Construction and Characterization of aSalmonella entericaSerovar Typhimurium Clone Expressing a Salivary Adhesin ofStreptococcus mutansunder Control of the Anaerobically InduciblenirBPromoter

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Abstract
AttenuatedSalmonella entericaserovar Typhimurium has been used for targeted delivery of recombinant antigens to the gut-associated lymphoid tissues. One potential problem associated with this vaccine approach is the likelihood of in vivo instability of the plasmid constructs caused by constitutive hyperexpression of the heterologous immunogen. The aim of this study was to generate and characterize an expression system encoding the saliva-binding region (SBR) ofStreptococcus mutansantigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the induciblenirBpromoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing ofSalmonella. The gene encoding the chimeric SBR-CTA2/B was amplified by PCR using primers containing appropriate restriction sites for subcloning into pTETnirB, which contains thenirBpromoter. The resulting plasmid was introduced into serovar Typhimurium by electroporation. Production of the SBR-CTA2/B chimeric protein under anaerobic conditions was verified by enzyme-linked immunosorbent assay of whole-cell lysates on plates coated with GM1ganglioside and developed with antibodies to SBR. Similar procedures were followed for cloning the gene encoding SBR in serovar Typhimurium undernirBcontrol. Anaerobic expression of SBR was confirmed by Western blotting of whole-cell lysates probed with anti-SBR antibodies. The resulting serovar Typhimurium strains were administered by either the oral or the intranasal route to mice, and colonization was assessed by microbiologic analysis of dissociated spleens, Peyer's patches (PP), and nasal tissues. High numbers of the recombinant strains persisted in PP and spleen for at least 21 days following oral challenge. A single intranasal administration of theSalmonellaclones to mice also resulted in the colonization of the nasal tissues by the recombinant bacteria. Salmonellae were recovered from nasal lymphoid tissues, superficial lymph nodes, internal jugular lymph nodes, PP, and spleens of mice for at least 21 days after challenge. This study provides quantitative evidence for colonization bySalmonellastrains expressing a recombinant protein under the control of the induciblenirBpromoter in PP or nasal tissues following a single oral or nasal administration of the bacteria, respectively.