Rhodotorula glutinis as a living cell liposome to deliver polypeptide drugs in vivo.

The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living cell liposomes to carry polypeptide drugs may be an ideal polypeptide oral drug delivery system. GM4-ΔTS was constructed by LFH-PCR from Rhodotorula glutinis GM4, which was screened and preserved in our laboratory, and then transferred into choline-phosphate cytidylyltransferase (CCT), which is a rate-limiting enzyme for lecithin synthesis. The results showed that the CCT gene was highly expressed in the GM4-ΔTS strain and could significantly increase fatty acid and lecithin contents in GM4-ΔTS-PGK1-CCT. Moreover, insulin, H22-LP, and α-MSH were successfully introduced into cells in vitro, and the strain no longer proliferated in vivo, for safe and controllable polypeptide drug delivery. In vivo, normal mice were intragastrically administered with recombinant strains carrying insulin and α-MSH, and different levels of polypeptide drugs were detected in serum and tissue, respectively. Then, recombinant strains carrying insulin were administered to type II diabetes mellitus mice. The results showed that the strains could effectively reduce blood glucose levels in mice, which indicated that the recombinant strains could carry insulin into the body, and the drug effect was remarkable. Therefore, recombinant GM4-ΔTS-PGK1-CCT strains were successfully used as living cell liposomes to carry insulin, H22-LP, and α-MSH peptides into the body for the first time; additionally, these strains have enhanced safety, controllability, and efficacy.

Rhodosporidium toruloides sir2-like genes remodelled the mitochondrial network to improve the phenotypes of ageing cells.

It is known that mitochondria are associated with the ageing process, and the eukaryotic sir2 family of genes significantly affects cellular lifespan. The mammalian sir2 family affects mitochondrial function by regulating targets in different pathways, including oxidative stress, oxidative phosphorylation, and mitochondrial biosynthesis. This study reports that Rt-sirtuin2 and Rt-sirtuin4 genes transfections significantly impacted the lifespan of Rhodosporidium toruloides, and they can significantly improve cellular responses to H2O2 treatment, which induces cell senescence, and restore mitochondrial function. The Rt-sirtuin2 and Rt-sirtuin4 genes increase the expression of the mitochondria-associated proteins Mfn1, Mfn2, and Drp1 and the autophagy-associated proteins LC3-II, LC3-I, Beclin-1 and Parkin and reconstitute mitochondrial networks. Overall, the phenotypic reversal of senescent cells is achieved by regulating mitochondrial viability and mitochondrial autophagy. In vivo experiments with animals also confirmed the improvement of various ageing indexes by the Rt-sirtuin2 and Rt-sirtuin4 genes. Strategies for remodelling mitochondria and improving mitochondrial quality and function can reverse the state of human cells from an ageing phenotype to an active metabolic phenotype. The R. toruloides Sir2 genes can be used to prevent and treat diseases of ageing or mitochondrial dysfunction.

Enterococcus faecalis sir2-like gene enhances aerobic metabolism of themselves and mitochondrial respiration of mammal cells to bring about improving metabolic syndrome through the PGC-1α pathway.

Recent studies showed that probiotics could improve metabolic syndrome, making the identification of factors affecting metabolic control more important than ever. The mammalian sirtuin protein family has received much attention for its regulatory role, especially in various mitochondrial ATP, glucose, and lipid metabolic pathways. However, compared with the mammalian sirtuin protein family, the function of prokaryotic sir2 protein is much less known. We studied the effects of probiotics sir2 protein on cell energy metabolize pathway, which showed that deletion of Enterococcus faecalis sir2 inhibited the aerobic oxidation of bacteria and increased the bacterial fermentation. The study of EF-sir2 (sir2 protein of E. faecalis) role of molecular targets demonstrated that deacetylation of EF-sir2 was via Rho upregulating in E. faecalis. When transfected into HEK293T cells, EF-sir2 could significantly facilitate aerobic oxidation of glucose, enhance the respiration to generate more ATP, and cause upregulation of NRF1 target gene. Then, we found EF-sir2 could increase activity of PGC-1α by deacetylation and PGC-1α inhibition decreased the expression of NRF1 target gene. Finally, we demonstrated that EF-sir2 could significantly improve the metabolic index of mammalian cells through insulin resistanced model in vitro and metabolic syndrome rat model in vivo. Our results first revealed that prokaryotic sir2 genes affect the molecular mechanism of cellular metabolism and the regulatory of cell homeostasis in prokaryotic and mammalian cells, suggesting that EF-sir2 has a positive regulatory effect on metabolic disturbance and may be used for the prevention and treatment of pathological processes related to metabolic syndrome.

Oral Bifidobacterium longum expressing GLP-2 improves nutrient assimilation and nutritional homeostasis in mice.

Bifidobacterium has been developed for the oral delivery of peptides and has the added beneficial effect on our bodies through its probiotic properties. Here, we utilize Bifidobacterium as a delivery system to orally deliver Glucagon like peptide-2 (GLP-2). We constructed vector derived from pET-31b(+) to construct a Bifidobacterium longum expressing GLP-2. We then determined the bioactivity of recombinant Bifidobacterium in Caco-2 cells. Finally, we quantified newly synthesized ApoB48 and chylomicron production in mice infused with exogenous GLP-2 or Bifidobacterium expressing GLP-2. Results based on secretion of the triglyceride (TG)-rich lipoprotein (TRL)-ApoB48 and secretion of chylomicron revealed that recombinant Bifidobacterium was efficient in treating intestinal dysfunction,suggesting an alternative way to use Bifidobacterium as a delivery system to deliver GLP-2 for gastrointestinal nutrition coordination.