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Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

Jiaxin Li^1,,

Cheryl Ryder^1,

Manas Mandal^1,,

¹ Center for Molecular Medicine and Infectious Diseases, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
² Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
³ Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA

Correspondence
Thomas J. Inzana
tinzana{at}vt.edu

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.

These authors contributed equally to this work.

Present address: The Institute for Genomic Research, Rockville, MD, USA.

Present address: College of Pharmacy, University of Southern Nevada, Henderson, NV, USA.

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