Editing of a Specific Strain of Escherichia coli in the Mouse Gut Using Native Phages.

There is a lack of methodological investigation of the in situ functions of bacterial species in microecosystems. Here, we used native phages as a microbial editing tool for eliminating Escherichia coli strain MG1655 labeled with green fluorescent protein (GFP) in the mouse gut. The virulent phages (W1 and W3) possessed host specificity at both the genus and species levels, resulting in an 8.8-log10 difference in the titer of viable bacteria after 12 h of phage treatment compared with that in the phage-free control in an in vitro test. In vivo, they reduced strain MG1655 colonizing the mouse gut at concentrations of 106 to 108 CFU g-1 to a 102 CFU g-1 level, which is almost undetectable by the plate colony-counting method. Moreover, the impact of phage treatment on the microbial community structure of the mouse gut was not significant (P > 0.05), indicating that native phages can effectively edit a target bacterium, with limited perturbation of microbial diversity and relative abundance. Therefore, we developed an engineering technique for investigation of the functions of a specific bacterium by depleting its abundance in microecosystems. IMPORTANCE This report describes a gut engineering technique for investigation of the functions of a specific bacterium. Native phages with host specificity can knock down the corresponding E. coli strain in the mouse gut with limited perturbation of microbial diversity and relative abundance, indicating that they, as a microbial editing tool, can effectively edit the abundance of a target bacterium. Such an approach is undoubtedly of interest in the context of lack of knowledge of how to methodologically study the in situ function of a specific species in a complex microecosystem.

Whole-cell biosynthesis of cytarabine by an unnecessary protein-reduced Escherichia coli that coexpresses purine and uracil phosphorylase.

Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

Bone marrow and non-bone marrow TLR4 regulates hepatic ischemia/reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is a highly coordinated process often observed during liver transplantation, liver surgery, and hemorrhagic shock. Signaling through toll-like receptor 4 (TLR4), which is widely expressed on all kinds of liver cells, appears critical in the pathogenesis of IRI. Although the role of TLR4 expressed on non-parenchymal cells (NPCs) of the liver, including Kupffer cells and neutrophils, in IRI has been widely studied, TLR4 signaling on liver sinusoidal endothelial cells (LSECs) or hepatocytes in the process of IRI, and their coordination with bone marrow derived TLR4 in the late reperfusion stage, is largely unknown. We produced TLR4 chimeric mice that received hepatic IRI, and examined the degree of liver injury and the underlying mechanisms of injury. Results indicated that mutation of TLR4 on bone-marrow or non-bone marrow derived cells reduced hepatic IRI in the late reperfusion stage via cytokine release and neutrophil infiltration, while non-bone marrow derived TLR4 regulated the expression of ICAM-1 on hepatocytes and LSECs, exacerbating their injury. In conclusion, both TLR4 on bone marrow derived and non-bone marrow derived cells were necessary in the process of hepatic IRI.

Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft.

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-gamma-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-gamma/CXCL9. It was reported that CXC chemokines IFN-gamma-inducible protein 10/CXCL10 and monokine induced by IFN-gamma/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 +/- 2.4 days) compared with physiological saline treatment (7.5 +/- 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 +/- 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.