RESUMO
MAP kinase kinase 1 (MKK1) is encoded by a single copy gene in Trypanosoma brucei. It has been shown recently that MKK1 is not essential for bloodstream forms [14]. To investigate the requirement for MKK1 in other life-cycle stages we generated null mutants in procyclic forms of a fly-transmissible strain. These grew normally in culture and were able to establish midgut infections in tsetse at normal rates and intensities, but were incapable of colonising the salivary glands. Transformation of null mutants with an ectopic copy of MKK1 enabled parasites to complete the life cycle in tsetse and infect mice. This is the first example of a gene that is indispensable for transmission of T. brucei. It also raises the possibility that activating the MKK1 signalling cascade in vitro might trigger the differentiation and proliferation of life-cycle stages of T. brucei that are currently refractory to culture.
Assuntos
Vetores de Doenças , MAP Quinase Quinase 1/metabolismo , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/transmissão , Moscas Tsé-Tsé/parasitologia , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Deleção de Genes , Teste de Complementação Genética , Intestinos/parasitologia , MAP Quinase Quinase 1/genética , Camundongos , Glândulas Salivares/parasitologia , Trypanosoma brucei brucei/genética , Fatores de Virulência/genéticaAssuntos
Resposta ao Choque Térmico , Temperatura , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Animais , Camundongos , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.
Assuntos
Patógenos Transmitidos pelo Sangue , Estágios do Ciclo de Vida/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Trypanosoma brucei brucei/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Contagem de Células , Diferenciação Celular , Células Cultivadas , Engenharia Genética , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Mutação , Filogenia , Sensibilidade e Especificidade , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: To determine the effects of overexpression of mutated BIGH3 in HeLa and human corneal epithelial (HCE) cells. METHODS: Six mutations known to be responsible for autosomal dominant corneal dystrophies linked to chromosome 5 were generated in a BIGH3 expression vector and transfected in HeLa and HCE cells. The expression and secretion of the various BIGH3-EGFP fusion proteins were measured by Western blot analysis. Apoptotic cells were identified by Hoechst/propidium iodide and annexin V staining. Lactate dehydrogenase (LDH) activity was measured in the medium of transfected cells. Truncated BIGH3 protein and site-specific mutations were generated to determine the exact region that mediated apoptosis. RESULT: The overexpressed BIGH3 fusion protein was secreted regardless of its mutation status and was clearly observed in the culture medium. Overexpression of mutated BIGH3 induced apoptosis in both cell lines through activation of caspase-3. Although all the disease-causing mutations tested in this experiment induced apoptosis, the strongest effect was observed with the R124C and R555W mutations. Overexpression of a carboxyl-truncated BIGH3 protein did not induce apoptosis, suggesting that a region located in the C-terminal domain was necessary to mediate cell death. In addition, mutation of the Pro-Asp-Ile (PDI) site at 616-618 was sufficient to prevent induction of apoptosis. CONCLUSIONS: Overexpression of mutated BIGH3 induces apoptosis in HeLa and HCE cells through activation of a pathway that uses the PDI domain of the fourth internal Fas domain and activation of caspase-3. Because DI is a known site of interaction with alpha 3 beta 1 integrins, it suggests that integrins play a role in mediating apoptosis in the system used in the current study. This work suggests that apoptosis is a key element in the pathophysiology of BIGH3-related corneal dystrophies.
