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.     

Diffusion of extracellular K+ can synchronize bursting oscillations in a model islet of Langerhans.

Electrical bursting oscillations of mammalian pancreatic beta-cells are synchronous among cells within an islet. While electrical coupling among cells via gap junctions has been demonstrated, its extent and topology are unclear. The beta-cells also share an extracellular compartment in which oscillations of K+ concentration have been measured (Perez-Armendariz and Atwater, 1985). These oscillations (1-2 mM) are synchronous with the burst pattern, and apparently are caused by the oscillating voltage-dependent membrane currents: Extracellular K+ concentration (Ke) rises during the depolarized active (spiking) phase and falls during the hyperpolarized silent phase. Because raising Ke depolarizes the cell membrane by increasing the potassium reversal potential (VK), any cell in the active phase should recruit nonspiking cells into the active phase. The opposite is predicted for the silent phase. This positive feedback system might couple the cells' electrical activity and synchronize bursting. We have explored this possibility using a theoretical model for bursting of beta-cells (Sherman et al., 1988) and K+ diffusion in the extracellular space of an islet. Computer simulations demonstrate that the bursts synchronize very quickly (within one burst) without gap junctional coupling among the cells. The shape and amplitude of computed Ke oscillations resemble those seen in experiments for certain parameter ranges. The model cells synchronize with exterior cells leading, though incorporating heterogeneous cell properties can allow interior cells to lead. The model islet can also be forced to oscillate at both faster and slower frequencies using periodic pulses of higher K+ in the medium surrounding the islet. Phase plane analysis was used to understand the synchronization mechanism. The results of our model suggest that diffusion of extracellular K+ may contribute to coupling and synchronization of electrical oscillations in beta-cells within an islet.     

Mechanistic studies on aromatase and related C---C bond cleaving P-450 enzymes

Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:

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It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed Fe^III---O---O^. species is converted into Fe^III---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe^V = O ↔ (+^.)Fe^IV = O ↔ Fe^IV---O^.. One of these, Fe^IV---O^. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce Fe^IV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe^IV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the Fe^III---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,

“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”.     

Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants.

Poliovirus RNA species with nucleotides 564 to 571 deleted or with a secondary structure domain (positions 564 to 629) replaced by a shorter irregular oligonucleotide have been engineered previously; these RNAs have been considered quasi-infectious (yielding a single late revertant plaque) and dead, respectively (E. Pilipenko, A. Gmyl, Y. Svitkin, S. Maslova, A. Sinyakov, and V. Agol, Cell 68:119-131, 1992). By using large amounts of these RNAs for transfections, revertant clones with a great variety of genetic changes (point mutations, insertions of foreign sequences, short or extended deletions) were isolated. The pattern of these changes supported the notion that an appropriately spaced oligopyrimidine-AUG tandem is important for efficient poliovirus RNA translation. Structural features within and around this tandem modulated the initiation efficiency. The functional and genetic plasticities of the poliovirus genome are briefly discussed.