Genome analysis and in vivo virulence of porcine extraintestinal pathogenic Escherichia coli strain PCN033

Background

Strains of extraintestinal pathogenic Escherichia coli (ExPEC) can invade and colonize extraintestinal sites and cause a wide range of infections. Genomic analysis of ExPEC has mainly focused on isolates of human and avian origins, with porcine ExPEC isolates yet to be sequenced. To better understand the genomic attributes underlying the pathogenicity of porcine ExPEC, we isolated two E. coli strains PCN033 and PCN061 from pigs, assessed their in vivo virulence, and completed and compared their genomes.

Results

Animal experiments demonstrated that strain PCN033, but not PCN061, was pathogenic in a pig model. The chromosome of PCN033 was 384 kb larger than that of PCN061. Among the PCN033-specific sequences, genes encoding adhesins, unique lipopolysaccharide, unique capsular polysaccharide, iron acquisition and transport systems, and metabolism were identified. Additionally, a large plasmid PCN033p3 harboring many typical ExPEC virulence factors was identified in PCN033. Based on the genetic variation between PCN033 and PCN061, corresponding phenotypic differences in flagellum-dependent swarming motility and metabolism were verified. Furthermore, the comparative genomic analyses showed that the PCN033 genome shared many similarities with genomic sequences of human ExPEC strains. Additionally, comparison of PCN033 genome with other nine characteristic E. coli genomes revealed 425 PCN033-special coding sequences. Genes of this subset included those encoding type I restriction-modification (R-M) system, type VI secretion system (T6SS) and membrane-associated proteins.

Conclusions

The genetic and phenotypic differences between PCN033 and PCN061 could partially explain their differences in virulence, and also provide insight towards the molecular mechanisms of porcine ExPEC infections. Additionally, the similarities between the genomes of PCN033 and human ExPEC strains suggest that some connections between porcine and human ExPEC strains exist. The first completed genomic sequence for porcine ExPEC and the genomic differences identified by comparative analyses provide a baseline understanding of porcine ExPEC genetics and lay the foundation for their further study.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1890-9) contains supplementary material, which is available to authorized users.     

Characterization and identification of Sox2+ radial glia cells derived from rat embryonic cerebral cortex

During the central nervous system (CNS) development, radial glia cells (RGCs) play at least two essential roles, they contribute to neuronal production and the subsequent guidance of neuronal migration, whereas its precise distribution and contribution to cerebral cortex remains less understood. In this research, we used Vimentin as an astroglial marker and Sox2 as a neural progenitor marker to identify and investigate RGCs in rat cerebral cortex at embryonic day (E) 16.5. We found that the Sox2+ progenitor cells localized in the germinal zone (GZ) of E16.5 cerebral cortex, ~95% Sox2+ cells co-localized with Vimentin+ or Nestin+ radial processes which extended to the pial surface across the cortical plate (CP). In vitro, we obtained RG-like cells from E16.5 cerebral cortex on adherent conditions, these Sox2+ Radial glia (RG)-like cells shared some properties with RGCs in vivo, and these Sox2+ RG-like cells could differentiate into astrocytes, oligodendrocytes and presented the radial glia—neuron lineage differentiation ability. Taken together, we identified and investigated some characterizations and properties of Sox2+ RGCs derived from E16.5 cerebral cortex, we suggested that the embryonic Sox2+ progenitor cells which located in the cortical GZ were mainly composed of Sox2+ RGCs, and the cortex-derived Sox2+ RG-like cells displayed the radial glia—neuron lineage differentiation ability as neuronal progenitors in vitro.     

Structural and functional insights into polymorphic enzymes of cytochrome P450 2C8

The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure–function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure–function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.     

Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates

Successful development of an ultrasensitive and highly specific electrochemical aptasensor for thrombin based on amplification of aptamer-gold nanoparticles-horseradish peroxidase (aptamer-AuNPs-HRP) conjugates was reported. In this electrochemical protocol, aptamer1 (Apt1) was immobilized on core/shell Fe(3)O(4)/Au magnetic nanoparticles (AuMNPs) and served as capture probe. Aptamer2 (Apt2) was dual labeled with AuNPs and HRP and used as detection probe. In the presence of thrombin, the sandwich format of AuMNPs-Apt1/thrombin/Apt2-AuNPs-HRP was fabricated. Remarkable signal amplification was realized by taking the advantage of AuNPs and catalytic reactions of HRP. Other proteins, such as human serum albumin, lysozyme, fibrinogen, and IgG did not show significant interference with the assay for thrombin. Linear response to thrombin concentration in the range of 0.1-60 pM and lower detection limit down to 30 fM (S/N=3) was obtained with the proposed method. This electrochemical aptasensor is simple, rapid (the whole detection period for a thrombin sample is less than 35 min), sensitive and highly specific, it shows promising potential in protein detection and disease diagnosis.     

Conditions optimized for the preparation of single-stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer

The generation of DNA aptamer by Systematic Evolution of Ligands by Exponential Enrichment requires a good method of ssDNA generation. There are various methods developed to generate ssDNA such as streptavidin-biotin based separation techniques, asymmetric PCR and strand separation of the PCR product containing primer with a terminator and an extension of 20 nucleotides on denaturing urea-polyacrylamide gel. In the present investigation, we have shown the possible improvements for the regular lambda nuclease digestion under optimized conditions. Optimization of the PCR cycles, time course studies on lambda nuclease digestion and purification of the ssDNA from the lambda exonuclease digestion mixture was found to be able to recover ssDNA amounting up to 39.19 ± 2.48 % of the starting amount of dsDNA. These strategies can be applied to the techniques involving essential usage of ssDNA.     

Efficient gene disruption in Saccharomyces cerevisiae using marker cassettes with long homologous arms prepared by the restriction-free cloning strategy

Here we report an improved method for targeted gene disruption with high efficiency in S. cerevisiae, where the selection markers with long homologous arms are defined by the choice of the primer binding sites at the target locus and the disruption cassettes are constructed by restriction-free (RF) cloning strategy. Three genes, SAM1, IDH1 and IDH2, were disrupted with this method and the disruption efficiencies of SAM1 was improved several folds with much lower false-positive rates compared to the conventional one-step PCR-based gene disruption method. This approach for gene disruption cassettes construction with long flanking homologous arms may be readily applicable to facilitate targeted gene disruption in other non-conventional yeasts and fungi.     

Nucleosome structural studies

Chromatin plays a fundamental role in eukaryotic genomic regulation, and the increasing awareness of the importance of epigenetic processes in human health and disease emphasizes the need for understanding the structure and function of the nucleosome. Recent advances in chromatin structural studies, including the first structures of nucleosomes containing the Widom 601 sequence and the structure of a chromatin protein-nucleosome assembly, have provided new insight into stretching of nucleosomal DNA, nucleosome positioning, binding of metal ions, drugs and therapeutic candidates to nucleosomes, and nucleosome recognition by nuclear proteins. These discoveries ensure promising future prospects for unravelling structural attributes of chromatin.