OspA, a lipoprotein antigen of the obligate intracellular bacterial pathogen Piscirickettsia salmonis.
No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate intracellular pathogen Piscirickettsia salmonis are presented. Genomic DNA isolated from Percoll density gradient purified P. salmonis, was used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against P. salmonis, with a bias toward P. salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the lac promoter was required to obtain a stable clone of a 4,983 bp insert in Escherichia coli due to insert toxicity exerted by the accompanying radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa outer membrane lipoprotein of Rickettsia prowazekii, previously thought confined to members of the genus Rickettsia. Palmitate incorporation demonstrated that OspA is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression, ospA was optimized for codon usage in E. coli by PCR synthesis. Expression of ospA was ultimately improved (approximately 13% of total protein) with a truncated variant lacking a signal sequence. High level expression (approximately 42% tot. prot.) was attained as an N-terminal fusion protein with the fusion product recovered as inclusion bodies in E. coli BL21. Expression of OspA in P. salmonis was confirmed by immunoblot analysis using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera from coho salmon (Oncorhynchus kisutch).
Kuzyk
M A
MA
Canadian Bacterial Diseases Network, Department of Biochemistry and Microbiology, University of Victoria, British Columbia.
Burian
J
J
Thornton
J C
JC
Kay
W W
WW
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001428:Bacterial Vaccines; D017123:Bacteriophage T7 / Q000235:genetics; D015152:Blotting, Northern / Q000379:methods; D003062:Codon; D015151:Immunoblotting / Q000379:methods; D007424:Intracellular Fluid / Q000382:microbiology; D008074:Lipoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D022123:Lyme Disease Vaccines / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D020560:Proteobacteria / Q000235:genetics* / Q000276:immunology; D012487:Salmonidae / Q000382:microbiology; D017421:Sequence Analysis; D014158:Transcription, Genetic
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D003062:Codon; D008074:Lipoproteins; D022123:Lyme Disease Vaccines; C072045:OspA protein; D021382:Protein Sorting Signals