Whole Genome Sequencing and Biological Characteristics of Two Strains of Porcine Escherichia coli Isolated from Saba Pigs.

Escherichia coli (E. coli) is an important pathogen that causes diarrhea and death in piglets. In this work, whole genome sequencing of two E. coli strains (ZB-1, ZWW-1) isolated from Saba pigs. And focus on the relationship between drug resistance, pathogenic phenotype and genotype of the two strains. This study analyzed the drug susceptibility of the two strains. The LD50 values, tissue bacterial load and intestinal pathological changes in mice infected with the two strains. The differences in gene functions such as drug resistance, virulence, and unique genes between the two strains, as well as the genetic evolutionary relationship of housekeeping genes were analyzed. The results showed that the two strains had the same resistance phenotype to most drugs. The LD50 value, tissue load, and pathological changes in mice infected with strain ZB-1 revealed that this strain was more virulent and pathogenic than strain ZWW-1. In addition, the housekeeping genes contained in the two strains are in the same large branch as E. coli of different species, and the genetic evolution is stable. All of them carry EPEC-type strain-specific virulence genes escV and ent, indicating that they are all new members of EPEC-type strains. This study laid the foundation for understanding the genetic background and biological characteristics of E. coli from Saba pigs.

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains.

To construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

High pathogenicity island is associated with enhanced autophagy in pathogenic Escherichia coli HPI - infected macrophages.

High pathogenicity island (HPI), which is widely distributed in Escherichia coli (E. coli), can enhance the pathogenicity of E. coli. Thus the HPI positive E. coli could pose a threat to human and animal health. It remains to be elucidated how HPI affects the virulence of pathogenic E. coli. Autophagy is an important mechanism to maintain cellular homeostasis and an innate immunity responses of organisms against pathogens. The interaction between pathogenic E. coli possessing HPI (E. coli HPI) and host autophagy system has not been reported. In this study, it was demonstrated that pathogenic E. coli induced autophagy in 3D4/21 macrophages and HPI was associated with enhanced autophagy through transmission electron microscopy, immunofluorescence and real-time PCR. The PI3K/Akt/mTOR pathway is an important negative regulatory pathway for autophagy. Through detecting the expression of key genes of PI3K/Akt/mTOR pathway, it was speculated that HPI enhanced the inhibition of the signaling pathway stimulated by pathogenic E. coli. Furthermore, HPI inhibited the secretion of IFN-γ, while the presence of HPI did not significantly affect the secretion of IL-1ß. This work is the first attempt to explore the interplay between HPI carried by pathogenic E. coli and host cell autophagy. The findings might enable better understanding of the contribution of HPI to pathogenicity.

Molecular cloning and expression analysis of the gene encoding proline dehydrogenase from Jatropha curcas L.

Proline dehydrogenase (ProDH) (EC 1.5.99.8) is a key enzyme in the catabolism of proline. The enzyme JcProDH and its complementary DNA (cDNA) were isolated from Jatropha curcas L., an important woody oil plant used as a raw material for biodiesels. It has been classified as a member of the Pro_dh superfamily based on multiple sequence alignment, phylogenetic characterization, and its role in proline catabolism. Its cDNA is 1674 bp in length with a complete open reading frame of 1485 bp, which encodes a polypeptide chain of 494 amino acids with a predicted molecular mass of 54 kD and a pI of 8.27. Phylogenetic analysis indicated that JcProDH showed high similarity with ProDH from other plants. Reverse transcription PCR (RT-PCR) analysis revealed that JcProDH was especially abundant in the seeds and flowers but scarcely present in the stems, roots, and leaves. In addition, the expression of JcProDH increased in leaves experiencing environmental stress such as cold (5 °C), heat (42 °C), salt (300 mM), and drought (30 % PEG6000). The JcProDH protein was successfully expressed in the yeast strain INVSc1 and showed high enzyme activity in proline catabolism. This result confirmed that the JcProDH gene negatively participated in the stress response.