ABSTRACT
Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.
Subject(s)
Entamoeba histolytica/immunology , Liver Abscess, Amebic/prevention & control , Protozoan Proteins/administration & dosage , Protozoan Vaccines/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Animals , Antibodies, Protozoan/blood , Disease Models, Animal , Entamoeba histolytica/genetics , Humans , Immunogenicity, Vaccine , Lectins/genetics , Lectins/immunology , Liver/immunology , Liver/parasitology , Liver/pathology , Liver Abscess, Amebic/blood , Liver Abscess, Amebic/parasitology , Liver Abscess, Amebic/pathology , Male , Mesocricetus , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Protozoan Vaccines/genetics , Protozoan Vaccines/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
OBJECTIVE: To generate an immunogenic chimeric protein containing the Entamoeba histolytica LC3 fragment fused to the retrograde delivery domains of exotoxin A of Pseudomonas aeruginosa and KDEL3 for use as an effective vaccine. RESULTS: A codon-optimized synthetic gene encoding the PEΔIII-LC3-KDEL3 fusion construct was designed for expression in Pichia pastoris. This transgene was subcloned into the plasmid pPIC9 for methanol-inducible expression. After transformation and selection of positive-transformed clones by PCR, the expression of the recombinant protein PEΔIII-LC3-KDEL3 was elicited. SDS-PAGE, protein glycosylation staining and western blot assays demonstrated a 67 kDa protein in the medium culture supernatant. The recombinant protein was detected with a polyclonal anti-6X His tag antibody and a polyclonal E. histolytica-specific antibody. A specific antibody response was induced in hamsters after immunization with this protein. CONCLUSIONS: We report for the first time the design and expression of the recombinant E. histolytica LC3 protein fused to PEΔIII and KDEL3, with potential application as an immunogen.
Subject(s)
ADP Ribose Transferases/genetics , Bacterial Toxins/genetics , Entamoeba histolytica/genetics , Exotoxins/genetics , Recombinant Fusion Proteins/genetics , Vaccines , Virulence Factors/genetics , ADP Ribose Transferases/immunology , Animals , Bacterial Toxins/immunology , Entamoeba histolytica/immunology , Exotoxins/immunology , Pichia/genetics , Recombinant Fusion Proteins/immunology , Virulence Factors/immunology , Pseudomonas aeruginosa Exotoxin AABSTRACT
Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia and is normally transmitted by aerosols and direct contact between animals. A. pleuropneumoniae has traditionally been considered an obligate pathogen of pigs and its presence in the environment has yet to be investigated. Here, the presence of A. pleuropneumoniae was detected in drinking water of pig farms in Mexico using a PCR specific for the RTX toxin gene, apxIV. The presence of A. pleuropneumoniae in farm drinking water was confirmed by indirect immunofluorescence using an A. pleuropneumoniae-specific polyclonal antibody and by fluorescent in situ hybridization. Viable bacteria from the farm drinking water were detected using the Live/Dead BacLight stain. Additionally, viable A. pleuropneumoniae was selected and isolated using the cAMP test and the identity of the isolated bacteria were confirmed by Gram staining, a specific polyclonal antibody and an A. pleuropneumoniae-specific PCR. Furthermore, biofilms were observed by scanning electron microscopy in A. pleuropneumoniae-positive samples. In conclusion, our data suggest that viable A. pleuropneumoniae is present in the drinking water of swine farms and may use biofilm as a strategy to survive in the environment.
