Human humoral immunity to hsp70 during Trypanosoma cruzi infection

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.     

Molecular Comparison of the Mitochondrial and Cytoplasmic hsp 70 of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major

ABSTRACT. We compared the expression and localization of the mitochondrial and cytoplasmic hsp70 of the protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The mitochondrial protein is encoded by multiple mRNA in all species, while the cytoplasmic protein is encoded by a single mRNA. In all three species, the mitochondrial hsp70 is concentrated in the kinetoplast, a submitochondrial structure that houses the unusual DNA (kDNA) that characterizes this group of organisms, while the cytoplasmic protein is distributed throughout the cell. These results suggest that, in all kinetoplastid species, mt-hsp70 has a specific function in kDNA biology, possibly in the processes of kDNA replication, RNA editing or kinetoplast structure.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

Minicircle organization and diversity in Trypanosoma cruzi populations

Trypanosoma cruzi strains and isolates can be divided into at least two groups using biochemical and molecular markers such as isoenzymes, ribosomal DNA, mini-exon gene spacers and some maxicircle genes. Despite the accumulating evidence that these major groups are phylogenetically distinct, their kinetoplast minicircle overall organization (i.e. number of conserved regions per length of minicircle molecule) remains conserved in all T. cruzi isolates studied so far, including the two T. cruzi major lineages -T. cruzi I and T. cruzi II - and a third group of uncertain taxonomic status, T. cruzi ZIII. Thus far, despite the extensive intra- and inter-minicircle sequence polymorphism, no group clustering has been observed.     

Comparison of genes encoding Trypanosoma cruzi antigens

Trypanosoma cruzi, the causative agent of Chagas disease, simultaneously expresses several different surface antigens. This contrasts sharply with blood-stream forms o f the African trypanosomes, which display only one variant surface glycoprotein at a time. Over the past few years, the genes coding for a number of T. cruzi proteins recognized by sera from patients have been cloned and at least partially sequenced. However, some of those discovered in more than one laboratory have been given different names. Here, Carlos Frasch, Juan Cazzulo, Lena Aslund and UIf Pettersson try to systematize the literature available on these antigens, including what is known about their localization and function.     

Xenopus hsp 70 genes are constitutively expressed in injected oocytes.

Xenopus heat-shock genes are transiently heat-inducible in somatic cells, but they are also subject to a long-term developmental control in oogenesis and early embryogenesis. In order to understand whether different genes or different promoter elements are involved in the two types of control, several genomic clones coding for Xenopus heat-shock proteins, hsp 70 and hsp 30, were isolated, characterised and tested for expression in oocytes and COS cells. Three isolated hsp 70 genes are nearly identical in their promoter and mRNA leader sequences, indicating that there is only one type of hsp 70 gene. These promoters contain a consensus sequence element (CT-GAA--TTC-AG) upstream of the TATA-box, which is presumably required for their transient heat-inducibility. The two isolated hsp 30 genes show 5'-flanking sequences similar to each other, except that one of them shows a homology disruption precisely around the consensus sequence element. The same gene contains a frameshift mutation in the protein coding part and, since it cannot be expressed after introduction into oocytes or COS cells, it is probably a pseudogene. The other hsp 30 gene is strongly heat-inducible in injected oocytes or transfected COS cells. In contrast, the hsp 70 genes are strongly heat-inducible in COS cells, but their expression is highly efficient in injected oocytes at the normal temperature and is not increased during heat shock. This represents correct cell type-specific regulation of a cloned reintroduced gene, since the endogenous hsp 70 genes are constitutively activated during oogenesis, leading to the accumulation of stored hsp 70 mRNA in oocytes.     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。     

Multiple phases of nucleosomes in the hsp 70 genes of Drosophila melanogaster.

The arrangement of the nucleosomes with respect to the DNA sequence has been examined in the genes coding for the major heat shock protein (hsp 70) in Drosophila. In the repressed state of the genes, the nucleosomes are precisely phased in at least three frames.     

A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania.

A Trypanosoma cruzi expression vector has been constructed using sequences derived from the flanking regions of the glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. The neomycin phosphotransferase (neor) gene was incorporated as a selectable marker. Using electroporation we have introduced this vector into both T. cruzi and Leishmania cells and conferred G418 resistance. Transformation is mediated by large extrachromosomal circular elements composed of head-to-tail tandem repeats of the vector. The transformed phenotype is stable for at least 6 months in the absence of G418 and can be maintained during passage through the T. cruzi life-cycle. Foreign genes inserted into an expression site within the vector (pTEX) can be expressed at high levels in transformed cells. To our knowledge this paper describes the first trypanosome shuttle vector and the first vector which functions in both trypanosomes and Leishmania.     

Evolutionary history of Trypanosoma cruzi according to antigen genes

Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.     

Molecular karyotype and chromosomal localization of genes encoding beta-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of beta-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of beta-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.     

Repertoire, genealogy and genomic organization of cruzipain and homologous genes in Trypanosoma cruzi, T. cruzi-like and other trypanosome species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.