Constitutive expression and anchoring of Mycobacterium tuberculosis antigens in Lactobacillus plantarum

Tuberculosis is the leading cause of death caused by a single agent worldwide. A new and effective vaccine against this infection is therefore imperative. This study is a part of a larger project where the long-term goal is to create an effective vaccine against tuberculosis using LAB as live vectors. Using LAB as a delivery vector for vaccines is highly desirable because of their GRAS status, their non-pathogenicity, probiotic properties, and their ability to deliver functional proteins to mucosal surfaces. These properties make LAB such as L. plantarum an ideal live vector for vaccine delivery. In this study, a constitutive expression system was constructed by replacing the inducible promoter psppA used in the pSIP vectors with constitutive promoters derived from Lactobacillus spp. Moreover, genes directly related to the inducible system, sppK (HK) and sppR (RR), were removed in an attempt to reduce the fitness cost of the vector. This study reveals the challenges of constructing a constitutive plasmid for heterologous protein production. E. coli TOP10 was utilized as a subcloning vector. The production of AgE6 fusion antigen indicated to elicit a toxic effect in E. coli as most of the constitutive promoter constructs only survived when selected for inactive mutants. The toxic effect in E. coli indicates that most of the Lactobacillus derived promoters were also functional in E. coli. Plasmids with constitutive protein expression which previously promoted antigen production were immobilized by the removal of the sppK and sppR genes. sppK and sppR were found to most likely be vital for constitutive protein expression utilizing the SIP system. L. plantarum strains harboring the SlpA or PgM promoter produced the most AgE6 anchored on the cell membrane. However, strains harboring the promoter PgM had a significantly higher growth rate. The constitutive AgE6 production is however not comparable to the inducible promoter production of AgE6, and more research is needed. The fluorescent protein mCherry was used to tag the promoters and was successfully cloned downstream of the inducible promoter psppA and the constitutive SlpA promoter. mCherry did not affect the overall fitness cost in L. plantarum and did not lose its ability to fluoresce over time, thus making it a promising candidate for tracking the vaccine through the GIT.Tuberkulose er den største årsaken til dødsfall forårsaket av en singulær infeksjon og en ny og effektiv vaksine mot tuberkulose er derfor betydningsfullt. Denne studien er en del at et større prosjekt der langtidsmålet er å lage en ny og effektiv vaksine mot tuberkulose ved å bruke LAB som levende vektor og leverandør av vaksinen. Bruk av LAB er meget gunstig på grunn av deres GRAS status, de er ikke-patogene, har probiotiske egenskaper og har evne til å levere funksjonelle proteiner til slimhinner. Disse egenskapene gjør at LAB, som L. plantarum, er ideelle som vaksinevektorer.submittedVersionM-BIOTE