Expression in fibroblasts and in live animals of Entamoeba histolytica polypeptides EhCP112 and EhADH112

Abstract

EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in Entamoeba histolytica's cytopathic effect, target-cell adherence and phagocytosis. The EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles. Here, the independent expression of EhCP112 and EhADH112 in fibroblasts and hamsters was analysed. Also investigated was the immunological response in animals independently inoculated with plasmid pcDNA-Ehcp112, which carries the complete cysteine protease-encoding gene, or with plasmid pcDNA-Ehadh112, which carries the C terminus of the adhesin-encoding gene, or with a mixture of both. Both proteins were expressed in the plasma membranes of the transfected fibroblasts. EhCP112 was toxic for the mammalian cells. Proteins were also independently expressed in hamsters after inoculation with the plasmids. Their expression was indirectly evaluated by the presence of antibodies in the inoculated animals. Remarkably, co-immunization of the animals with the two DNA plasmids resulted in an earlier and higher anti-E. histolytica IgG induction than immunization with separate plasmids. In contrast, the cellular immune response was not noticeably improved by the plasmid mixture. Interestingly, protection against liver abscesses was detected only in animals that received the plasmid mixture and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112.

A 176 amino acid polypeptide derived from the mumps virus HN ectodomain shows immunological and biological properties similar to the HN protein

“…Lower levels of IFNγ and IL2 were detected in the pcDNA3.1-immunized and challenged hamsters (Group E). This unspecific induction by pcDNA3.1 immunization in hamsters has been previously reported [35]. Plasmid DNA vaccines, when injected intramuscularly or intradermally, induce a Th1 response due to the vector CpG motifs that stimulate the production of IL-12, which favors the activation of Th1 lymphocytes [41].…”

“…However, these results support the proposal that the pcDNAHN176-transfected cells were capable of expressing the HN176 polypeptide in a correct folding structure and exporting it to the surface such that it was accessible to the erythrocytes or the antibodies to inhibit the HD reaction. We do not know the mechanism by which the peptide could be expressed on the cell surface, but other groups using the same pcDNA3.1 vector have reported that some proteins lacking the signal peptide are sorted into the cellular membrane [35,36]. Some of those proteins have shown the RXLXEQ motif, which has been associated with ER exportation [37].…”

Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica

“…EhCP112 and EhADH112 have been independently characterized [24,26,27,37]. In particular, the study and characterization of EhCP112 suggest that this protein could play an important role in the virulence of E. histolytica [24,26,27,38], but there is not a conclusive study about the role of EhCP112 in the E. histolytica pathogenesis.…”

Proteases fromEntamoebaspp. and Pathogenic Free-Living Amoebae as Virulence Factors

“…Antibodies against this complex inhibit adherence, phagocytosis, and destruction of MDCK monolayers by live trophozoites and extracts. Antibodies also greatly reduce the ability of amoebae to produce liver abscesses in hamsters [59, 60, 97–99]. …”