Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica.

A 125-kD surface antigen of Entamoeba histolytica is recognized by 73% of immune sera from patients with amoebic liver abscesses. Using pooled human immune sera a cDNA clone (lambda cM17) encoding this antigen (M17) has been isolated from a lambda gt11 expression library of the virulent stain E. histolytica HM1:IMSS. Monospecific antibodies, purified by binding to phage lysate of lambda cM17, and mAb FA7 reacted exclusively with the 125-kD antigen by Western blot analysis. Surface binding and cap formation are observed with patient sera, purified monospecific antiserum, and mAb FA7. Corresponding genomic clones (pBSgM17-1/2/3) were isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp, which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with lambda cM17. The inferred amino acid sequence predicts a 125,513 dalton protein that contains 17 potential N-linked glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic NH2-terminal region may serve as membrane anchor or signal sequence. In contrast to conservation of an immunodominant epitope recognized in pathogenic and nonpathogenic strains by monoclonal FA7 and human immune sera, amplification and sequence analysis of a 1,4000-bp fragment of this gene from a fresh nonpathogenic isolate by use of the PCR demonstrate regions of significant sequence divergence in this antigen. A 1% sequence variability among different isolates of the pathogenic strain HM1:IMSS and a 12-13% variability between pathogenic and nonpathogenic strains are revealed by comparison to published partial amino acid sequences (Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Proc. Natl. Acad. Sci. USA. 86:5118). Some restriction enzymes were found that allowed PCR diagnosis of nonpathogenic and pathogenic isolates with the exclusion of E. histolytica-like Laredo, suggesting that a detailed study of nonpathogenic and pathogenic isolates in relation to the M17 antigen sequence will provide a basis of differentiating isolates.

A major immunogen in Schistosoma mansoni infections is homologous to the heat-shock protein Hsp70.

A 70,000 mol wt protein of Schistosoma mansoni was shown to be a major immunogen that invariably elicited an antibody response in infected humans. The universality of the response to this abundant antigen was confirmed in experimental animals and included the antibody response associated with the protective irradiated cercarial vaccine. We identified the 70,000 mol wt antigen as an S. mansoni homologue of the major eukaryotic heat-shock protein hsp70 by DNA sequence analysis of a cDNA insert from a lambda gt11 clone expressing the antigen and located the immunodominant epitope near the COOH-terminus of the molecule. The antigenic relationship of hsp70 to schistosome infections suggested an important role for this protein in parasite development and pathogenesis.

Cell surface patterning and morphogenesis: biogenesis of a periodic surface array during Caulobacter development.

Shape changes, extended processes, and other surface elaborations are associated with cellular differentiation, and the cell membranes involved with these developmental changes often are reshaped without a major alteration in biochemical composition. Caulobacter crescentus produces a hexagonally-packed periodic surface layer that covers the entire cell and further, mimics some of the membrane-mediated changes of higher organisms by forming a membranous stalk during its distinctive life cycle. Growth of the surface layer was examined during the cell cycle by treating synchronously growing cells with surface layer antibody, continuing growth, and then labeling for electron microscopy with a protein A-colloidal gold conjugate. Three regions of distinctive surface array biogenesis were resolved. The periodic surface layer on the main cell body was enlarged by insertion of new material at numerous uniformly distributed points. In contrast, the surface layer on the stalk appeared as entirely new synthesis. In examining growth of the stalk in subsequent generations, we noted that growth of stalk surface persisted at the stalk-cell body junction. The region of cell division also showed a pattern of entirely new surface layer production at late stages in division, similar to the stalk. The immunocytological method also facilitated a careful examination of stalk initiation and growth. Although initiation was under precise temporal and spatial regulation, the rate of stalk elongation was variable from cell to cell and apparently no longer under cell cycle control. The similarity of surface layer biogenesis on the stalk and the site of cell division may be a significant reflection of other events occurring at the cell pole. A model suggested by this and other studies that can account for the temporal pattern of polar morphogenesis is discussed, as is the potential relationship between the geometrically ordered surface array and the formation or maintenance of the stalk.

Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria.

The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.

Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes.

Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.

Three small nucleolar RNAs identified from the spliced leader-associated RNA locus in kinetoplastid protozoans.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

Schistosome elastases: biological importance, structure, function and stage-specific expression.

Larval schistosomes (Digenea: Trematoda) invade their definitive host by directly penetrating the skin. During the process they secrete a number of macromolecules, ostensibly to facilitate their entry. Among these we have identified and characterized a dominant proteolytic species: a serine protease capable of fragmenting keratin, types IV and VIII collagen, proteoglycan, fibronectin, laminin, and elastin. The enzyme exhibits the specificity characteristic of elastases, has a molecular mass of 30,000 Da and pI of 7.8, and is potently immunogenic in its native form. Specificity of the active site has been analysed, tetrapeptides having large hydrophobic or aromatic amino acids at size P1 serving as best substrates. The amino terminal 20 amino acids of the mature enzyme have been sequenced and the information derived has been used to construct an oligonucleotide (22-mer) complement of its corresponding mRNA. The latter has been used to establish, by Northern analysis, that expression of the enzyme is stage specific (differing in this respect from most schistosome immunogens), and under transcriptional control. Transcripts are encoded by a multigene family. Several cDNAs hybridizing to the oligonucleotide have been isolated, subcloned into bacteriophage M-13, and sequenced by the di-deoxy method. It is our expectation that this line of investigation will lead towards: (i) an anti-infection vaccine; (ii) a means for chemically preventing infection (using enzyme inhibitors), and/or (iii) a rapid diagnostic assay of prepatent infection.

The identification and tracking of Candida albicans isolates from oral lesions in HIV-seropositive individuals.

Restriction fragment polymorphism analysis was used to investigate the identity and genotypic relatedness of Candida albicans strains isolated from human immunodeficiency virus (HIV)-infected patients with or without oral candidiasis and from some of their sexual partners. Use of the species-specific DNA probe Ca3 revealed that most subjects carried a single distinct C. albicans strain throughout the course of the study, during both symptomatic and asymptomatic periods. Sexual partners were more likely to carry the same or similar C. albicans isolates than unrelated subjects, raising the possibility of transmission via intimate contact. One patient appeared to acquire his partner's isolate, which then became predominant in both partners in subsequent isolations. These findings indicate that recurrent oral candidiasis is usually caused by a single persistent strain unique to each patient, but that in some cases transmission via intimate contact may occur between sexual partners.

Human and fungal 3' splice sites are used by Trypanosoma brucei for trans splicing.

In Trypanosoma brucei, pre-mRNAs are joined to a 5' 39 nt spliced leader sequence by trans splicing, a process that has not been well characterized. We have asked whether the 3' splice site regions of human and yeast introns are able to substitute in vivo for the 3' spliced leader acceptor regions of trypanosome pre-mRNA sequences. The ability of heterologous sequences to participate in trans splicing in trypanosomes was assayed by chloramphenicol acetyltransferase (CAT) enzyme activity and/or the detection of spliced CAT mRNA. Four out of the six heterologous 3' splice site regions (human beta-globin intervening sequence (IVS)2, human c-myc IVS2, human factor-VIII IVS1, and yeast actin IVS) functioned as 3' spliced leader acceptor regions in T. brucei, while two did not show significant or detectable levels of CAT activity (human beta-globin IVS1 and human c-myc IVS1). In the case of the human beta-globin IVS1 however, lengthening of the polypyrimidine tract as a result of single purine to pyrimidine transversions produced an active acceptor in which the spliced leader addition site coincides with the 3' splice site of the beta-globin exon 2. These studies indicate that some, but not all 3' acceptor regions in humans can function as spliced leader addition sites in trypansomes.

A Trypanosoma brucei small RNP particle containing the 5S rRNA.

In mammalian cells, approximately 50% of the 5S rRNA is found in ribosomes, and the remainder in a small particle, the 5S rRNA/ribosomal protein L5 complex, which is thought to be a precursor in ribosome assembly. Trypanosoma brucei, an African trypanosome, is one of the most primitive eukaryotic organisms which have been studied, and it likewise possesses a 5S rRNA species, a small proportion of which is found in an apparent ribonucleoprotein-(RNP) complex. Like the mammalian RNP particle, the T. brucei particle has a sedimentation coefficient of about 7S in sucrose gradients; unlike its mammalian counterpart, the complex is not disrupted by high salt and can be fractionated in cesium sulfate density gradients at a density characteristic of RNP complexes (1.45 g ml-1). Our studies demonstrate the the T. brucei 7S RNP contains 5S rRNA in association with a 36-kDa rRNA binding protein which not only shares molecular size, but also immunological determinants, with the yeast ribosomal protein YL3, and its mammalian homologue, L5. These results indicate that the RNP complex formed between the 5S rRNA and the 36-kDa ribosomal protein is conserved throughout great evolutionary distances between eukaryotic species.

A transcriptional analysis of the Trypanosoma brucei hsp83 gene cluster.

Ten to twelve copies of the 83-kDa heat-shock protein gene (hsp83) from Trypanosoma brucei are arranged in a head-to-tail tandem array of 2.8-kb repeat units, which are transcribed to give 2.6-kb mature mRNAs. We have cloned and sequenced one of the repeat units. The gene encodes a putative protein of 81 kDa which is highly homologous to Hsp83 of Drosophila melanogaster (75%), Hsp90 of Saccharomyces cerevisiae (72%) and the C62.5 protein of Escherichia coli (61%). The 5' end of the mature mRNA was mapped by primer extension sequence analysis and shown to contain the spliced leader. The mapping of the 3' poly(A) addition sites by S1 analysis indicated that there is 218 nt of intergenic sequence linking the boundaries encoding the mature mRNA. Within this sequence are a number of elements conserved with the trypanosome hsp70 intergenic region, including a 14-nt sequence that also has homology to the Drosophila heat-shock consensus element.

Bloodstream and metacyclic variant surface glycoprotein gene expression sites of Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense is the causative agent of chronic human sleeping sickness. Previous studies have indicated that T. b. gambiense isolates expressed the antigens U1 or L2 in both the metacyclic and early bloodstream form of the parasite life cycle. These studies suggested that L2 and U1 were likely to be metacyclic variant surface glycoproteins (mVSG). The basic copies of the genes encoding the VSGs L2 and U1 are present in single copy in non-expressing isolates of T. b. gambiense. Furthermore, they have been found to be maintained stably in a large number of stocks isolated from a wide geographic area over a 30-year period. The genomic DNA comprising the upstream 5' flanking regions of the U1 and L2 putative mVSG gene expression sites have been cloned from bloodstream forms of T. b. gambiense. The L2 expression site clone, containing 12.5 kb of sequences 5' to the VSG gene, was found to lack the 72/76-bp repeat unit generally found in the 'barren' region upstream of bloodstream form expression sites. The U1 expression site clone, containing 13.5 kb of the 5' flanking region, appeared to have the repeats, which were localized to 2 kb of DNA immediately 5' to the U1 mVSG gene. Neither the U1 nor the L2 clone was found to have ESAG2 or ESAG3 gene sequences, but both were found to have ESAG1 genes. The ESAG1 genes from the putative metacyclic expression sites and from the U1 and L2 bloodstream form expression sites (in the form of cDNA clones) were sequenced and compared to all other published ESAG1 sequences.

The 7SL RNA homologue of Trypanosoma brucei is closely related to mammalian 7SL RNA.

In eukaryotes, protein translocation across the endoplasmic reticulum is mediated by a signal recognition particle, a small ribonucleoprotein (RNP) containing 7SL RNA. We have cloned and sequenced the gene coding for the Trypanosoma brucei 7SL RNA homologue and found that its sequence shows the highest degree of similarity to the human 7SL RNA sequence. In keeping with the prototype secondary structure of eukaryotic 7SL RNA, the trypanosome 7SL RNA secondary structure can be folded into four domains. The 7SL RNP, which sediments at approximately 11S on sucrose density gradients, was partially purified using column chromatography. A particle containing a 76-nucleotide-long RNA co-purified with the 7SL RNP; however, these particles did not co-fractionate by non-denaturing polyacrylamide gel electrophoresis.

Schistosome heat-shock proteins are immunologically distinct host-like antigens.

Constitutively expressed schistosome homologues of heat-shock protein Hsp70 elicit a dominant antibody response in humans infected with either Schistosoma japonicum or Schistosoma mansoni; in each case the parasite antigens are immunologically distinct and noncrossreactive. The antigenic site of the homologues is located near their carboxyl terminus where phylogenetic divergence between Hsp70 proteins is greatest. Nucleotide sequence comparison between these regions predicts very few amino acid differences between the schistosome protein and that of their human host. Thus strikingly limited diversity is sufficient to elicit a discriminatory antibody response to these parasite host-like antigens.

Two variant surface glycoprotein genes distinguish between different substrains of Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense differs from other T. brucei subspecies in the stability and conservation of its bloodstream form antigenic repertoire. Two variant surface glycoprotein (VSG) cDNA clones corresponding to the antigens U1 and L2 were isolated from T. b. gambiense bacteriophage lambda gt11 expression libraries and characterized. A third VSG cDNA clone, P1, was also examined. The L2 and U1 VSG genes are present in a large number of T. b. gambiense stocks isolated over a thirty-year period from different geographical areas of Africa, suggesting that they are stably maintained in the T. b. gambiense genome. These probes may be useful in epidemiological studies of Gambian sleeping sickness to differentiate between T. b. gambiense isolates.

Expression of a Trypanosoma brucei brucei variant antigen in Escherichia coli.

A cDNA library derived from antigenically homogeneous bloodstream stage Trypanosoma brucei brucei was screened with an antiserum directed against the variant surface antigen (VSA) using an enzyme-linked filter immunoassay. Several recombinant clones were detected and the clone giving the most intense reaction was further analyzed. It contained a VSA-specific cDNA insert and synthesized a protein of the expected molecular weight bearing VSA determinants. The nucleotide sequence of the insert was determined and shown to have the unusual codon bias characteristic of T. brucei VSAs, frequently employing codons specifying tRNAs rare in Escherichia coli. These results indicate that a codon bias very different from that of E. coli does not preclude the expression of a cloned sequence to detectable levels in this heterologous host.