title: Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine.
abstract: Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.
authors.forename: Marcos
authors.initials: M
authors.lastname: Mancilla
authors.forename: María-Jesús
authors.initials: MJ
authors.lastname: Grilló
authors.forename: María-Jesús
authors.initials: MJ
authors.lastname: de Miguel
authors.forename: Ignacio
authors.initials: I
authors.lastname: López-Goñi
authors.forename: Beatriz
authors.initials: B
authors.lastname: San-Román
authors.forename: Ana
authors.initials: A
authors.lastname: Zabalza-Baranguá
authors.affiliation: Departamento de Microbiología y Parasitología e Instituto de Salud Tropical, Universidad de Navarra, C/ Irunlarrea, 1, 31008 Pamplona, Spain. imoriyon@unav.es.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Moriyón
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001428:Bacterial Vaccines / Q000235:genetics* / Q000276:immunology; D015139:Blotting, Southern / Q000662:veterinary; D017347:Brucella melitensis / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D002006:Brucellosis / Q000382:microbiology / Q000628:therapy / Q000662:veterinary*; D002876:Chromosomes, Bacterial; D005260:Female; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D044404:Genomic Islands; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary
chemical_list: D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D016695:Glycosyltransferases; D019426:Integrases