Refinement of a Therapeutic Shiga Toxin–Binding Probiotic for Human Trials

Abstract

We have previously constructed a recombinant bacterium expressing a modified lipopolysaccharide (LPS) mimicking the Shiga toxin receptor, which binds toxin with high avidity. This involved cloning Neisseria galactosyl transferase genes (lgtC and lgtE) in pK184 in a derivative of Escherichia coli R1 (CWG308). Such constructs have considerable potential for prevention of disease caused by Shiga toxin-producing E. coli (STEC). However, neither the E. coli host strain nor the expression plasmid is suitable for human use, because the former is derived from a clinical isolate and the latter contains a kanamycin-resistance gene. We have constructed, as a prelude to human trials, a nonpathogenic E. coli K-12 C600 derivative with deletions in waaO and waaB, such that it has the same LPS core structure as CWG308. We also deleted the thyA gene from this strain, rendering it thymine dependent. The kanamycin-resistance gene was also deleted from pK184 and was replaced with Salmonella typhimurium thyA. Neisseria lgtCE was then cloned into this plasmid and transformed into C600 Delta waaOB Delta thyA. The plasmid was stably maintained, and the construct produced a modified LPS and neutralized Stx1 and Stx2c. Moreover, mice challenged with an otherwise fatal dose of STEC were completely protected by oral administration of the novel construct.

Bioengineered bugs expressing oligosaccharide receptor mimics: Toxin-binding probiotics for treatment and prevention of enteric infections

“…15 This system also uses a host strain derived from E. coli K-12, which was subjected to comprehensive human safety trials in the early 1980s. First, we deleted both the waaO and waaB genes from the E. coli K-12 strain C600, such that it has the same LPS core structure as CWG308.…”

“…Moreover, like the prototype Gb 3 mimic, it fully protected mice against otherwise fatal challenge with highly virulent STEC strains. 15 Receptor mimic probiotics are likely to be most effective when administered early in the course of disease, preferably prophylactically. In humans, the severe Stx-mediated systemic complications of STEC disease usually occur 7-10 days after onset of gastrointestinal symptoms.…”

Protection against Shiga-Toxigenic Escherichia coli by Non-Genetically Modified Organism Receptor Mimic Bacterial Ghosts

“…Minicell-and blebshedding host strains were constructed in the E. coli C600⌬waaOB strain, which, like CWG308, has a truncated LPS core terminating in Glc (13). For the generation of minicells (14), the minB operon (consisting of the minC, minD, and minE genes) and the msbB gene were deleted using the method described by Datsenko and Wanner (15); PCR primers used for mutagenesis are listed in Table 2.…”

Pathogenesis and treatment of Shiga toxin-producing Escherichia coli infections

“…Paton et al [82,83] have constructed a recombinant bacterium which displays globotriose (the Stx receptor) on its surface and adsorbs and neutralizes these toxins with very high efficiency. Oral administration of the live bacterium, or formaldehyde killed bacteria given three times per day, completely protected mice from challenge with virulent STEC.…”