PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage Aa⌽23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.
E. coli is a target for polyclonal Th1 responses in healthy individuals. The impairment of these responses in CD and AS patients might be due to recruitment of enterobacteria-specific Th1 cells to the gut or might reflect inadequate priming of adaptive immune response.
Background: Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies.
The normal intestinal flora is required for the development of intestinal inflammation in animal models of inflammatory bowel disease (IBD). In humans, several studies indicated a potential association of Escherichia coli (E. coli) with IBD. In addition, we have shown that T-cell clones of IBD patients cross react toward different enteric bacterial species and thus likely respond to conserved bacterial antigens. Therefore, we hypothesized that highly conserved E. coli proteins might be a reasonable candidate to screen for abnormal T-cell responses in IBD. We used high-throughput techniques for cloning, expression, and purification under native conditions of a set of 271 conserved proteins of E. coli, of which 196 were used for whole blood stimulations to assess peripheral T helper (T(H))-cell responses. In addition, because of the association of an adherent-invasive E. coli with Crohn's disease (CD), we included 13 pathogenicity factors of E. coli in the study. We observed that pools of these conserved E. coli proteins less frequently induced interferon-gamma (IFNgamma) production in peripheral T(H) cells in patients with CD and ankylosing spondylitis (AS) compared with healthy controls. In addition, lower percentage of patients with CD and AS responded toward single proteins. The reason for the decreased frequency of an in vitro T(H)-cell IFNgamma response toward E. coli proteins in peripheral blood of CD and AS patients, e.g., increased suppression needs to be clarified.
The systematic structural analysis of many target proteins involves generating expression clones in high throughput. This requires robust laboratory procedures and benefits from laboratory automation and data management systems. This chapter gives an overview of the Protein Structure Factory, a structural genomics project focusing on human proteins, and presents the authors' method for cloning bacterial expression clones with the restriction enzymes BamHI and NotI and compatible enzymes. PCR amplification, product purification and digestion and vector ligation were adapted to the 96-well microtiter plate format.
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