TY - JOUR
T1 - Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica
AU - Ruiz-Olvera, Patricia
AU - Ruiz-Pérez, Fernando
AU - Sepulveda, Nicolás Villegas
AU - Santiago-Machuca, Araceli
AU - Maldonado-Rodríguez, Rogelio
AU - Garcia-Elorriaga, Guadalupe
AU - González-Bonilla, César
N1 - Funding Information:
The authors thank Dr. Leopoldo Aguilar Faisal for his technical assistance. This work was in part supported by CONACYT Grant M28958 (P.I: C.G.B) and IMSS Grant FP-2001/056, Reg.2000-693-0012 (P.I: C.G:B.). P.R.O., A.S:M., and F.R.P. were recipients of fellowship from CONACYT and IMSS.
PY - 2003/7
Y1 - 2003/7
N2 - 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 β 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 β-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.
AB - 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 β 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 β-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.
KW - Autodisplay
KW - Circumsporozoite protein
KW - Live vector vaccines
KW - MisL
KW - Plasmodium falciparum
KW - Salmonella enterica
UR - http://www.scopus.com/inward/record.url?scp=0038121598&partnerID=8YFLogxK
U2 - 10.1016/S0147-619X(03)00047-7
DO - 10.1016/S0147-619X(03)00047-7
M3 - Artículo
SN - 0147-619X
VL - 50
SP - 12
EP - 27
JO - Plasmid
JF - Plasmid
IS - 1
ER -