Construction of stable LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses

Abstract

The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified 13-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, SalmoneUla typhimurium, and attenuated antigen carrier strains of S. typhimurium (aroA mutants). The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes. S. typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane. High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities. The lower levels of gene expression observed with the tl-lactamase and aerobactin promoters did not have this effect. Immunization of mice with S. typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses. This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxinor Shiga-like toxin-producing pathogens. Bacillary dysentery is an invasive disease of the colon in humans and higher primates and is transmitted via the fecal-oral route. The disease is associated with poor hygiene, overcrowding, and stress, and hence, the majority of cases occur in regions of developing countries that have suboptimal sanitation. However, outbreaks have also been associated with war zones and mental institutions in developed countries. The causative agents are various species of Shigella and enteroinvasive strains of Escherichia coli. The current pandemic of bacillary dysentery caused by Shigella dysenteriae serotype 1 is characterized by high fatality rates * Corresponding author. t This paper is dedicated to the memory of J.-P. Lecocq, acclaimed scientist and friend, who is sorely missed.

Inhibitors of Intravesicular Acidification Protect Against Shiga Toxin in a pH‐Independent Manner

“…The recombinant protein was expressed by inducing cultures of transformed E. coli strain BL21 with 1 m m isopropyl β‐ d ‐thiogalactoside for 3 h. Bacteria were harvested by 15‐min centrifugation at 6000 × g , before extracting the periplasmic fraction by osmotic shock treatment as previously described . The protein was purified by ion exchange chromatography using a MonoQ column and gel filtration using a Superdex 200 column, before inspection by SDS–PAGE.…”

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

“…We constructed two versions of the b-lactamase promoter (blaP) using synthetic oligonucleotides (Figure 8). The ®rst, blaP 1 , was adapted from Su et al (1992), and contained the ±35 and ±10 promoter signals, a ribosome-binding site, an EcoRI site and 5¢ overhangs that allowed cloning into BglII and NdeI sites. These oligonucleotides were cloned into the BglII±NdeI sites of pBEF198, which replaced the T7 promoter with the blaP promoter.…”

“…The ±35 and ±10 promoter signals, the start of transcription (+1) and the ribosome-binding site (RBS) are indicated. The sequence of blaP 1 was adapted from Su et al (1992). In blaP 2 , the ®rst base in the ±10 region was changed from G to T (arrow), and 5 bp were inserted immediately after the transcription start site to create a SacI site that was used to distinguish easily between the two promoters.…”

The A‐subunit of surface‐bound Shiga toxin stimulates clathrin‐dependent uptake of the toxin

“…The StxB containing a tandem of sulfation sites in the C-terminus (StxB-Sulf 2 ) was produced in E. coli BL21 (DE3) cells essentially as described previously [39]. Briefly, a 10 mL overnight bacterial culture grown at 37°C was inoculated in 500 mL of LB medium and further grown to an attenuance at 600 nm of 0.6.…”