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Infection and Immunity, June 2003, p. 3261-3271, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3261-3271.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Brucella abortus O-Polysaccharide and Core Lipopolysaccharide Mutants and Demonstration that a Complete Core Is Required for Rough Vaccines To Be Efficient against Brucella abortus and Brucella ovis in the Mouse Model

D. Monreal,1 M. J. Grilló,2 D. González,1 C. M. Marín,2 M. J. De Miguel,2 I. López-Goñi,1 J. M. Blasco,2 A Cloeckaert,3 and I. Moriyón1*

Departamento de Microbiología, Universidad de Navarra, Pamplona,1 Servicio de Investigación Agroalimentaria, Diputación General de Aragón, Zaragoza, Spain,2 Unité BioAgresseurs, Santé et Environnement, Institut National de la Recherche Agronomique, 37380 Nouzilly, France3

Received 19 December 2002/ Returned for modification 31 January 2003/ Accepted 19 February 2003

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manBcore to distinguish it from the wbk manBO-Ag. The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.

* Corresponding author. Mailing address: Departamento de Microbiología, Universidad de Navarra, Aptdo. 177, 31080 Pamplona, Spain. Phone: 34-948-425600. Fax: 34-948-425649. E-mail: imoriyon{at}unav.es.

Editor: J. T. Barbieri

Infection and Immunity, June 2003, p. 3261-3271, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3261-3271.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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