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The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro cultured cancer cell lines. Gene encoding PE24 was isolated from P. aeruginosa isolates, cloned into pET22b( +) plasmid, and expressed in E. coli BL21 (DE3) under IPTG induction. Genetic recombination was confirmed by colony PCR, the appearance of insert post digestion of engineered construct, and protein electrophoresis using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The chemical compound NBAG has been used to confirm PE24 extract ADP-ribosyl transferase action through UV spectroscopy, FTIR, c 13 -NMR, and HPLC before and after low-dose gamma irradiation (5, 10, 15, 24 Gy). The cytotoxicity of PE24 extract alone and in combination with paclitaxel and low-dose gamma radiation (both 5 Gy and one shot 24 Gy) was assessed on adherent cell lines HEPG2, MCF-7, A375, OEC, and Kasumi-1 cell suspension. Expressed PE24 moiety ADP-ribosylated NBAG as revealed by structural changes depicted by FTIR and NMR, and the surge of new peaks at different retention times from NBAG in HPLC chromatograms. Irradiating recombinant PE24 moiety was associated with a reduction in ADP-ribosylating activity. The PE24 extract IC50 values were < 10 μg/ml with an acceptable R 2 value on cancer cell lines and acceptable cell viability at 10 μg/ml on normal OEC. Overall, the synergistic effects were observed upon combining PE24 extract with low-dose paclitaxel demonstrated by the reduction in IC50 whereas antagonistic effects and a rise in IC50 values were recorded after irradiation by low-dose gamma rays. KEY POINTS: • Recombinant PE24 moiety was successfully expressed and biochemically analyzed. • Low-dose gamma radiation and metal ions decreased the recombinant PE24 cytotoxic activity. • Synergism was observed upon combining recombinant PE24 with low-dose paclitaxel., (© 2023. The Author(s).)

Colibactin and cytotoxic necrotizing factor 1 (Cnf 1) are cyclomodulins secreted by uropathogenic E. coli. In this study, uropathogenic E. coli expressing colibactin and Cnf 1 was exposed to antibiotics subMICs and gamma radiation to investigate their effects on its cytotoxicity and expression of colibactin. The test isolate was exposed to three subMIC levels of levofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole and ceftriaxone and irradiated with gamma rays at 10 and 24.4 Gy. The cytotoxicity for either antibiotic or gamma rays treated cultures was measured using MTT assay and the expression of colibactin encoding genes was determined by RT-PCR. Treatment with fluoroquinolones nearly abolished the cytotoxicity of E. coli isolate and significantly downregulated clbA gene expression at the tested subMICs (P ≤ 0.05) while trimethoprim/sulfamethoxazole treated cultures exerted significant downregulation of clbA and clbQ genes at 0.5 MIC only (P ≤ 0.05). Ceftriaxone treated cultured exhibited reduction in the cytotoxicity and insignificant effects on expression of clbA, clbQ and clbM genes. On contrast, significant upregulation in the expression of clbA and clbQ genes was observed in irradiated cultures (P ≤ 0.05). Fluoroquinolones reduced both the cytotoxicity of UPEC isolate and colibactin expression at different subMICs while ceftriaxone at subMICs failed to suppress the expression of genotoxin, colibactin, giving an insight to the risks associated upon their choice for UTI treatment. Colibactin expression was enhanced by gamma irradiation at doses resembling these received during pelvic radiotherapy which might contribute to post-radiotherapy complications.