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Infection and Immunity, July 2002, p. 3768-3776, Vol. 70, No. 7
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.7.3768-3776.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

H2O2, Which Causes Macrophage-Related Stress, Triggers Induction of Expression of Virulence-Associated Plasmid Determinants in Rhodococcus equi

Stephanie Benoit,1,2 Abdellah Benachour,1 Said Taouji,2 Yanick Auffray,1 and Axel Hartke1*

Laboratoire de Microbiologie de l'Environnement, IRBA, EA 956 USC INRA, Université de Caen, 14032 Caen Cedex,1 Institut de Pathologie du Cheval, Association Française de Sécurité Sanitaire des Aliments-Dozulé, 14430 Dozulé, France2

Received 15 October 2001/ Returned for modification 11 December 2001/ Accepted 4 March 2002

The response of the intracellular pathogen Rhodococcus equi to H2O2 treatment, a situation potentially encountered after the oxidative burst of alveolar macrophages, was analyzed. Compared to other bacteria, including Deinococcus radiodurans, R. equi showed exceptionally high resistance to this stress. A proteomic approach showed that four polypeptides present in the wild-type strain (85F) are missing in the plasmid-cured strain 85F(P-), and by using a DNA macroarray, we identified two plasmid-encoded vap genes, vapA and vapG, whose expression was highly induced by H2O2 treatment. Whereas the transcript size of vapA was compatible with a monocistronic mRNA, the transcript of vapG was considerably longer. Rapid amplification of cDNA ends PCRs showed that the transcriptional start sites of the two operons were 69 and 269 nucleotides (nt) upstream of the start codon, respectively. Analysis of these leader sequences revealed the presence of a small open reading frame named podG, which encodes a sequence of 55 amino acids preceded by a putative ribosome binding site sequence in the vapG transcript. Taking this result into account, the untranslated leader of the podG/vapG operon is 87 nt. Alignment of this sequence with the leader sequences of vapA and vapD, genes previously shown to be induced by acid, revealed significant homologies. Since our results showed that vapA, vapD, and vapG are genes highly induced by macrophage-related stresses, their gene products may, within the Vap protein family, play a dominant role inside these phagocytic cells and may be the most promising candidates for vaccination strategies.

* Corresponding author. Mailing address: Laboratoire de Microbiologie de l'Environnement, IRBA, EA 956 USC INRA, Université de Caen, 14032 Caen Cedex, France. Phone: 33 (0)2 31 56 54 04. Fax: 33 (0)2 31 56 53 11. E-mail: hartke{at}ibba.unicaen.fr.

Editor: V. J. DiRita

Infection and Immunity, July 2002, p. 3768-3776, Vol. 70, No. 7
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.7.3768-3776.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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