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Applied and Environmental Microbiology, February 2002, p. 910-916, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.910-916.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis

Luciana A. Ribeiro,1,2 Vasco Azevedo,2 Yves Le Loir,1 Sergio C. Oliveira,2 Yakhya Dieye,1 Jean-Christophe Piard,1 Alexandra Gruss,1 and Philippe Langella1*

Laboratoire de Génétique Appliquée, Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France,1 Institute of Biological Sciences, Federal University of Minas Gerais (UFMG-ICB), Belo Horizonte, Minas Gerais, Brazil2

Received 11 May 2001/ Accepted 12 October 2001

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

* Corresponding author. Mailing address: Laboratoire de Génétique Appliquée, Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. Phone: 33 1 34 65 20 83. Fax: 33 1 34 65 20 65. E-mail: langella{at}biotec.jouy.inra.fr.

Applied and Environmental Microbiology, February 2002, p. 910-916, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.910-916.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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