Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS.
Francisella tularensis is a zoonotic, Gram-negative coccobacillus that causes tularemia in humans and animals. F. tularensis subspecies tularensis (type A) and F. tularensis subspecies holarctica (type B) are antigenically similar and more virulent than Francisella novicida in humans. The genetic locus that encodes the LPS O antigen was found to be substantially different between the type B live vaccine strain (LVS) and F. novicida. One LVS-specific gene with homology to a galactosyl transferase was selected for allelic replacement using a sacB-chloramphenicol expression suicide plasmid, and recombinants were screened for colony morphology on Congo red agar that matched that of F. novicida. Two mutants (WbtI(S187Y) and WbtI(G191V)) were isolated that contained substitutions in conserved motifs in the sugar transamine/perosamine synthetase (WbtI) of the O-antigen locus, and the latter mutant was extensively tested and characterized. WbtI(G191V) grew at the same rate as the parent strain in Chamberlain's defined medium, completely lacked O antigen, was serum-sensitive but could grow in a mouse macrophage cell line, had increased resistance to sodium deoxycholate, and was highly attenuated in mice. Complementation of WbtI(G191V) with the wild-type wbtI gene in trans restored normal LPS synthesis, phenotypic properties similar to the parent, and virulence in mice. Immunization with WbtI(G191V) protected mice against a relatively low-dose intraperitoneal challenge with LVS, but was less protective against a high-dose challenge. These results indicate that complete loss of O antigen alters the surface phenotype and abrogates virulence in F. tularensis, but also compromises the induction of full protective immunity against F. tularensis infection in mice.
Li
Jiaxin
J
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Ryder
Cheryl
C
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Mandal
Manas
M
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Ahmed
Farzana
F
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Azadi
Parastoo
P
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.
Snyder
D Scott
DS
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.
Pechous
Roger D
RD
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.
Zahrt
Thomas
T
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.
Inzana
Thomas J
TJ
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
D000818:Animals; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000276:immunology; D002460:Cell Line; D019295:Computational Biology; D005604:Francisella tularensis / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000472:pathogenicity; D006801:Humans; D007114:Immunization; D008070:Lipopolysaccharides / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D019081:O Antigens / Q000737:chemistry / Q000235:genetics*; D014406:Tularemia / Q000382:microbiology / Q000517:prevention & control*; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000276:immunology; D014774:Virulence
D001428:Bacterial Vaccines; D008070:Lipopolysaccharides; D019081:O Antigens; D014613:Vaccines, Attenuated