Construction and Characterization of a Salmonella enterica Serovar Typhimurium Clone Expressing a Salivary Adhesin of Streptococcus mutans under Control of the Anaerobically Inducible nirB Promoter
AUTOR(ES)
Huang, Yan
FONTE
American Society for Microbiology
RESUMO
Attenuated Salmonella enterica serovar 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) of Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the inducible nirB promoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing of Salmonella. The gene encoding the chimeric SBR-CTA2/B was amplified by PCR using primers containing appropriate restriction sites for subcloning into pTETnirB, which contains the nirB promoter. 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 GM1 ganglioside and developed with antibodies to SBR. Similar procedures were followed for cloning the gene encoding SBR in serovar Typhimurium under nirB control. 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 the Salmonella clones 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 by Salmonella strains expressing a recombinant protein under the control of the inducible nirB promoter in PP or nasal tissues following a single oral or nasal administration of the bacteria, respectively.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=97314Documentos Relacionados
- Induction of Protective Immunity against Streptococcus mutans Colonization after Mucosal Immunization with Attenuated Salmonella enterica Serovar Typhimurium Expressing an S. mutans Adhesin under the Control of In Vivo-Inducible nirB Promoter
- High level heterologous expression in E. coli using the anaerobically-activated nirB promoter.
- Effect of Attenuated Salmonella enterica Serovar Typhimurium Expressing a Streptococcus mutans Antigen on Secondary Responses to the Cloned Protein
- Construction, Characterization, and Immunogenicity of an Attenuated Salmonella enterica Serovar Typhimurium pgtE Vaccine Expressing Fimbriae with Integrated Viral Epitopes from the spiC Promoter
- Oral Vaccination against Tetanus: Comparison of the Immunogenicities of Salmonella Strains Expressing Fragment C from the nirB and htrA Promoters