Small Heat-Shock Proteins,IbpAB, Protect Non-Pathogenic Escherichia coli from Killing by Macrophage-Derived Reactive Oxygen Species

Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn’s disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox^-/-) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox^-/- macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains.     

Distribution and function of prophage phiRv1 and phiRv2 among Mycobacterium tuberculosis complex

Mycobacterium tuberculosis complex (MTBC) is notorious for causing diseases, such as tuberculosis. Tuberculosis caused by M. tuberculosis remains a global public health concern. Two prophages, phiRv1 and phiRv2, can be found among most MTBC genomes. However, no precise functions have been assigned for the two prophages. In this paper, to find out the function of these two prophages, the distribution and function of phiRv1 and phiRv2 in MTBC genomes were analyzed from multiple omics data. We found that complex insertion, deletion, and reorganization appeared on the locus of two prophages in MTBC genomes; some genes of the two prophages can be translated and are functional from proteomic data; the expression of other prophage genes, such as Rv1577c, Rv2650c, Rv2652c, Rv2659c, and Rv2658c, can vary with environmental stresses and might enhance the fitness of MTBC. These data will facilitate our in-depth understanding of their function.     

Analysis and assessment of heavy metals pollution in soils around a Pb and Zn smelter in Baoji City,Northwest China

Soil heavy metal pollution from mining activities is potentially harmful to human health through the food chain. In this study, a total of 43 soil samples were collected from a depth of 0–20 cm from fields close to a Pb and Zn smelter. The samples were used to: 1) analyze the pollution level of heavy metals (Pb, Zn, Cr, and Cu) and spatial distribution pattern; 2) evaluate the degree of accumulation and enrichment, potential ecological risk, and human health risk; and 3) perform source apportionment in Fengxiang County, Shaanxi Province of China. The results showed that the concentration ranged from 43.67 to 189.55, 131.43 to 239.53, 74.77 to 112.25, and 24.69 to 37.71 mg·kg^?1 for Pb, Zn, Cr, and Cu, respectively, and the mean concentration for Pb, Zn, Cr, and Cu was 129.46, 192.85, 91.98 and 31.67 mg·kg^?1, respectively. The concentrations were greater than the Shaanxi Province background values, while they were lower than the second-level limits of Environmental Quality Standard for Soils of China (EQSS). The spatial distribution of heavy metal contents showed a banded in soil except Cu. The spatial distribution pattern and pollution assessment indexes (I_geo, EF) indicated that the investigated metals had been accumulated in the study areas, and implied significant influences from anthropogenic activities, local meteorological situation, and soil properties. The ecological risk assessment showed that the risks were relatively low (RI<150). Compared with the exposure risk for adults, that for children was significantly greater. The ingestion of heavy metals in the soils by humans was the main exposure pathway compared with the dermal exposure. There may be a risk of noncarcinogenic adverse health effects (HQ < 1, 0.377 ≤ HI≤1.553) on children, but the adults were unlikely to experience obvious adverse health effects (HQ < 1, HI < 1). The carcinogenic risk of Cr for adults and children was at an unacceptable level. The carcinogenic and noncarcinogenic risks were in the order of children > adults. The correlation analysis showed that Pb, Cr, and Cu have identical anthropogenic and natural sources, while Zn has another identical source. This study could provide a basis for the sustainable management of this region by reducing metal inputs and to protect soils from long-term heavy metal accumulation.     

Immunoassay employing surface-enhanced Raman spectroscopy

Surface-enhanced Raman scattering (SERS) was used to measure binding between biomolecules with mutual affinity, including antigen-antibody interactions. The conjugation of nitro groups onto bovine serum albumin enhanced their specific SERS activity 10(4)-fold. A dye, 2-[4'-hydroxyphenylazo]benzoic acid (HABA), with a major absorption at the Raman excitation frequency, demonstrated surface-enhanced resonance Raman scattering (SERRS) when captured from solution by avidin-coated silver films. Individual peak intensities showed a logarithmic relationship to the HABA concentration in solution over the range 10(-8) to 10(-5) M. Another resonance dye, p-dimethylaminoazobenzene (DAB) was covalently attached to an antibody directed against human thyroid stimulating hormone (TSH), without loss of antibody activity. The resultant conjugate was used in a sandwich immunoassay for TSH antigen: silver surfaces coated with anti-TSH antibody captured TSH antigen which in turn captured the DAB-anti-TSH antibody conjugate. A linear relationship was observed between the intensity of the resultant SERRS signals and the TSH antigen concentration over a range of from 4 to 60 microIU/ml. These results demonstrate the potential utility of the SERRS effect as a readout in a one-step, no wash immunoassay system.     

Processing of lipid-modified prolipoprotein requires energy and sec gene products in vivo.

The kinetics of processing of glyceride-modified prolipoprotein that accumulated in globomycin-treated Escherichia coli has been found to be affected by sec mutations, i.e., secA, secE, secY, secD, and secF, and by metabolic poisons which affect proton motive force (PMF). The effect of sec mutations on processing of glyceride-modified prolipoprotein in vivo was not due to a secondary effect on PMF. Neither a secF mutation nor metabolic poisons affected the processing of previously accumulated proOmpA protein in vivo, suggesting that the requirements for functional sec gene products and PMF are specific to the processing of lipoprotein precursors by signal peptidase II.     

Principles and pitfalls in designing site-directed peptide ligands

An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallo-graphically refined structures of 14 peptide–protein complexes at 2.7 Å resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change inconformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program anend-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligandswere diffused into preexisting crystals. In sucha large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints. © 1993 Wiley-Liss, Inc.