Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor

The micrographic phytopathogen Botrytis cinerea causes gray mold diseases in a large number of dicotyledonous crop plants and ornamentals. Colonization of host tissue is accompanied by rapid killing of plant cells ahead of the growing hyphen, probably caused by secretion of nonspecific phytotoxins, e.g., the sesquiterpene botrydial. Although all pathogenic strains tested so far had been shown to secrete botrydial and although the toxin causes comparable necrotic lesions as infection by the fungus, the role of botrydial in the infection process has not been elucidated so far. Here, we describe the functional characterization of bcbot1, encoding a P450 monooxygenase and provide evidence that it is involved in the botrydial pathway, i.e., it represents the first botrydial biosynthetic gene identified. We show that bcbot1 is expressed in planta and that expression in vitro and in planta is controlled by an alpha-subunit of a heterotrimeric GTP-binding protein, BCG1. Deletion of bcbot1 in three standard strains of B. cinerea shows that the effect on virulence (on several host plants) is strain-dependent; only deletion in one of the strains (T4) led to reduced virulence.     

Neamine dimers targeting the HIV-1 TAR RNA

Natural aminoglycoside antibiotics, such as neomycin, target bacterial ribosomal RNA. Neomycin also binds strongly to HIV TAR and RRE RNA through the predominant interactions of its neamine core. In the search for antiviral agents targeting multiple binding sites for aminoglycosides in RNA, we report here the synthesis of new neamine dimers and a trimer in which the neamine cores are connected by different linking chains attached at the 4'- and/or 5-positions. Inhibition of TAR-Tat complexation by these oligomers was studied via fluorimetric binding assays performed under two ionic strengths. All dimers strongly inhibit TAR-Tat association, with IC50 values 17-85 times better than the value obtained with neomycin. These results demonstrate that modifying neamine at the 4'- or the 5-position is a promising strategy in the search for antiviral agents.     

Implications of the effects of viscosity, macromolecular crowding, and temperature for the transient interaction between cytochrome f and plastocyanin from the cyanobacterium Phormidium laminosum

The reaction between cytochrome f and plastocyanin is a central feature of the photosynthetic electron-transport system of all oxygenic organisms. We have studied the reaction in solution to understand how the very weak binding between the two proteins from Phormidium laminosum can nevertheless lead to fast rates of electron transfer. In a previous publication [Schlarb-Ridley, B. G., et al. (2003) Biochemistry 42, 4057-4063], we suggested that the reaction is diffusion-controlled because of a strong effect of viscosity of the medium. The effects of viscosity and temperature have now been examined in detail. High molecular mass viscogens (Ficoll 70 and Dextran 70), which might mimic in vivo conditions, had little effect up to a relative viscosity of 4. Low molecular mass viscogens (ethane diol, glycerol, and sucrose) strongly decreased the bimolecular rate constant (k(2)) over a similar viscosity range. The effects correlated well with the viscosities of the solutions of the three reagents but not with their dielectric constants or molalities. A power law dependence of k(2) on viscosity suggested that k(2) depends on two viscosity-sensitive reactions in series, while the reverse reactions are little affected by viscosity. The results were incompatible with diffusion control of the overall reaction. Determination of the effect of temperature on k(2) gave an activation enthalpy, DeltaH(++) = 45 kJ mol(-)(1), which is also incompatible with diffusion control. The results were interpreted in terms of a model in which the stable form of the protein-protein complex requires further thermal activation to be competent for electron transfer.     

The plasticity of p19 ARF null hepatic stellate cells and the dynamics of activation

In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.     

Interference of hepatitis A virus replication by small interfering RNAs

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.