RESUMO
The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.
Assuntos
Temperatura Alta/efeitos adversos , Leishmania mexicana/metabolismo , Estresse Oxidativo , Proteínas de Protozoários/fisiologia , Animais , Antiprotozoários/farmacologia , Western Blotting , Imunofluorescência , Regulação da Expressão Gênica , Leishmania mexicana/química , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Macrófagos Peritoneais/parasitologia , Meglumina/farmacologia , Antimoniato de Meglumina , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Mutação , Novobiocina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Compostos Organometálicos/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
The META1 gene of Leishmania is upregulated in metacyclic promastigotes and encodes a 12 kDa virulence-related protein, conserved in all Leishmania species analysed. In this study, the genomic region adjacent to the Leishmania amazonensis META1 gene was characterised and compared to the Leishmania major META1 locus as well as to syntenic loci identified in Trypanosoma brucei and Trypanosoma cruzi. Three new genes expressed with increased abundance of steady state mRNA in L. amazonensis promastigotes were identified, two of which are upregulated in stationary phase promastigotes, sharing the pattern of expression previously described for the META1 mRNA. One of these new genes, named META2, encodes a polypeptide of 444 amino acid residues with a repetitive structure showing three repeats of the META domain (defined as a small domain family found in the Leishmania META1 protein and in bacterial proteins hypothetically secreted and/or implicated in motility) and a carboxyl-terminal region similar to several putative calpain-like proteins of Trypanosoma and Leishmania.