title: Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica.
abstract: The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL beta translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp-Ala-Asp-Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL beta-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A(-)) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP)(3). Higher expression of the (NANP)(8) and (NANP)(53) fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP)(53) in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP)(8) and (NANP)(53), respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.
authors.affiliation: Unidad de Investigación Médica en Inmunología e Infectología, Hospital de Infectología "Dr. Daniel Méndez Hernández," Centro Médico "La Raza," IMSS, D.F., Mexico.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ruiz-Olvera
authors.forename: Fernando
authors.initials: F
authors.lastname: Ruiz-Pérez
authors.forename: Nicolás Villegas
authors.initials: NV
authors.lastname: Sepulveda
authors.forename: Araceli
authors.initials: A
authors.lastname: Santiago-Machuca
authors.forename: Rogelio
authors.initials: R
authors.lastname: Maldonado-Rodríguez
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Garcia-Elorriaga
authors.forename: César
authors.initials: C
authors.lastname: González-Bonilla
mesh_terms: D018830:Adhesins, Escherichia coli / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics*; D015202:Protein Engineering; D015800:Protozoan Proteins / Q000235:genetics*; D019779:Salmonella enterica / Q000235:genetics*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism
chemical_list: C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D001426:Bacterial Proteins; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C460865:MisL protein, Salmonella enterica; D015800:Protozoan Proteins; C041595:circumsporozoite protein, Protozoan; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease