High-level transient expression of the N-terminal domain of IpaD from Shigella dysenteriae in four plant species transformed with different construct configurations

Abstract

The Ipa operon contains four genes (IpaA, IpaB, IpaC, and IpaD), all assumed to be mandatory factors in the invasion process of Shigella spp. Of these, IpaD is argued to play the most critical role in invasion of Shigella dysenteriae into epithelial cells of the human gut, which often results in shigellosis disease. The potential expression of S. dysenteriae invasion plasmid antigen D (IpaD) was examined in the leaves of four plant hosts—Nicotiana tabacum, N. benthamiana, lettuce (Lactuca sativa), and soybean (Glycine max)—transformed with constructs containing different expression elements. The highest yields of IpaD, obtained by targeting the protein to ER-derived protein bodies, were more than 1.21-fold (in soybean) and 1.48-fold (in N. benthamiana) higher than when protein was targeted to apoplastic compartments, based on total soluble protein (TSP). IpaD production in N. benthamiana was more abundant than in N. tabacum, lettuce, or soybean, averaging 2.2 ng of IpaD μg−1 TSP and with a maximum of 3.4 ng IpaD μg−1 TSP with the best-performing construct (pCAMBIA-ZCIpaD), corresponding to 15.7 μg IpaD g−1 fresh leaf weight. Quantitative RT-PCR suggested that the Cucumber mosaic virus 2b silencing suppressor could efficiently enhance transient expression of IpaD mRNA approximately 4-fold in N. benthamiana at 96 h after infiltration. Finally, with pCAMBIA-ZCIpaD, containing Cowpea mosaic virus UTRs as possible expression enhancers, the accumulation of IpaD in agroinfiltrated N. benthamiana was nearly 2-fold that was obtained with constructs not containing UTRs. This is the first report describing the effects of several important signal peptides and translational enhancers on IpaD antigen production in four different plant hosts. These results seem to indicate a promising route for the production of therapeutic IpaD antigen in vitro, as a candidate vaccine for human shigellosis.