Cholera toxin B: one subunit with many pharmaceutical applications

Abstract

Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

Figure 1

Cholera toxin (CT) crystal structure. (A) CT (side view; Protein Data Bank [PDB] ID: 1XTC). The CTA subunit is shown in red (CTA1 in dark red and CTA2 in light red) and the CTB subunit is shown in blue; (B) CTB (top view; PDB ID: 1XTC with CTA subunit removed). Each monomer of the B subunit is show in a different color. Images were created in Accelrys Discovery Studio Visualizer 2.5.

Figure 2

CT, not rCTB, inhibits the release of TNF-α by Raw 264.7 cells stimulated with LPS. (A) Commercial non-recombinant CTB containing a trace amount of CT (CTB^+CT) significantly reduces the production of TNF-α due to LPS stimulation. Raw 264.7 cells were pretreated with 10 μg/mL rCTB (produced in E. coli [100]), CTB^+CT (Sigma‑Aldrich, St. Louis, MO, USA; catalog no. C9903), or PBS, and a final concentration of 1 μg/mL LPS was added and incubated for 24 h. TNF-α levels in cell supernatants were determined using a commercial ELISA kit (eBioscience, San Diego, CA, USA). Data represent the mean ± SEM (n = 4). a: p < 0.001, compared to PBS; b: p < 0.05, compared to PBS + LPS and rCTB + LPS (one-way ANOVA with Bonferroni multiple comparison tests); (B) Picomolar levels of CT inhibit the production of TNF-α. Raw 264.7 cells were pretreated for 2 h with varying concentration of CT, and a final concentration of 0.1 μg/mL LPS was added and incubated for 6 h. The 50% inhibitory concentration (IC₅₀) of CT was determined by non-linear regression analysis (GraphPad Prism 5.0, GraphPad Software, Inc., La Jolla, CA, USA) to be 0.49 pM. Data represent the mean ± SEM (n = 2). The TNF-α level of PBS + LPS was 4516.8 ± 791.1 pg/mL (mean ± SEM; n = 2).