the splice acceptor dinucleotide and containing three potential branch point A residues was highly beneficial for reported gene activity. In contrast, a 30-nt pyrimidine-rich sequence immediately upstream of the splice acceptor site did not by itself enhance luciferase activity. Luciferase activity was associated with the presence of trans-spliced luciferase mRNA. The results suggest that sequences 31-69 bp upstream of gp63 genes enhance gene expression through provision of signals for efficient trans-splicing.

Animals↗

PubMed1996

The promoter for the ribosomal RNA genes of Leishmania chagasi.

A promoter for the rRNA genes of Leishmania chagasi was found to be located about 1 kb upstream of the 18S rRNA coding region and immediately downstream of 64 bp tandem repeats. Its approximate boundaries and corresponding transcription start site were determined by transient transfections and primer extension assays. This promoter for RNA polymerase I has differing activities when transfected into various Leishmania species and no activity in Trypanosoma cruzi. Its sequence has no obvious similarities with other known rRNA promoters in Trypanosomatids. Depending on the species, this promoter can be used to increase expression of a protein from a plasmid in Leishmania by as much as 45-fold over that from a plasmid lacking a promoter.

Animals↗

PubMed1996

Genome research and evolution in trypanosomes.

Trypanosoma brucei and Trypanosoma cruzi cause different human diseases. As strategies for immune evasion, T. brucei undergone antigenic variation whereas T. cruzi becomes an intracellular organism. This fundamental difference is reflected by major differences in their genome organizations. Recent comparisons of their gene sequences indicate that these two trypanosome species are highly divergent evolutionarily.

Animals↗

PubMed1996

Developmental changes in the expression of Leishmania chagasi gp63 and heat shock protein in a human macrophage cell line.

The ability of the protozoan Leishmania chagasi to infect a vertebrate host depends on its ability to survive intracellularly in a mammalian macrophage. Novel patterns of gene expression are probably important for conversion from the extracellular promastigote to the obligate intracellular amastigote parasite form. We found that the human macrophage-like cell line U937 provided an in vitro model of phagocytosis of L. chagasi promastigotes and intracellular conversion to amastigotes, allowing examination of parasite protein and RNA expression. The Leishmania surface protease gp63 assumed three isoforms during stage conversion, and a 64-kDa form of gp63 not present in promastigotes became the most prominent form in amastigotes. gp63 RNAs derived from the three different classes of msp genes (mspS, mspL, and mspC) were also differentially expressed. Infectious promastigotes contained mRNAs from mspS and mspC genes, whereas converting parasites expressed only mspL and mspC mRNAs. Sequence analysis of clones from an amastigote cDNA library confirmed the presence of gp63 mRNAs only from mspL and mspC class genes in tissue-derived amastigotes. Finally, 24 h after phagocytosis, there was a transient increase in the level of hsp70 and hsp90 proteins that subsequently decreased to baseline; this increase was not due to heat shock alone. We conclude that a unique pattern of selected L. chagasi proteins and RNAs is induced following phagocytosis by macrophages.

Animals↗

PubMed1995

Post-transcriptional elements regulating expression of mRNAs from the amastin/tuzin gene cluster of Trypanosoma cruzi.

The genome of Trypanosoma cruzi contains tandemly arrayed copies of the gene encoding amastin, an abundant protein on the surface of the amastigote stage of the parasite. The transcription rate of the amastin genes is the same in the different developmental stages, but the steady state level of the 1.4-kilobase amastin mRNA is 50-85 times higher in amastigotes than in epimastigotes or trypomastigotes (1). Here we show that the amastin genes alternate with genes encoding another protein, called tuzin, whose 1.7-kilobase mRNA is much less abundant in amastigotes. The 3'-untranslated region (UTR) of tuzin mRNA is only a few nucleotides in length or even nonexistent, in contrast with the 630-nucleotide 3'-UTR of amastin mRNA. No promoter elements were found upstream or within the amastin/tuzin gene cluster. However, in amastigotes, the protein synthesis inhibitor cycloheximide caused a 3-fold decrease in amastin mRNA and a 7-fold increase in tuzin mRNA. Furthermore, when the amastin 3'-UTR plus its downstream intergenic region were fused behind the luciferase coding region in a chimeric plasmid for transient transfections, luciferase activity increased 7-fold in amastigotes and decreased 5-fold in epimastigotes. Thus, developmental expression of these alternating genes is regulated by different mechanisms.

Amino Acid Sequence↗

PubMed1995

Inhibition of protein phosphatase 1 and 2A down-regulates beta-tubulin gene expression in Trypanosoma rhodesiense.

We studied the effect of okadaic acid, a specific inhibitor of serine/threonine protein phosphatase types 1 and 2A, on the expression of beta-tubulin and procyclin genes in Trypanosoma rhodesiense. Okadaic acid was found to decrease the steady-state level of beta-tubulin mRNA about 5-fold in differentiating bloodstream trypanosomes and about 3-fold in established procyclic trypanosomes. No effect was observed on the expression of the procyclin gene. The down-regulation of beta-tubulin gene expression by okadaic acid in procyclic trypanosomes occurs at the post-transcriptional level. These results demonstrate the involvement of protein phosphatase 1 and/or 2A activity in maintaining the steady-state level of beta-tubulin mRNA in African trypanosomes.

Animals↗