A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.     

Role of an internal and two 3'-terminal RNA elements in assembly of tombusvirus replicase

Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3'-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.     

Purification of the cucumber necrosis virus replicase from yeast cells: role of coexpressed viral RNA in stimulation of replicase activity

Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for Cucumber necrosis tombusvirus (CNV), which is a plus-stranded RNA virus, from Saccharomyces cerevisiae. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), including the ability (i). to initiate cRNA synthesis de novo on both plus- and minus-stranded templates, (ii). to generate replicase products that are shorter than full length by internal initiation, and (iii). to perform primer extension from the 3' end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.     

A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity

Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.     

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.     

Ectopic expression of LIM-nebulette (LASP2) reveals roles in cell migration and spreading

LIM-nebulette (LASP2) is a small focal adhesion protein and a member of the nebulin family of actin binding proteins. This recently identified splice variant of the nebulette locus is widely expressed and highly enriched in neuronal tissue. Other than that LIM-nebulette is a focal adhesion protein and interacts with zyxin, nothing is known about its function. Given that LIM-nebulette has an identical modular organization and overlapping tissue distributions to that of LASP1, we have analyzed the role of LIM-nebulette in comparison with that of LASP1. We find that LIM-nebulette is a dynamic focal adhesion protein that increases the rate of attachment and spreading of fibroblasts on fibronectin coated surfaces. Additionally, LIM-nebulette is recruited from the cortical cytoskeleton in non-motile cells to focal adhesions at the leading edge of stimulated cells. In confluent cultures of HeLa and NIH3T3 cells, LIM-nebulette co-localizes with alpha-catenin in putative adherens junctions, whereas LASP1 is devoid of these areas. Interestingly, overexpression of LIM-nebulette in PC6 cells inhibits neurite outgrowth in response to growth factors. Collectively, our data indicate that LIM-nebulette and LASP1 have distinct roles in the actin cytoskeleton.     

Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release,a Thrombotic Mechanism in Hemolytic Uremic Syndrome

Background

Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)–bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF.

Methodology/Principal Findings

The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF–expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery.

Conclusions/Significance

By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.     

Single molecule detection of DNA looping by NgoMIV restriction endonuclease

Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.     

Yellow stripe1. Expanded roles for the maize iron-phytosiderophore transporter

Graminaceous monocots, including most of the world's staple grains (i.e. rice, corn, and wheat) use a chelation strategy (Strategy II) for primary acquisition of iron from the soil. Strategy II plants secrete phytosiderophores (PS), compounds of the mugineic acid family that form stable Fe(III) chelates in soil. Uptake of iron-PS chelates, which occurs through specific transporters at the root surface, thus represents the primary route of iron entry into Strategy II plants. The gene Yellow stripe1 (Ys1) encodes the Fe(III)-PS transporter of maize (Zea mays). Here the physiological functions performed by maize YS1 were further defined by examining the pattern of Ys1 mRNA and protein accumulation and by defining YS1 transport specificity in detail. YS1 is able to translocate iron that is bound either by PS or by the related compound, nicotianamine; thus, the role of YS1 may be to transport either of these complexes. Ys1 expression at both the mRNA and protein levels responds rapidly to changes in iron availability but is not strongly affected by limitation of copper or zinc. Our data provide no support for the idea that YS1 is a transporter of zinc-PS, based on YS1 biochemical activity and Ys1 mRNA expression patterns in response to zinc deficiency. YS1 is capable of transporting copper-PS, but expression data suggest that the copper-PS uptake has limited significance in primary uptake of copper.     

UVA, UVB and incidence of cutaneous malignant melanoma in Norway and Sweden

Latitudinal dependencies of UVA and UVB were studied together with relevant epidemiological data for squamous cell carcinoma (SCC) and cutaneous malignant melanoma (CMM) in Norway and Sweden. Our data support the hypothesis that solar UVA radiation may play a role for CMM induction. The etiologies of SCC and CMM are different according to a latitudinal dependency and differences in age curves. Sun exposure patterns, age-related decay rates of repair of UV damage and sex hormones may play different roles for the two skin cancers. Also, UVB induction of vitamin D may be involved. CMM incidence rates among young people have decreased or been constant since about 1990 in Norway and Sweden. All reasons for UVA contributing to CMM will be discussed.