Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration

Staphylococcus aureus Panton-Valentine leukocidin is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored. Here, we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and present indirect evidence for receptor recycling that allows multiple toxin pores to be formed close together. With the use of live cell single-molecule super-resolution imaging, Forster resonance energy transfer and nanoscale total internal reflection fluorescence colocalization microscopy, we visualized toxin pore formation in the presence of its natural docking ligand. We demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyperinflammatory reactions in early stages of microbial infections and have implications for several other similar bicomponent toxins and the design of new antibiotics.Haapasalo, K., Wollman, A. J. M., de Haas, C. J. C., van Kessel, K. P. M., van Strijp, J. A. G., Leake, M. C. Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration.Peer reviewe

Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis

Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His(6))-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His(6)-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His(6)-tag. This seems to be due to an influence of the His(6)-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins

Влияние фенилгидразина и алкилсульфатов на осмотическую чувствительность эритроцитов млекопитающих

Показано, что чувствительность эритроцитов млекопитающих к гипертоническому стрессу при 37 °С после обработки фенилгидразином зависит от видовой принадлежности клеток. Установлено, что модификация фенилгидразином эритроцитов млекопитающих приводит к снижению антигемолитической активности алкилкилсульфатов в условиях гипертонического стресса.Показано, що чутливість еритроцитів ссавців до гіпертонічного стресу при 37 °С після обробки фенілгідразином залежить від видової приналежності клітин. Встановлено, що модифікація фенілгідразином еритроцитів ссавців призводить до зниження антигемолітичної активності алкілсульфатів в умовах гіпертонічного стресу.It is shown that the sensitivity of mammalian erythrocytes to hypertonic stress at 37 °C after the treatment with phenylhydrazine depends on the species of a cell. We have established that the modification with phenylhydrazine of mammalian erythrocytes leads to a decrease of the alkyl sulfates antihemolytic activity under hypertonic stress

Microenvironment involved in FPR1 expression by human glioblastomas

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.</p

Under-reporting bicycle accidents to police in the COST TU1101 international survey: Cross-country comparisons and associated factors

Police crash reports are often the main source for official data in many countries. However, with the exception of fatal crashes, crashes are often underreported in a biased manner. Consequently, the countermeasures adopted according to them may be inefficient. In the case of bicycle crashes, this bias is most acute and it probably varies across countries, with some of them being more prone to reporting accidents to police than others. Assessing if this bias occurs and the size of it can be of great importance for evaluating the risks associated with bicycling. This study utilized data collected in the COST TU1101 action “Towards safer bicycling through optimization of bicycle helmets and usage”. The data came from an online survey that included questions related to bicyclists' attitudes, behaviour, cycling habits, accidents, and patterns of use of helmets. The survey was filled by 8655 bicyclists from 30 different countries. After applying various exclusion factors, 7015 questionnaires filled by adult cyclists from 17 countries, each with at least 100 valid responses, remained in our sample. The results showed that across all countries, an average of only 10% of all crashes were reported to the police, with a wide range among countries: from a minimum of 0.0% (Israel) and 2.6% (Croatia) to a maximum of a 35.0% (Germany). Some factors associated with the reporting levels were type of crash, type of vehicle involved, and injury severity. No relation was found between the likelihood of reporting and the cyclist's gender, age, educational level, marital status, being a parent, use of helmet, and type of bicycle. The significant under-reporting – including injury crashes that do not lead to hospitalization – justifies the use of self-report survey data for assessment of bicycling crash patterns as they relate to (1) crash risk issues such as location, infrastructure, cyclists' characteristics, and use of helmet and (2) strategic approaches to bicycle crash prevention and injury reduction.Fil: Shinar, D.. Ben Gurion University of the Negev; IsraelFil: Valero Mora, Pedro. Universidad de Valencia; EspañaFil: van Strijp Houtenbos, M.. Institute For Road Safety Research; Países BajosFil: Haworth, N.. Queensland University of Technology; AustraliaFil: Schramm, A.. Queensland University of Technology; AustraliaFil: de Bruyne, G.. Universiteit Antwerp; BélgicaFil: Cavallo, V.. No especifíca;Fil: Chliaoutakis, J.. No especifíca;Fil: Pereira Dias, Joao. Instituto Superior Tecnico; PortugalFil: Ferraro, Ottavia Eleonora. Universita Degli Studi Di Pavia; ItaliaFil: Fyhri, Aslak. No especifíca;Fil: Sajatovic, Anika Hursa. No especifíca;Fil: Kuklane, Kalev. Lund University; SueciaFil: Ledesma, Ruben Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Psicología Básica, Aplicada y Tecnología. Universidad Nacional de Mar del Plata. Facultad de Psicología. Instituto de Psicología Básica, Aplicada y Tecnología.; ArgentinaFil: Calvé Mascarell, Oscar. Ben Gurion University of the Negev; IsraelFil: Morandi, A.. Universita Degli Studi Di Pavia; ItaliaFil: Muser, Markus. No especifíca;Fil: Otte, Diettmar. No especifíca;Fil: Papadakaki, M.. No especifíca;Fil: Sanmartín, J.. Universidad de Valencia; EspañaFil: Dulf, D.. No especifíca;Fil: Saplioglu, M.. No especifíca;Fil: Tzamalouka, Georgia. No especifíca

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30–60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell–extracellular matrix interactions

Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro and in vivo biofilm

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus.

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component