RESUMO
A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8(+) lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4(+) lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice.
Assuntos
Cisteína Proteases/imunologia , Leishmania mexicana/enzimologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Animais , Western Blotting , Cricetinae , Cisteína Proteases/genética , Cisteína Proteases/isolamento & purificação , DNA de Protozoário/química , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação Enzimológica da Expressão Gênica , Leishmania mexicana/genética , Linfonodos/citologia , Linfonodos/imunologia , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Linfócitos T/imunologiaRESUMO
Trypanosoma cruzi metacyclic trypomastigotes express the developmentally regulated GP82 glycoprotein, which is implicated in host cell invasion. Although GP82 mRNA and protein are not present and the mRNAs barely detectable in epimastigotes, nuclear run-on analysis showed that it is transcribed in both stages. This result indicates that accumulation of transcripts in metacyclic forms is not due to increased transcription of the GP82 gene. To investigate whether mRNA stability may be responsible for the differences in the steady-state levels of this mRNA, parasites were treated with actinomycin D or cycloheximide. When treated with actinomycin D, the half-lives estimated for GP82 transcripts were about 6h in metacyclic trypomastigotes and 0.5h in epimastigotes. In the presence of cycloheximide, the levels of GP82 mRNA decayed slightly after 8h in metacyclic trypomastigotes, whereas in epimastigotes the levels of this mRNA increased. This effect suggests a stabilizing mechanism acting in metacyclic trypomastigotes and a destabilizing mechanism in epimastigotes which could be mediated by an element present in the 3'-UTR of the transcripts. Consistent with this finding, northern blot analysis showed that GP82 mRNAs were mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes.
Assuntos
Regulação da Expressão Gênica , Proteínas de Protozoários/biossíntese , Estabilidade de RNA , Trypanosoma cruzi/fisiologia , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Regiões 3' não Traduzidas , Animais , Polirribossomos/metabolismo , Biossíntese de Proteínas , Trypanosoma cruzi/genéticaRESUMO
We report the cloning of a Paracoccidioides brasiliensis cDNA, here named PbCnx, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin. Calnexin specifically recognizes monoglucosylated glycoproteins in the endoplasmic reticulum, thus being an essential component of the complex that interacts with the folded state of nascent secreted glycoproteins. The PbCnx open reading frame was found in a 1701 base pair (bp) fragment that encodes a 567 amino acid protein with an estimated mass of 62 680 Da. Northern and Southern blot hybridizations showed that PbCnx is encoded by a single, or a low number of, gene copies. PbCnx contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family proteins. A cDNA-encoding PbCnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant PbCnx was recognized by 6 out of 10 sera from PCM patients, a result that rules out its possible consideration for further use in diagnosis. Using confocal microscopy with anti-PbCnx mouse serum against yeast forms, a cytoplasmic staining pattern was observed.
Assuntos
Calnexina/genética , Paracoccidioides/genética , Paracoccidioides/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Calnexina/biossíntese , Calnexina/imunologia , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/química , DNA Fúngico/genética , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Dados de Sequência Molecular , Paracoccidioidomicose/sangue , Paracoccidioidomicose/imunologia , RNA Fúngico/química , RNA Fúngico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia de SequênciaRESUMO
A recombinant protein, rLdccys1, which was produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used for detection of antibodies in sera from patients with active visceral leishmaniasis (VL) in enzyme-linked immunosorbent assays. Analysis of the predicted amino acid sequence of rLdccys1 showed that it contains all the characteristics of a cysteine proteinase. The ability of the protein to react with sera from humans with VL was also shown by Western blotting. The sensitivity for detection of specific antibodies to L. (L.) chagasi bodies using rLdccys1, L. (L.) chagasi promastigote lysates, and amastigote lysates was 80%, 98%, and 99%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and there was little positive reactivity with sera from patients with cutaneous leishmaniasis and tuberculosis, compared with promastigote and amastigote extracts. Our findings indicate that rLdccys1 from L. (L.) chagasi constitutes a potential tool for the diagnosis of American VL.
Assuntos
Anticorpos Antiprotozoários/sangue , Cisteína Endopeptidases/genética , Leishmania/genética , Leishmania/imunologia , Leishmaniose Visceral/diagnóstico , Sequência de Aminoácidos , Animais , Western Blotting , Estudos de Casos e Controles , Cricetinae , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leishmaniose Visceral/sangue , Mesocricetus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Testes SorológicosRESUMO
The present study describes the cloning and characterisation of a gene encoding a cysteine proteinase isoform, Llacys1, expressed in amastigote forms of Leishmania (L.) amazonensis. Recombinant clones containing the Llacys1 gene were isolated from genomic DNA by PCR amplification and screening of an amastigote cDNA library. Sequence analysis of the Llacys1 gene showed a high identity to sequence of Leishmania (L.) pifanoi Lpcys1, Leishmania (L.) major cpa, Leishmania (L.) mexicana LCPa, and Leishmania (L.) chagasi Ldccys2. The Llacys1 gene is present in a single copy per L. (L.) amazonensis haploid genome and was mapped on a chromosome of approximately 700 kb. Two transcripts of the Llacys1 gene were identified, one of 2.4 kb transcribed in both forms of L. (L.) amazonensis, and another of 1.6 kb weakly expressed in amastigotes. Related forms of Llacys1 gene exist in other species of Leishmania genus, including L. (L.) major, L. (L.) mexicana, L. (L.) chagasi and Leishmania (V.) braziliensis. The Llacys1 expression in Escherichia coli was obtained when the nucleotide sequence corresponding to the signal sequence was deleted, suggesting that this signal sequence was recognised by Escherichia coli and cleaved, generating a truncated protein.
Assuntos
Cisteína Endopeptidases/genética , Genes de Protozoários/genética , Leishmania mexicana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA de Protozoário/genética , Expressão Gênica , Dados de Sequência Molecular , RNA de Protozoário/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.
