Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1

Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, intercepts material from host cell membrane transport pathways to create a specialized vacuolar compartment that supports bacterial replication. Delivery of bacterial effector proteins into the host cell requires the Dot/Icm type IV secretion system. Several effectors, including SidM, SidD, and LepB, were shown to target the early secretory pathway by manipulating the activity of the host GTPase Rab1. While the function of these effectors has been well characterized, the role of another Rab1-interacting protein from L. pneumophila, the effector protein LidA, is poorly understood. Here, we show that LidA binding to Rab1 stabilized the Rab1-guanosine nucleotide complex, protecting it from inactivation by GTPase-activating proteins (GAPs) and from nucleotide extraction. The protective effect of LidA on the Rab1-guanine nucleotide complex was concentration dependent, consistent with a 1:1 stoichiometry of the LidA-Rab1 complex. The central coiled-coil region of LidA was sufficient for Rab1 binding and to prevent GAP-mediated inactivation or nucleotide extraction from Rab1. In addition, the central region mediated binding to phosphatidylinositol 3-phosphate and other phosphoinositides. When bound to Rab1, LidA interfered with the covalent modification of Rab1 by phosphocholination or AMPylation, and it also blocked de-AMPylation of Rab1 by SidD and dephosphocholination by Lem3. Based on these findings, we propose a role for LidA in bridging the membrane of the Legionella-containing vacuole (LCV) with that of secretory transport vesicles surrounding the LCV.     

Efficient Production of Bioactive Recombinant Human Flt3 Ligand in <Emphasis Type="Italic">E. coli</Emphasis>

Flt3 ligand (FL) is an early-acting hematopoietic cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells by activating its cognate receptor, Flt3. Recently, FL was shown to potently contribute to the development and expansion of antigen-presenting dendritic cells and CD34⁺ natural killer cell progenitors in vivo. Here, we report a comprehensive method for the production of bioactive recombinant human FL (rhFL) in E. coli, suitable for structural, biophysical and physiological studies. A soluble form of human FL capable of binding to the Ftl3 receptor could be overexpressed in the E. coli strain Rosetta-gami(DE3) as inclusion bodies. We have established protocols for the efficient in vitro refolding and ensuing purification of rhFL to homogeneity (>95%), with yields approaching 5 mg of pure rhFL per liter of culture. The ability of rhFL to adopt a bioactive conformation was confirmed via a cell-proliferation assay and the activation of the Flt3 receptor in the human leukemic cell line, OCI-AML3.     

Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity

Botulinum neurotoxins (BoNTs) are among the most lethal biological substances to have been weaponized and are listed as biodefense category A agents. Currently, no small molecule (non-peptidic) therapeutics exist to counter this threat; hence, identifying and developing compounds that inhibit BoNTs is a high priority. In the present study, a high-throughput assay was used to identify small molecules that inhibit the metalloprotease activity of BoNT serotype A light chain (BoNT/A LC). All inhibitors were further verified using a HPLC-based assay. Conformational analyses of these compounds, in conjunction with molecular docking studies, were used to predict structural features that contribute to inhibitor binding and potency. Based on these results, a common pharmacophore for BoNT/A LC inhibitors is proposed. This is the first study to report small molecules (non-peptidics) that inhibit BoNT/A LC metalloprotease activity in the low microM range.     

Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization

Adult bone marrow is a rich reservoir of hematopoietic and vascular stem and progenitor cells. Mobilization and recruitment of these cells are essential for tissue revascularization. Physiological stress, secondary to tissue injury or tumor growth, results in the release of angiogenic factors, including vascular endothelial growth factor (VEGF), which promotes mobilization of stem cells to the circulation, contributing to the formation of functional vasculature. VEGF interacts with its receptors, VEGFR2 and VEGFR1, expressed on endothelial and hematopoietic stem cells, and thereby promotes recruitment of these cells to neo-angiogenic sites, accelerating the revascularization process. The mobilization of stem cells from marrow is a dynamic process, regulated by shear stress imparted by blood flow, and the activation of metalloproteinases that induce the release of 'Kit ligand', facilitating egress from the marrow to the circulation. Identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells will open up new avenues for the design of clinical trials to accelerate tissue vascularization and organogenesis.     

Specific and non specific interactions involving Le X determinant quantified by lipid vesicle micromanipulation

Carbohydrate-carbohydrate recognition is emerging today as an important type of interaction in cell adhesion. One Ca(2+)mediated homotypic interaction between two Lewis( X ) determinants (Le( X )) has been proposed to drive cell adhesion in murine embryogenesis. Here, the adhesion energies of lipid vesicles functionalized with glycolipids bearing monomeric or dimeric Le( X ) determinants were measured in NaCl or CaCl(2) media with the micropipette aspiration technique. These experiments on Le( X ) with an environment akin to that provided by biological membrane confirmed the existence of this specific calcium dependent interaction of monomeric Le( X ). In contrast, dimeric Le( X ) produced a repulsive contribution. By using a simple model involving the various contributions to the adhesion free energy, specific and non specific interactions could be separated and quantified. The involvement of calcium ions has been discussed in the monomeric and dimeric Le( X ) lipids.     

Identification of small molecule inhibitors of anthrax lethal factor

The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have K(i) values in the 0.5-5 microM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.     

Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis

Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhalational anthrax because they initiate germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign particles and migrate to lymph nodes. However, the participation of DCs in phagocytosis and dissemination of spores has not been investigated previously. We found that human DCs readily engulfed fully pathogenic Ames and attenuated B. anthracis spores predominately by coiling phagocytosis. Spores provoked a loss of tissue-retaining chemokine receptors (CCR2, CCR5) with a concurrent increase in lymph node homing receptors (CCR7, CD11c) on the membrane of DCs. After spore infection, immature DCs displayed a mature phenotype (CD83(bright), HLA-DR(bright), CD80(bright), CD86(bright), CD40(bright)) and enhanced costimulatory activity. Surprisingly, spores activated the MAPK cascade (ERK, p38) within 30 min and stimulated expression of several inflammatory response genes by 2 h. MAPK signaling was extinguished by 6 h infection, and there was a dramatic reduction of secreted TNF-alpha, IL-6, and IL-8 in the absence of DC death. This corresponded temporally with enzymatic cleavage of proximal MAPK signaling proteins (MEK-1, MEK-3, and MAP kinase kinase-4) and may indicate activity of anthrax lethal toxin. Taken together, these results suggest that B. anthracis may exploit DCs to facilitate infection.     

Inhibition of Yersinia tyrosine phosphatase by furanyl salicylate compounds

To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar Ki values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopH-induced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis.