Co-expression of a Chimeric Protease Inhibitor Secreted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation.

Abstract

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anti-cancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

Tryptophanase Expressed by Salmonella Halts Breast Cancer Cell Growth In Vitro and Inhibits Production of Immunosuppressive Kynurenine

“…This approach may synergize with epidermal growth factor receptor (EGFR)-targeted toxins [ 30 ], which have been shown to kill the MDA-MB-468 cell line that expresses the EGFR. This effect was shown to be further enhanced by the co-expression of a protease inhibitor [ 48 ]. Because amino acid degrading enzymes have also been coupled with the autophagy inhibitor chloroquine that is already known to enhance Salmonella therapy [ 49 ], exploring these therapeutic properties in combination is a promising avenue for in vivo studies.…”