Strain-Dependent Effects of Azurin-Producing Probiotic Escherichia coli on HCT116 Colorectal Carcinoma Cells

Probiotic bacteria are increasingly being explored as engineering platforms for local production of bioactive proteins, but the outcome may depend on both the recombinant protein and the bacterial strain. Azurin-producing derivatives of Escherichia coli Nissle 1917 (EcN) and E. coli M17 were generated using the same lacUV5-pelB-azu cassette and evaluated in HCT116 colorectal carcinoma cells. Western blotting confirmed azurin production in both strains, predominantly in cell-associated fractions and at lower levels in concentrated culture supernatants. A separately produced recombinant azurin preparation obtained from E. coli NiCo21(DE3) reduced HCT116 cell biomass after 48 h, with an estimated IC50 of 320 µg/mL. In bacterial co-culture, the azurin-producing derivatives showed lower mean SRB-assessed HCT116 biomass than their corresponding wild-type strains, but these differences did not reach statistical significance. In contrast, YO-PRO-1/PI analysis at a bacteria-to-cell ratio of 500 identified a significant overall increase in the total apoptosis-associated fraction in the azurin-producing groups, whereas the necrosis-associated fraction remained low. The overall response was strain-dependent: EcN showed strong baseline inhibitory activity, whereas the lower baseline effect of M17 allowed the azurin-associated contribution to be distinguished more clearly. Thus, both strains supported recombinant azurin production, but the host–cell response was strongly influenced by the bacterial background. These findings highlight strain selection as an important factor in the development and evaluation of engineered probiotic systems for bioactive protein production.