Trypanosoma cruzi: accumulation of polycistronic hsp70 RNAs during severe heat shock.
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Animals↗
Biomedical subjects
L V Kirchhoff
Publications and source records attributed to L V Kirchhoff.
Trypanosoma cruzi: interruption of both alleles of a gene encoding a protein containing 14-amino-acid repeats by targeted insertion of NEOr and HYGr.
In Trypanosoma cruzi approximately 90% of the 121- and 176-kDa cytoskeletal proteins encoded by the two alleles of the TCR27 gene is composed of 14-amino-acid repeats. To gain insight into the function of the TCR27 proteins we replaced the corresponding regions of 42-nucleotide repeats in the two alleles with the NEOr and HYGr genes. Analyses of DNAs and RNAs from four clones resistant to both G418, a neomycin analogue, and hygromycin showed that in both cases the repetitive regions had in fact been deleted. In addition, the absence of expression of the 14-amino-acid repeats was confirmed in Western blots. In axenic cultures growth rates of the morphologically unchanged, doubly resistant organisms were not different from those of wild-type parasites. However, the doubly resistant organisms proliferated more slowly in cultured mammalian cells than did wild-type parasites. These findings indicate that the absence of the TCR27 repetitive regions is detrimental, but not fatal, to the parasites.
Alleles↗
Repetitive protein antigens of Trypanosoma cruzi have diverse intracellular locations.
We previously identified by immunoscreening Trypanosoma cruzi cDNA libraries a group of proteins containing long stretches of tandem repeats. The goal of the current project was to gain insight into the functions of these proteins through ultrastructural analyses consisting of western blotting and electron microscopic localization studies. By comparing western blots of total parasite lysate and different fractions of T. cruzi, we found that 3 of the repetitive antigens are exclusively associated with the parasite membrane, or cytoskeleton, or both. One of the 4 repetitive antigens studied has some association with the membrane or cytoskeleton but also appears to be free in the cytosol. In immunoelectron microscopic studies, the 4 repetitive antigens were detected in different intracellular locations. One of the proteins is located between the flagellum and parasite body, the second has a nuclear distribution, the third is associated with the cell membrane, and the fourth is dispersed throughout the cytoskeletal network. These findings suggest that despite the general structure similarities of these repetitive proteins, they may serve different cellular functions.
Amino Acid Sequence↗
Trypanosoma cruzi infection in Walker hounds from Virginia.
Trypanosomiasis has been reported in dogs from Texas, Oklahoma, Louisiana, and South Carolina. We describe the first isolation and characterization of Trypanosoma cruzi from a Walker Hound pup in Virginia that also had postvaccinal distemper. The mother of the pup and 7 of its 8 siblings also were found to be infected with T cruzi, suggesting that the parasite had been transmitted transplacentally or through lactation. Parasitologic, serologic, histologic, and molecular methods were used to establish the diagnosis of T cruzi infection in these dogs. In a serologic survey of 12 dogs (including the sire of the pups) from the area in which the index case occurred, none were found to have antibodies to T cruzi. However, 2 of a further 52 dogs from different areas (to the index case), but in the same county, were seropositive to T cruzi. These findings indicate that canine trypanosomiasis is present in an area of the United States not previously known to be enzootic.
Animals↗
A differentially expressed gene family encoding "amastin," a surface protein of Trypanosoma cruzi amastigotes.
A new family of closely related glycoproteins, collectively called amastins, has been found on the surface of the amastigote form of Trypanosoma cruzi. The gene family encoding these amastigote-specific proteins was identified by differentially screening an amastigote cDNA library with reverse transcribed poly(A)+ RNA from amastigote and epimastigote stages of the parasite. Amastins are encoded by eight or more tandem genes, at least five of which are distinguished by nucleotide point changes. The 1.4-kilobase amastin mRNAs are 50 times more abundant in amastigotes than in epimastigotes or trypomastigotes. The amastin genes are transcribed to an equal extent in both amastigotes and epimastigotes, indicating that the stage-specific amastin mRNA levels are determined by a post-transcriptional mechanism. Sequence determination of full-length cDNAs reveals an open reading frame encoding 174 amino acids and a 700-base pair 3'-untranslated region. Nascent amastins contain four distinct hydrophobic regions of 20-30 amino acids each, 2 at internal locations and 1 each at the N and C termini.
Amino Acid Sequence↗
Transcription and editing of cytochrome oxidase II RNAs in Trypanosoma cruzi.
The cytochrome oxidase subunit II (COII) gene is one of the maxicircle cryptogenes of kinetoplastids whose primary transcripts are sometimes modified by RNA editing to produce mature mRNAs. We determined the sequence of the COII gene in three strains of Trypanosoma cruzi (Y, Corpus Christi, and Tulahuén) and examined its developmental expression. Comparison of the RNA and DNA sequences encoding COII indicated that in the three strains of T. cruzi, four uridines are inserted in the pre-mRNA at the same positions as they are in the COII pre-mRNAs of Trypanosoma brucei, Leishmania tarentolae, and Crithidia fasciculata. The putative guide RNA (gRNA) sequence that serves as a template for the four uridine insertions is located in the 3'-untranslated region of the T. cruzi COII mRNA. Analysis of editing intermediates demonstrates that the COII gRNA remains attached to the pre-mRNA while participating in the formation of chimeric RNAs. Northern blots used to investigate stage-specific expression of the COII gene revealed RNAs of 800 and 900 nucleotides, similar in size to those present in T. brucei. In contrast to the differential expression observed in T. brucei, no difference occurs between the COII mRNA levels of insect and mammalian stages of T. cruzi.
Amino Acid Sequence↗
Characterization of a Trypanosoma cruzi poly(A)-binding protein and its genes.
We have characterized the biochemical properties of a 66-kDa poly(A)-binding protein (PABP1) in the protozoan Trypanosoma cruzi and isolated two classes of cDNAs encoding the protein. In concordance, Southern blots showed the presence of 2 gene copies. The two cDNA classes differ in the length of adenosine-rich segments in the 5' untranslated region and in point changes scattered throughout the sequence, but their 1650-bp open reading frames encode identical proteins. A single mRNA of 5.5 kb was detected, indicating that the noncoding regions are unusually long. Both the mRNA and the protein are constitutively expressed in all stages of T. cruzi life cycle. The biochemical properties and sequence comparisons show that the T. cruzi PABP1 is similar to the PABP1 of other eukaryotic organisms. These results indicate that PABP1 has been conserved throughout eukaryotic evolution.
Amino Acid Sequence↗
Characterization of kinetoplast DNA minicircles in Trypanosoma rangeli.
Kinetoplast DNA, the mitochondrial DNA of kinetoplastid protozoans, is a network of interlocked minicircles and maxicircles. We analyzed the sequence organization of minicircle DNAs in the El Tocuyo strain and the San Augustin clone B6 of Trypanosoma rangeli. The frequencies of different minicircle types, as defined by the number of 136-bp conserved regions (CRs), are different in the two strains. About half of the 1.7-kb T. rangeli El Tocuyo minicircles have 1 CR and most of the others have 2. In contrast, most of the 1.6-kb T. rangeli San Augustin minicircles have 2 CRs, while some have four. The CR contains a replication origin at one end and is conserved both within and between the two strains. Comparisons of the T. rangeli El Tocuyo and T. rangeli San Augustin minicircle CRs with minicircle CRs of other kinetoplastid species reveal that they are most similar to those of Trypanosoma cruzi.
Animals↗
Kinetic analysis of antigen-specific immune responses in resistant and susceptible mice during infection with Trypanosoma cruzi.
The Tulahuén strain of Trypanosoma cruzi has been shown previously to cause higher parasitemias and greater mortality in BALB/c mice compared with C57BL6/J mice. The goal of our study was to determine whether different cytokine responses to parasite Ag during T. cruzi infection correlate with the susceptible and resistant phenotypes identified in these mice. At several time points after initial infection with insect-derived metacyclic trypomastigotes, lymph node and spleen cell suspensions were prepared from animals of each mouse strain. These lymphocyte suspensions were stimulated with Ag prepared from cultured parasites and the production of IFN-gamma, IL-4, IL-2, and IL-5 was measured. Lymphocytes from C57BL6/J mice produced 10-fold more IFN-gamma than BALB/c lymphocytes. However, this enhanced response occurred only for a limited time preceding peak parasitemias. Ag-induced secretion of IL-4 from BALB/c lymphocytes was detectable by 2 wk of infection and increased during the 2nd and 3rd mo of infection. Most C57BL6/J culture supernatants did not contain measurable levels of IL-4. Lymphocytes from both murine strains produced levels of IL-2 and IL-5 indistinguishable from uninfected controls. These results indicate that increased numbers or potency of lymphocytes that produce Ag-specific IFN-gamma responses are present in resistant mice during T. cruzi infection. This phenomenon may be responsible for the lower parasitemias seen in C57BL6/J mice. However, even these relatively resistant mice become chronically infected with T. cruzi, and spleen cells from infected mice can suppress IFN-gamma induced by heterologous Ag. Our data suggest that IL-4 production is a marker for the T. cruzi susceptible phenotype. Differential production of IL-2 or IL-5 was not found, suggesting that these cytokines are not important factors in T. cruzi resistance or susceptibility.
Animals↗
American trypanosomiasis (Chagas' disease)--a tropical disease now in the United States.
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Chagas Disease↗
Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene.
In Trypanosoma cruzi, the cause of Chagas' disease in Latin America, a large proportion of the antigenic proteins described to date have repetitive domains. In earlier work we identified a partial length cDNA, designated TCR27, encoding approx. 26 copies of a 14-amino acid repeat and a unique 61-amino acid C-terminal region. The goal of the current project was to replace the repetitive region of a TCR27 gene with the neomycin phosphotransferase gene (NEOr). A pBluescript-based vector was constructed in which the 0.9-kb NEOr coding region replaced the 2.9-kb internal repetitive segment of a TCR27 gene and was in frame with its nonrepetitive 5' coding sequence (pTCR27-2::NEO). Epimastigotes were electroporated in the presence of linearized pTCR27-2::NEO and transfected clones were selected on solid medium containing G418. Southern and Northern analyses of DNAs and RNAs from four G418-resistant clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by NEOr. The absence of the native TCR27-2 protein in the transfected clones was confirmed by Western blot. In axenic cultures growth rates of epimastigotes bearing an interrupted TCR27-2 gene were not different from those of wild-type parasites. In addition, there was no relative impairment of the four transfected clones' ability to proliferate in cultured mammalian cells. The fact that the clones having the interrupted TCR27-2 gene were not impaired biologically suggests that the length of the repetitive region of the TCR27 protein is not a critical factor for survival.
Amino Acid Sequence↗
Use of a questionnaire to identify potential blood donors at risk for infection with Trypanosoma cruzi.
Trypanosoma cruzi is the protozoan parasite that causes American trypanosomiasis (Chagas' disease). Chagas' disease is endemic in Latin America. The infection is usually seen in poor people who live in rural areas in substandard housing, where they are bitten by infected reduviid bugs. Transmission also can occur by blood transfusion. Infected individuals who immigrate to the United States might donate blood if they are asymptomatic and unaware of their infection. This study evaluated the usefulness of a questionnaire for identifying T. cruzi-infected individuals among prospective blood donors who met all American Association of Blood Banks, Food and Drug Administration, and State of California criteria for donor eligibility. Seventy-two of 3492 otherwise eligible donors were disqualified because of their answers on the questionnaire. Forty-five of these 72 agreed to be tested serologically, and 2 were positive for T. cruzi antibodies. One of six autologous blood donors tested also was positive for T. cruzi antibodies. We conclude that the questionnaire selected a subgroup of Latin Americans at high risk for T. cruzi infection. The deferral of these high-risk individuals clearly reduced the risk of transmission of T. cruzi by transfusion, without intolerably decreasing the supply of donated blood.
Blood Donors↗
Chagas disease. American trypanosomiasis.
Chagas disease, caused by the protozoan parasite, Trypanosoma cruzi, is a major source of morbidity and death in Latin America. Many infected immigrants from that region now reside in the United States, posing a risk of transfusion-associated transmission of the organism. Serologic testing is the cornerstone of diagnosing chronic T. cruzi infections, and improved assays are needed. Drug treatment is problematic because the two available drugs can have severe side effects and lack efficacy. T. cruzi infection can be particularly severe in immunosuppressed patients.
Animals↗
Chagas' disease.
Chagas' disease, caused by Trypanosoma cruzi, is an important cause of morbidity in many countries in Latin America. The important modes of transmission are by the bite of the reduviid bug and blood transfusion. The organism exists in three morphological forms: trypomastigotes, amastigotes, and epimastigotes. The mechanism of transformation and differentiation is currently being explored, and signal transduction pathways of the parasites may be involved in this process. Parasite adherence to and invasion of host cells is a complex process involving complement, phospholipase, penetrin, neuraminidase, and hemolysin. Two clinical forms of the disease are recognized, acute and chronic. During the acute stage pathological damage is related to the presence of the parasite, whereas in the chronic stage few parasites are found. In recent years the roles of tumor necrosis factor, gamma interferon, and the interleukins in the pathogenesis of this infection have been reported. The common manifestations of chronic cardiomyopathy are arrhythmias and thromboembolic events. Autoimmune, neurogenic, and microvascular factors may be important in the pathogenesis of the cardiomyopathy. The gastrointestinal tract is another important target, and "mega syndromes" are common manifestations. The diagnosis and treatment of this infection are active areas of investigation. New serological and molecular biological techniques have improved the diagnosis of chronic infection. Exacerbations of T. cruzi infection have been reported for patients receiving immuno-suppressive therapy and for those with AIDS.
Animals↗
Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas' disease patients.
Approximately 70% of the cDNA clones identified by immunoscreening Leishmania donovani expression libraries with serum from a patient with visceral leishmaniasis (kala-azar) were found to encode the highly conserved Hsp90 and Hsp70 members of the heat shock protein family. Recombinant fusion proteins containing the C-terminal portions of L. donovani Hsp90 and Hsp70 were used as target antigens in enzyme-linked immunosorbent assays of various sera. Sera from four patients with visceral leishmaniasis recognized recombinant Leishmania Hsp90 and Hsp70, while sera from seven patients with Chagas' disease did not, despite the fact that Trypanosoma cruzi Hsp90 and Hsp70 share more than 80% amino acid identity with their counterparts in Leishmania spp. Thus, Leishmania Hsp90 and Hsp70 elicit strong humoral responses and are potential candidates for specific serodiagnostic assays capable of distinguishing between L. donovani and T. cruzi infections.
Amino Acid Sequence↗
Immunization and challenge of mice with insect-derived metacyclic trypomastigotes of Trypanosoma cruzi.
Infection with Trypanosoma cruzi was established in the reduviid vector, Dipetalogaster maximus, by repeated feedings on mice with high parasitaemias, and metacyclic trypomastigotes (IMT) were collected in insect urine after blood meals. The infectivity of IMT in mice was assessed by placing varying numbers of organisms, ranging from 5 to 5000, on to the conjunctivae or oral mucosa of anaesthetized animals. Half of the mice exposed to as few as 20 IMT by either route became parasitized, and the minimum inoculum size that resulted in all mice becoming infected was 640 IMT by the ocular route and 1250 IMT in the mice given parasites by mouth. Mice were immunized by tail vein infusion of irradiated IMT. Animals in the immunized group and in two control groups were then challenged by deposition of IMT on to the oral mucosa. Two of five immunized mice and nine of 10 comparison animals developed acute T. cruzi infection after challenge. These results indicate that IMT produced in this system are highly infective and that inocula containing 500-1000 IMT applied to the conjunctivae or oral mucosa constitute a gentle contaminative challenge. Moreover, our findings suggest that sterile protection against a contaminative challenge may be inducible by immunization with IMT, but experiments involving larger numbers of animals must be performed to resolve this question.
Animals↗
Is Trypanosoma cruzi a new threat to our blood supply?
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Canada↗
A novel flagellar Ca2+-binding protein in trypanosomes.
A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.
Amino Acid Sequence↗