RESUMO
Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.
Assuntos
Antígenos de Protozoários/sangue , Esterases/sangue , Leishmania donovani/enzimologia , Leishmaniose Visceral/parasitologia , África Oriental , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Sequência de Bases , Southern Blotting , Brasil , Linhagem Celular , Cromatografia de Afinidade , Cães , Esterases/imunologia , Esterases/metabolismo , Humanos , Imunoprecipitação , Índia , Leishmania donovani/genética , Leishmania donovani/imunologia , Leishmania donovani/isolamento & purificação , Leishmania infantum/enzimologia , Leishmania infantum/genética , Leishmaniose Visceral/imunologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release approximately >2-4-fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal expression system was devised and used to express an epitope-tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by reverse transcription-PCR, Western blots, and indirect immunofluorescence analyses. Further, results of coupled immunoprecipitation/enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen.
Assuntos
Quitinases/genética , Quitinases/metabolismo , Leishmania mexicana/enzimologia , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Quitinases/química , Humanos , Leishmania mexicana/patogenicidade , Macrófagos/parasitologia , Camundongos , Dados de Sequência Molecular , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , VirulênciaRESUMO
Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.