Streptococcus sobrinus is considered one of the principal agents of dental caries (17,20,26,41), and the treatment of this infectious disease is among the world's most costly health problems, due to its wide distribution (26, 34). The polyclonallymphocyte activation of the host, observed in several microbial infections, has been described as a general survival strategy of the infecting microorganism (9,21,30,48). Several microbial molecules have been described as B-and T-cell mitogens (8,16,19,25,27,31,35,39,40,49,50). It has been reported that S. sobrinus produces a protein that activates murine lymphocytes polyclonally, suppresses specific antibody response to S. sobrinus antigens, and potentiates the growth of the microorganism in the host (18).The vital and ubiquitous enzyme NAD ϩ synthetase belongs to the amidotransferase family. This enzyme catalyzes the synthesis of NAD ϩ from either NH 3 or glutamine and nicotinic acid adenine dinucleotide (29,53), and NAD ϩ is involved in an enormous variety of biochemical processes, such as the synthesis of various essential molecules and antibiotics, oxidationreduction reactions, and DNA repair and recombination (37). The NAD ϩ synthetase of Bacillus subtilis is a member of a B -dependent general stress regulon (5). NAD ϩ synthetases are essential for viability in Escherichia coli (3) and Salmonella enterica serovar Typhimurium (23).In this work, we identify and characterize for the first time the NAD ϩ synthetase of S. sobrinus as a murine B-cell-stimulatory protein. Our results provide information on the involvement of NAD ϩ synthetase in the modulation of the host immune system induced by S. sobrinus. As previously suggested and reported for several microorganisms (6,10,30,42,47), the isolation and the characterization of molecules involved in microbial pathogenicity may be useful in the identification of targets for vaccination.
MATERIALS AND METHODSStrains and plasmids. S. sobrinus strain 6715, able to induce caries in rats (15, 43), was a kind gift of B. Klausen from the Department of Oral Diagnosis and Microbiology, Royal Dental College, University of Copenhagen, Denmark. The strain was stored at Ϫ70°C in Todd-Hewitt broth medium (Difco Laboratories, Detroit, Mich.) with 10% glycerol. E. coli strain DH-5␣ (38) and the plasmid pGEM-T Easy vector (Promega Corp., Madison, Wis.) were used for cloning PCR fragments, while E. coli strain M-15 (38) was used as the host for the plasmid pQE-31 (Qiagen, Oslo, Norway) in the protein expression experiments. For immunobiological experiments, 6-to 8-week-old male C57BL/6 mice were bred at the Gulbenkian Institute of Science, Oeiras, Portugal. We followed the guidelines of the European Community for animal studies.Purification of the protein secreted by S. sobrinus. S. sobrinus was cultured in Todd-Hewitt broth medium for 2 days at 37°C with an initial inoculum of 10 8 cells/ml. The cultures were centrifuged at 12,000 ϫ g for 30 min. The supernatants were filtered through 0.22-m-pore-size filters (Schleicher & Schuell, Dassel...