Biogenesis of Yersinia pestis PsaA in recombinant attenuated Salmonella Typhimurium vaccine (RASV) strain.
Yersinia pestis PsaA is an adhesin important for the establishment of bacterial infection. PsaA synthesis requires the products of the psaEFABC genes. Here, by prediction analysis, we identified a PsaA signal sequence with two signal peptidase (SPase) cleavage sites, type-I and type-II (SPase-I and SPase-II). By Edman degradation and site-directed mutagenesis, the precise site for one of these Spase-I PsaA cleavage sites was located between alanine and serine at positions 31 and 32, respectively. Yersinia pestis psaA expression and the role of the PsaB and PsaC proteins were evaluated in recombinant attenuated Salmonella Typhimurium vaccine strains. PsaA was detected in total extracts as a major 15-kDa (mature) and 18-kDa (unprocessed) protein bands. PsaA synthesis was not altered by a DeltaA31-DeltaS32 double-deletion mutation. In contrast, the synthesis of PsaA (DeltaA31-DeltaS32) in Y. pestis and delivery to the supernatant was decreased. Otherwise, substitution of the amino acid cysteine at position 26 by valine involved in the SPase-II cleavage site did not show any effect on the secretion of PsaA in Salmonella and Yersinia. These results help clarify the secretion pathway of PsaA for the possible development of vaccines against Y. pestis.
Torres-Escobar
Ascención
A
Center for Infectious Diseases and Vaccinology, The Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ, USA.
Juárez-Rodríguez
María Dolores
MD
Curtiss
Roy
R
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D005822:Genetic Vectors; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010931:Plague Vaccine / Q000235:genetics* / Q000276:immunology; D021382:Protein Sorting Signals / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008565:Membrane Proteins; D010931:Plague Vaccine; D021382:Protein Sorting Signals; D014614:Vaccines, Synthetic; C081814:pH 6 antigen, Yersinia; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D016282:Aspartic Acid Endopeptidases; C095652:signal peptidase II