Mechanostability of the Fibrinogen Bridge between Staphylococcal Surface Protein ClfA and Endothelial Cell Integrin αVβ3

Binding of the Staphylococcus aureus surface protein clumping factor A (ClfA) to endothelial cell integrin αVβ3 plays a crucial role during sepsis, by causing endothelial cell apoptosis and loss of barrier integrity. ClfA uses the blood plasma protein fibrinogen (Fg) to bind to αVβ3 but how this is achieved at the molecular level is not known. Here we investigate the mechanical strength of the three-component ClfA-Fg-αVβ3 interaction on living bacteria, by means of single-molecule experiments. We find that the ClfA-Fg-αVβ3 ternary complex is extremely stable, being able to sustain forces (∼800 pN) that are much stronger than those of classical bonds between integrins and the Arg-Gly-Asp (RGD) tripeptide sequence (∼100 pN). Adhesion forces between single bacteria and αVβ3 are strongly inhibited by an anti-αVβ3 antibody, the RGD peptide, and the cyclic RGD peptide cilengitide, showing that formation of the complex involves RGD-dependent binding sites and can be efficiently inhibited by αVβ3 blockers. Collectively, our experiments favor a binding mechanism involving the extraordinary elasticity of Fg. In the absence of mechanical stress, RGD572-574 sequences in the Aα chains mediate weak binding to αVβ3, whereas under high mechanical stress exposure of cryptic Aα chain RGD95-97 sequences leads to extremely strong binding to the integrin. Our results identify an unexpected and previously undescribed force-dependent binding mechanism between ClfA and αVβ3 on endothelial cells, which could represent a potential target to fight staphylococcal bloodstream infections

Mechanical Forces Guiding Staphylococcus aureus Cellular Invasion

Staphylococcus aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. The current model for cellular invasion involves the interaction between the bacterial cell surface located fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5β1 integrin in the host cell membrane. While it is believed that the extracellular matrix protein Fn serves as a bridging molecule between FnBPs and integrins, the fundamental forces involved are not known. Using single-cell and single-molecule experiments, we unravel the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA-Fn-integrin interaction. We show that FnBPA mediates bacterial adhesion to soluble Fn via strong forces (∼1500 pN), consistent with a high-affinity tandem β-zipper, and that the FnBPA-Fn complex further binds to immobilized α5β1 integrins with a strength much higher than that of the classical Fn-integrin bond (∼100 pN). The high mechanical stability of the Fn bridge favors an invasion model in which Fn binding by FnBPA leads to the exposure of cryptic integrin-binding sites via allosteric activation, which in turn engage in a strong interaction with integrins. This activation mechanism emphasizes the importance of protein mechanobiology in regulating bacterial-host adhesion. We also find that Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes. Collectively, our results provide a molecular foundation for the ability of FnBPA to trigger host cell invasion by S. aureus and offer promising prospects for the development of therapeutic approaches against intracellular pathogens

Single-cell fluidic force microscopy reveals stress- dependent molecular interactions in yeast mating

Sexual agglutinins of the budding yeast Saccharomyces cerevisiae are proteins mediating cell aggregation during mating. Complementary agglutinins expressed by cells of opposite mating types “a” and “α” bind together to promote agglutination and facilitate fusion of haploid cells. By means of an innovative single-cell manipulation assay combining fluidic force microscopy with force spectroscopy, we unravel the strength of single specific bonds between a- and α-agglutinins (~100 pN) which require pheromone induction. Prolonged cell–cell contact strongly increases adhesion between mating cells, likely resulting from an increased expression of agglutinins. In addition, we highlight the critical role of disulfide bonds of the a- agglutinin and of histidine residue H273 of α-agglutinin. Most interestingly, we find that mechanical tension enhances the interaction strength, pointing to a model where physical stress induces conformational changes in the agglutinins, from a weak-binding folded state, to a strong-binding extended state. Our single-cell technology shows promises for under- standing and controlling the complex mechanism of yeast sexuality

Search for Neutrinos in Super-Kamiokande Associated with the GW170817 Neutron-star Merger

We report the results of a neutrino search in Super-Kamiokande (SK) for coincident signals with the first detected gravitational wave (GW) produced by a binary neutron-star merger, GW170817, which was followed by a short gamma-ray burst, GRB170817A, and a kilonova/macronova. We searched for coincident neutrino events in the range from 3.5 MeV to ~100 PeV, in a time window ±500 s around the gravitational wave detection time, as well as during a 14-day period after the detection. No significant neutrino signal was observed for either time window. We calculated 90% confidence level upper limits on the neutrino fluence for GW170817. From the upward-going-muon events in the energy region above 1.6 GeV, the neutrino fluence limit is ${16.0}_{-0.6}^{+0.7}$ (${21.3}_{-0.8}^{+1.1}$) cm−2 for muon neutrinos (muon antineutrinos), with an error range of ±5° around the zenith angle of NGC4993, and the energy spectrum is under the assumption of an index of −2. The fluence limit for neutrino energies less than 100 MeV, for which the emission mechanism would be different than for higher-energy neutrinos, is also calculated. It is 6.6 × 107 cm−2 for anti-electron neutrinos under the assumption of a Fermi–Dirac spectrum with average energy of 20 MeV