Abstract
The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
Highlights
Mycobacterium bovis BCG is currently the world’s most widely used vaccine and has been given to more than three billion people, making it a very attractive prospect for the development of a live recombinant BCG multivaccine [1]
A study in which expression of M. tuberculosis antigen 85B (Ag85B) in recombinant BCG (rBCG) was placed under the control of a limited set of promoters found that increasing promoter activity caused a skewing of the immune response to Ag85B in mice from a mixed Th1/Th2 response to a predominantly Th1 response [6]
Our aim was to generate a set of mycobacterial promoters that can be used to obtain a predictable range of gene expression levels in both M. smegmatis and M. bovis BCG
Summary
Mycobacterium bovis BCG is currently the world’s most widely used vaccine and has been given to more than three billion people, making it a very attractive prospect for the development of a live recombinant BCG (rBCG) multivaccine [1]. Stability of the heterologous (or native) gene(s) in BCG was usually obtained by cloning it on a plasmid or chromosomally integrative vector with expression achieved by placing it under the control of a range of mycobacterial promoters [2]. In another study, increasing expression of M. tuberculosis 19-kDa lipoprotein led to complete abrogation of the protective efficacy of BCG by polarizing the host immune responses to the Th2 subtype [7]. Another important factor for any vaccine vector is stability and the associated metabolic burden, which can lead to loss of antigen expression and/or premature elimination of the vector in the host because of loss of fitness [8].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
