Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

Abstract

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.

FIG. 1

Genetic maps of a representative set of isogenic expression plasmids, carrying an ori15A origin of replication, which differ only in the introduction of increasingly complex combinations of maintenance functions. The plasmids pGEN111, pGEN121, pGEN193, and pGEN222 contain maintenance functions encoded by the hok-sok, hok-sok plus par, hok-sok plus parA, and hok-sok plus par plus parA loci, respectively. Other expression plasmids were constructed by replacing each ori15A origin of replication cassette (copy number, ∼15) with an oriE1 origin (copy number, ∼60); a list of the plasmids used in this work is given in Table 2. The key restriction sites shown represent unique sites in the expression plasmids. P_ompC1, modified osmotically controlled ompC promoter from E. coli; gfpuv, gene encoding the prokaryotic codon-optimized GFPuv; T1, transcriptional terminator from the rrnB rRNA operon of E. coli; par, passive partitioning system from pSC101; bla, β-lactamase gene conferring resistance to carbenicillin; hok-sok, postsegregational killing locus from the multiple antibiotic resistance R plasmid pR1; parA, active partitioning system from pR1.

FIG. 2

Flow cytometry histograms of GFPuv fluorescence for populations of attenuated S. typhi CVD 908-htrA containing expression plasmids with the ori15A origin of replication. Histograms are arranged in rows indicating analysis of bacterial populations grown under inducing conditions with either 50, 150, or 300 mM NaCl. The histogram columns indicate CVD 908-htrA carrying the expression plasmid listed at the bottom of the column, which contains the maintenance function(s) listed at the top of the column. Results are plotted as the log of GFPuv relative fluorescence intensity versus the number of fluorescing bacteria. The mean fluorescence intensity for a given subpopulation is indicated under each curve; these data are fully quantitated in Table 4.