Traditional cancer therapies, such as surgery treatment, radiotherapy, and chemotherapy, often fail to completely eliminate tumor cells in an anaerobic microenvironment of tumor regions. In contrast to these traditional cancer therapies, the use of targeted delivery vectors to deliver anticancer genes or antitumor drugs to hypoxic areas in tumors is the most clinically promising cancer treatment with rapid development in recent years. In this study, E.coli Nissle 1917 (EcN), an intestinal probiotic, was utilized as a targeted transport vector to deliver p53 and Tum-5 protein to tumor hypoxic regions. The tumor-targeting characteristics of EcN were investigated using luciferase Lux CDABE operon, and the results demonstrated that EcN could specifically accumulate in the solid tumor areas of SMMC-7721 tumor-bearing BALB/c nude mice. The Tum 5-p53 bifunctional proteins were initially constructed and then delivered to solid tumor regions by using the targeted transporter EcN for cancer therapy. The antitumor effect and safety of three engineered bacteria, namely, EcN (Tum-5), EcN (p53), and EcN (Tum 5-p53), were also examined. The calculated tumor volume and tumor weight indicated that these three engineered bacteria could inhibit the growth of human hepatoma SMMC-7721 cells, and the antitumor effect of EcN (Tum 5-p53) expressing the Tum 5-p53 fusion protein was significantly better than those of EcN (Tum-5) and EcN (p53) alone. Immunofluorescence demonstrated that the expression of Ki-67, a nuclear proliferation-related protein, was inhibited in the tumor areas of the groups treated with the engineered bacteria, whereas the expression of caspase-3 was upregulated. The expression trends of Ki-67 and caspase-3 were consistent with the different antitumor efficacies of these three engineered bacteria. EcN did not elicit obvious side effects on mice. This research not only provids a foundation for tumor-targeted therapy but also contributes greatly to the development of antitumor agents and anticancer proteins. Electronic supplementary material The online version of this article (10.1186/s13036-019-0189-9) contains supplementary material, which is available to authorized users.
Probiotic-feeding continues to be a promising strategy to control the bacterial pathogens in aquaculture. A new Pediococcus pentosaceus strain (SL001) was isolated from 1000s of soil samples, which exhibited wide antimicrobial spectrum of against fish pathogens, involving Aeromonas hydrophila , Aeromonas veronii , Aeromonas sobria , Edwardsiella tarda , Lactococcus garvieae , and Plesiomonas shigelloide . The challenge test against A. hydrophila showed that the survival rate of SL001-supplemented group was significantly higher than that of control group ( P < 0.05). Moreover, SL001 could stably colonize in gut of grass carp and increased mucus-secreting goblet cells and extended intestinal villi could be observed in SL001-supplemented group ( P < 0.05). Feeding with SL001 supplemented diet could significantly enhance the growth rate ( P < 0.05) and markedly affect gut microbiota structure of grass carps, resulting in reduced potential pathogens and increased potential probiotics. Furthermore, feeding grass carps with SL001 caused the up-regulated expression of insulin-like growth factor (IGF-1 and IGF-2) and down-regulated expression of myostatin (MSTN-1 and MSTN-2) ( P < 0.05), which probably also account for the increased growth rate of SL001-fed group. Meanwhile, relative mRNA expression levels of immune-related genes in liver, spleen, and head kidney were analyzed in grass carps after feeding for 30 days with SL001 supplemented diets. In all three immune organs, the expression levels of immunoglobulin M (IgM) and complement 3 (C3) were significantly increased ( P < 0.05), whereas the interleukin-8 (IL-8) was down-regulated ( P < 0.05). Besides, whole genome sequencing revealed several probiotics properties of SL001, including organic acid synthesis, bacteriocin synthesis (coagulin), superoxide dismutase, and digestive enzymes. In conclusion, P. pentosaceus SL001 which could enhance immunity and promoter growth rate of grass carps, is prospective to be used as a dietary probiotic in freshwater fish aquaculture.
Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. pogona limit its broad application and metabolic engineering. Here, we showed that S. pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69-and 3.03fold, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.
Summary Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.
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