RESUMO
We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.
Assuntos
Entamoeba histolytica/química , Fagocitose/fisiologia , Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , DNA Complementar/química , DNA de Protozoário/química , Densitometria , Eletroforese em Gel de Poliacrilamida , Entamoeba/química , Entamoeba/genética , Entamoeba/imunologia , Entamoeba histolytica/genética , Entamoeba histolytica/imunologia , Dados de Sequência Molecular , Mutação , Fagocitose/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.
