A novel receptor – ligand pathway for entry of Francisella tularensis in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

<p>Abstract</p> <p>Background</p> <p><it>Francisella tularensis</it>, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for <it>F. tularensis </it>internalization has been suggested.</p> <p>Results</p> <p>We show here that cell-surface expressed nucleolin is a receptor for <it>Francisella tularensis </it>Live Vaccine Strain (LVS) and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu), a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin.</p> <p>Discussion</p> <p>Altogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in <it>Francisella tularensis </it>adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of LVS in infected monocytes are very similar to those of human pathogenic <it>F. tularensis </it>ssp <it>tularensis</it>, the mechanism of entry into monocyte-like THP-1 cells, involving interaction between EF-Tu and nucleolin, might be similar in the two subspecies. Thus, the use of either nucleolin-specific pseudopeptide HB-19 or recombinant EF-Tu could provide attractive therapeutic approaches for modulating <it>F. tularensis </it>infection.</p

Investigating the evolution of major Northern Hemisphere ice sheets during the last glacial-interglacial cycle

A 2.5-dimensional climate model of intermediate complexity, CLIMBER-2, fully coupled with the GREMLINS 3-D thermo-mechanical ice sheet model is used to simulate the evolution of major Northern Hemisphere ice sheets during the last glacial-interglacial cycle and to investigate the ice sheets responses to both insolation and atmospheric CO&lt;sub&gt;2&lt;/sub&gt; concentration. This model reproduces the main phases of advance and retreat of Northern Hemisphere ice sheets during the last glacial cycle, although the amplitude of these variations is less pronounced than those based on sea level reconstructions. At the last glacial maximum, the simulated ice volume is 52.5&amp;times;10&lt;sup&gt;15&lt;/sup&gt; m&lt;sup&gt;3&lt;/sup&gt; and the spatial distribution of both the American and Eurasian ice complexes is in reasonable agreement with observations, with the exception of the marine parts of these former ice sheets. &lt;br&gt; A set of sensitivity studies has also been performed to assess the sensitivity of the Northern Hemisphere ice sheets to both insolation and atmospheric CO&lt;sub&gt;2&lt;/sub&gt;. Our results suggest that the decrease of summer insolation is the main factor responsible for the early build up of the North American ice sheet around 120 kyr BP, in agreement with benthic foraminifera &amp;delta;&lt;sup&gt;18&lt;/sup&gt;O signals. In contrast, low insolation and low atmospheric CO&lt;sub&gt;2&lt;/sub&gt; concentration are both necessary to trigger a long-lasting glaciation over Eurasia

Mixed quantum state detection with inconclusive results

We consider the problem of designing an optimal quantum detector with a fixed rate of inconclusive results that maximizes the probability of correct detection, when distinguishing between a collection of mixed quantum states. We develop a sufficient condition for the scaled inverse measurement to maximize the probability of correct detection for the case in which the rate of inconclusive results exceeds a certain threshold. Using this condition we derive the optimal measurement for linearly independent pure-state sets, and for mixed-state sets with a broad class of symmetries. Specifically, we consider geometrically uniform (GU) state sets and compound geometrically uniform (CGU) state sets with generators that satisfy a certain constraint. We then show that the optimal measurements corresponding to GU and CGU state sets with arbitrary generators are also GU and CGU respectively, with generators that can be computed very efficiently in polynomial time within any desired accuracy by solving a semidefinite programming problem.Comment: Submitted to Phys. Rev.

Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes

Familial hematuria

Hematuria is a common presenting complaint in pediatric nephrology clinics and often has a familial basis. This teaching article provides an overview of causes, diagnosis, and management of the major forms of familial hematuria, Alport syndrome, and thin basement membrane nephropathy

Novel Roles of cAMP Receptor Protein (CRP) in Regulation of Transport and Metabolism of Carbon Sources

CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons

A Putative P-Type ATPase Required for Virulence and Resistance to Haem Toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem