Tumor-associated macrophages (TAMs) promote tumor development and metastasis and are categorized into M1-like macrophages, suppressing tumor cells, and M2-like macrophages. M2-like macrophages, occupying a major role in TAMs, can be repolarized into anti-tumoral phenotypes. In this study, outer membrane vesicles (OMVs) secreted by Escherichia coli Nissle 1917 carry perhexiline (OMV@Perhx) to explore the influence of OMVs and perhexiline on TAM repolarization. OMV@Perhx was internalized by macrophages and regulated the phenotype of TAMs from M2-like to M1-like efficiently to increase the level of tumor suppressor accordingly. Re-polarized macrophages promoted apoptosis and inhibited the mobility of tumor, cells including invasion and migration. The results indicate that OMVs improve the efficacy of perhexiline and also represent a promising natural immunomodulator. Combining OMVs with perhexiline treatments shows powerfully synergistic anti-tumor effects through co-culturing with re-polarized macrophages. This work is promising to exploit the extensive applications of OMVs and chemical drugs, therefore developing a meaningful drug carrier and immunomodulator as well as expanding the purposes of traditional chemical drugs.

Probiotic Escherichia coli Nissle 1917 (EcN) possesses excellent antibacterial effects on pathogenic enterobacteria. The microcins MccM and MccH47 produced in EcN played critical roles, but they are understudied and poorly characterized, and the individual antibacterial mechanisms are still unclear. In this study, three EcN mutants (ΔmcmA, ΔmchB, and ΔmcmAΔmchB) were constructed and compared with wild-type EcN (EcN wt) to test for inhibitory effects on the growth of Escherichia coli O157: H7, Salmonella enterica (SE), and Salmonella typhimurium (ST). The antibacterial effects on O157: H7 were not affected by the knockout of mcmA (MccM) and mchB (MccH47) in EcN. However, the antibacterial effect on Salmonella declined sharply in EcN mutants ΔmcmA. The overexpressed mcmA gene in EcN::mcmA showed more efficient antibacterial activity on Salmonella than that of EcN wt. Furthermore, the EcN::mcmA strain significantly reduced the abilities of adhesion and invasion of Salmonella to intestinal epithelial cells, decreasing the invasion ability of ST by 56.31% (62.57 times more than that of EcN wt) while reducing the adhesion ability of ST by 50.14% (2.41 times more than that of EcN wt). In addition, the supernatant of EcN::mcmA culture significantly decreased the mRNA expression and secretion of IL-1β, TNF-α, and IL-6 on macrophages induced by LPS. The EcN::mcmA strain generated twice as much orange halo as EcN wt by CAS agar diffusion assay by producing more siderophores. MccM was more closely related to the activity of EcN against Salmonella, and MccM-overproducing EcN inhibited Salmonella growth by producing more siderophores-MccM to compete for iron, which was critical to pathogen growth. Based on the above, EcN::mcmA can be developed as engineered probiotics to fight against pathogenic enterobacteria colonization in the gut.

Synthetic nanocarriers are a promising therapeutic delivery strategy. However, these systems are often hampered by inherent disadvantages such as strong biotoxicity and poor biocompatibility. To overcome these issues, biological carriers with commonly used chemotherapy drugs have been developed. In this work, engineered bacterial ghosts (BGs) originated from probiotic Escherichia coli Nissle 1917 (EcN) were devised to specifically target acidic extracellular environments of tumor tissue. To improve the production efficiency and safety, a novel lysis protein E from phage α3 was applied to produce EcN BGs under high growth densities in high quality. In addition, the acidity-triggered rational membrane (ATRAM) peptides were displayed in EcN BGs to facilitate specific cancer cell internalization within the acidic tumor microenvironment before drug release. In conclusion, the engineered EcN BGs offer a promising means for bionic bacteria construction for hepatocellular carcinoma therapy.