Efficient Cytotoxicity of Recombinant Azurin in Escherichia coli Nissle 1917-Derived Minicells against Colon Cancer Cells.

Compared to chemical drugs, therapeutic proteins exhibit higher specificity and activity and are generally well-tolerated by the human body. However, the limitations, such as poor stability both in vivo and in vitro as well as difficulties in penetrating cell membranes, hinder their widespread application. To overcome the challenges, a highly efficient protocol was developed and implemented for the recombinant expression of the therapeutic protein azurin and secretion into minicells derived from probiotic Escherichia coli Nissle 1917. The novel coupled production with a delivery system of therapeutic proteins based on minicells was obtained through purification to enhance protein activity, circulation characteristics, and targeting specificity. This protein drug carrier integrates the production of carrier materials and the loading of expression proteins. The drug carrier also protects the encapsulated polypeptide drugs from enzymatic or gastric acid degradation until they are released. Escherichia coli Nissle 1917-derived minicells have natural targeting to colon cancer cells, low toxicity, and can accumulate for a long time after penetrating tumor tissue. This self-produced protein drug delivery system simplified the process of protein preparation, and its inhibitory effect on different types of colon cancer cells was verified by CCK-8 cytotoxicity assay, cancer cell invasion, and migration assay. This work provided a simple method to prepare minicell drug delivery systems for protein drug production and a novel approach for the transport of recombinant protein drugs.

Bacterial Outer Membrane Vesicles Loaded with Perhexiline Suppress Tumor Development by Regulating Tumor-Associated Macrophages Repolarization in a Synergistic Way.

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.

Efficient Robust Yield Method for Preparing Bacterial Ghosts by Escherichia coli Phage ID52 Lysis Protein E.

Bacterial ghosts (BGs) are nonliving empty bacterial shells without cytoplasm retaining original morphology and identical antigenicity of natural bacteria, making them high potential and promising vaccine candidates and delivery vehicles. However, the low yield of commonly used BGs preparation methods limits its mass production and widely application. In order to improve BGs production, E. coli phage ID52 lysis protein E was introduced to generating BGs for the first time. Above all, we compared the lysis activity of lysis protein of E. coli phage φX174 and E. coli phage ID52 as well as the effects of promoters on the lysis activity of ID52-E, which shown that the lysis activity and BGs formation rate of protein ID52-E was significantly higher than protein φX174-E. Further, the lysis activity of ID52-E was significantly improved under the control of L-arabinose inducible promoter which initial induction OD600 reached as high as 2.0. The applicability of lysis protein ID52-E induced by L-arabinose was proved by preparing probiotic E. coli Nissle 1917 BGs and pathogenic Salmonella typhimurium BGs in mass production. This paper introduced a novel and highly efficient method for BGs preparation depending on recombinant expression of E. coli phage ID52-E under eco-friendly and reasonable price inducer L-arabinose.