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* ICBAS-Instituto de Ciências Biomédicas de Abel Salazar and
IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal; and
Unité de Biologie des Bactéries Pathogènes à Gram-Positif, Centre National de la Recherche Scientifique, Unité de Recherche Associée, Institut Pasteur, Paris, France
Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.
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1 This work was supported by Fundação para a Ciência e a Tecnologia and Fonds Européen de Développement Régional Grant POCTI/SAU-IMI/60014/2004. Research in P.T.-C.’s laboratory was funded by the Institut Pasteur (Grants PTR 17 and GPH 09).
2 P.M. and M.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Paula Ferreira, Laboratory of Immunology, Instituto Ciências Biomédicas Abel Salazar, Largo Prof. Abel Salazar 2, 4099-003 Porto, Portugal. E-mail address: pauferr{at}icbas.up.pt
4 Abbreviations used in this paper: GBS, group B streptococci; VIP, virulence-associated immunomodulatory protein; rGAPDH, recombinant GAPDH; oeGAPDH, overexpressing GAPDH; F1,6bisP, fructose 1, 6-bisphosphate; IPTG, isopropyl-
-D-thiogalactopyranoside; WT, wild type.
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