How Bacteria Bind More Strongly Under Mechanical Force: The Catch-Bond FimH

2003 ◽  
Author(s):  
Wendy E. Thomas ◽  
Evgeni V. Sokurenko ◽  
Viola Vogel
Keyword(s):  
Shear Stress ◽  
Structural Model ◽  
Intestinal Bacteria ◽  
Mechanical Force ◽  
The Body ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Structural Variations ◽  
Catch Bond ◽  
Static Conditions

We study a protein that responds to mechanical force in most striking manner. We demonstrate that Escherichia coli bacteria need shear stress to bind to certain tissues and model surfaces; they bind strongest precisely when the body tries to wash them off. We have determined that the protein responsible for this behavior is FimH, a ubiquitous adhesion protein in intestinal bacteria that mediates adhesion to host cells via the carbohydrate mannose. Although mechanical force noramlly decreases bond lifetimes, we have shown that the bond betweeen FimH and simple mono-mannose receptors is s “catch-bond” that lasts longer under shear stress. In contrast, structural variations in either FimH or the receptor cause a stronger mode of adhesion in static conditions with little or no activation under force. We derive a structural for how mechanical force switches FimH to a strong binding mode by using steered molecular dynamics simulations, and validate the predictions with subsequent site-directed mutagenesis. The physiological consequences as well as the engineering principles suggested by the structural model will be discussed.

scholarly journals Plasma fibronectin stabilizes Borrelia burgdorferi–endothelial interactions under vascular shear stress by a catch-bond mechanism

2017 ◽  
Vol 114 (17) ◽  
pp. E3490-E3498 ◽  
Author(s):  
Alexandra F. Niddam ◽  
Rhodaba Ebady ◽  
Anil Bansal ◽  
Anne Koehler ◽  
Boris Hinz ◽  
...  
Keyword(s):  
Shear Stress ◽  
Borrelia Burgdorferi ◽  
Live Cell Imaging ◽  
Host Cells ◽  
Catch Bond ◽  
Bacterial Dissemination ◽  
Bacterial Interaction ◽  
Blood Constituent ◽  
Formed Part ◽  
Bond Mechanism

Bacterial dissemination via the cardiovascular system is the most common cause of infection mortality. A key step in dissemination is bacterial interaction with endothelia lining blood vessels, which is physically challenging because of the shear stress generated by blood flow. Association of host cells such as leukocytes and platelets with endothelia under vascular shear stress requires mechanically specialized interaction mechanisms, including force-strengthened catch bonds. However, the biomechanical mechanisms supporting vascular interactions of most bacterial pathogens are undefined. Fibronectin (Fn), a ubiquitous host molecule targeted by many pathogens, promotes vascular interactions of the Lyme disease spirochete Borrelia burgdorferi. Here, we investigated how B. burgdorferi exploits Fn to interact with endothelia under physiological shear stress, using recently developed live cell imaging and particle-tracking methods for studying bacterial–endothelial interaction biomechanics. We found that B. burgdorferi does not primarily target insoluble matrix Fn deposited on endothelial surfaces but, instead, recruits and induces polymerization of soluble plasma Fn (pFn), an abundant protein in blood plasma that is normally soluble and nonadhesive. Under physiological shear stress, caps of polymerized pFn at bacterial poles formed part of mechanically loaded adhesion complexes, and pFn strengthened and stabilized interactions by a catch-bond mechanism. These results show that B. burgdorferi can transform a ubiquitous but normally nonadhesive blood constituent to increase the efficiency, strength, and stability of bacterial interactions with vascular surfaces. Similar mechanisms may promote dissemination of other Fn-binding pathogens.

scholarly journals Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

2015 ◽  
Vol 112 (22) ◽  
pp. 6991-6996 ◽  
Author(s):  
Takashi Suzuki ◽  
Miho Suzuki ◽  
Shinji Ogino ◽  
Ryo Umemoto ◽  
Noritaka Nishida ◽  
...  
Keyword(s):  
Shear Stress ◽  
Conformational Change ◽  
Mechanical Force ◽  
Binding Domain ◽  
Terminal Region ◽  
Hematopoietic Progenitor Cell ◽  
High Shear ◽  
Cell Rolling ◽  
High Affinity ◽  
C Terminus

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

2004 ◽  
Vol 287 (5) ◽  
pp. H2287-H2294 ◽  
Author(s):  
Akinori Ueda ◽  
Manabu Shimomura ◽  
Mariko Ikeda ◽  
Ryuhei Yamaguchi ◽  
Kazuo Tanishita
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Laser Scanning ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Interface Barrier ◽  
Uptake Process ◽  
Static Conditions

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.

scholarly journals Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity

2005 ◽  
Vol 187 (8) ◽  
pp. 2582-2591 ◽  
Author(s):  
Yasuo Mitani ◽  
XianYing Meng ◽  
Yoichi Kamagata ◽  
Tomohiro Tamura
Keyword(s):  
Cell Wall ◽  
Culture Media ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Wall Structure ◽  
Synthetase Activity ◽  
Glutamine Amidotransferase ◽  
Glutaminase Activity

ABSTRACT The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH4Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH4Cl, indicating that NH3 can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.

Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription

2006 ◽  
Vol 291 (1) ◽  
pp. G26-G34 ◽  
Author(s):  
Hideki Nakatsuka ◽  
Takaaki Sokabe ◽  
Kimiko Yamamoto ◽  
Yoshinobu Sato ◽  
Katsuyoshi Hatakeyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shear Stress ◽  
Consensus Sequence ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Consensus Sequences ◽  
Static Conditions ◽  
Stress Dependent ◽  
Pai 1

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5′-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

scholarly journals Mapping the endothelial cell S-Sulfhydrome highlights the crucial role of integrin sulfhydration in vascular function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
D R Bibli ◽  
D R Hu ◽  
D R Looso ◽  
D R Weigert ◽  
D R Wittig ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Vascular Disease ◽  
Disulfide Bond ◽  
Mesenteric Arteries ◽  
Human Endothelial Cells ◽  
Β3 Integrin ◽  
Static Conditions ◽  
Protein Disulfide

Abstract Background In vascular endothelial cells, cysteine metabolism by the cystathionine-γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the “S-sulfhydrome” i.e. the spectrum of proteins targeted by H2Sn in human endothelial cells. Methods LC-MS/MS was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intra-protein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements and flow-induced vasodilatation in endothelial cell-specific CSE knock out mice and a small collective of patients with endothelial dysfunction. Results Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low), (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression, and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell “S-sulfhydrome” consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S-sulfhydration affected intra-protein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between β3 integrin and Gα13, resulting in the constitutive activation of RhoA and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation and a failure to detect β3 integrin S-sulfhydration, all of which were rescued following the administration of an H2S supplement. Conclusions Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

scholarly journals Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3792-3799 ◽  
Author(s):  
Hilde Depraetere ◽  
Nadine Ajzenberg ◽  
Jean-Pierre Girma ◽  
Catherine Lacombe ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
Rotational Viscometer ◽  
Induce Platelet Aggregation ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

scholarly journals Pathogenetic Substantiation of the Therapeutic Impact on Microbiota in Irritable Bowel Syndrome

2019 ◽  
Vol 29 (4) ◽  
pp. 7-14
Author(s):  
Yu. O. Shulpekova ◽  
G. H. Babaeva ◽  
V. Yu. Rusyaev
Keyword(s):  
Irritable Bowel Syndrome ◽  
Intestinal Microbiota ◽  
Intestinal Bacteria ◽  
Host Cells ◽  
Intestinal Microbiome ◽  
Bacterial Strains ◽  
Blastocystis Hominis ◽  
Therapeutic Impact ◽  
Irritable Bowel ◽  
Positive Effect

Aim. This review aims to describe the nature of changes in the intestinal microbiota in irritable bowel syndrome (IBS) and provide a pathogenetic justification of the feasibility of a therapeutic impact on microbiota. General findings. An important aspect of the interaction of intestinal bacteria with the “host” cells is their contact with pattern recognition receptors of enterocytes, dendritic cell receptors, as well as a transcellular transport of antigens in the region of Peyer’s patches. The area of interaction of intestinal bacteria and the human body is not limited to the intestines. Intestinal bacteria demonstrate a significant humoral effect due to signalling molecules, some of which exhibit neurotransmitter properties. The study of the bacterial cross-feeding for various species, i.e. mutual use of nutrient substrates produced by bacteria of various species, is of a great interest. The development of a lowactivity inflammation in IBS can partly be explained by the increased interaction of flagellin with the corresponding receptor, as well as the influx of excess bacteria from the small intestine. The majority of studies on IBS have demonstrated the predominance of intestinal bacteria with pro-inflammatory potential (Enterobacteriaceae) and the lack of bacteria with a pronounced anti-inflammatory, antimicrobial and enzymatic action (Lactobacillus and Bifidobacterium), as well as increased mucus degradation. Similar changes are observed in inflammatory bowel diseases. Reduced microbial diversity increases susceptibility to intestinal infections and parasitoses, including those caused by protozoa conditionally pathogenic for adults, such as Blastocystis hominis hominis, Dientamoeba fragilis, Giardia lamblia. With the help of nutrition correction, the use of probiotics and functional foods containing certain probiotic strains, plant fibres (primarily psyllium) and, in some cases, nonabsorbable antibiotics, a positive effect can be achieved in a significant number of IBS patients. Recent works have shown that clinical improvement is accompanied by a change in the composition of the intestinal microbiota.Conclusion. For the pathogenetic treatment of irritable bowel syndrome, the use of non-drug treatment is justified, such as diet optimization and prescription of plant fibres and probiotic bacterial strains. The positive effect of such an approach is largely determined by modification of the intestinal microbiota composition. This opens up prospects for a further, more targeted impact on the intestinal microbiome.

scholarly journals Biomechanical Characterization of Endothelial Cells Exposed to Shear Stress Using Acoustic Force Spectroscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Giulia Silvani ◽  
Valentin Romanov ◽  
Charles D. Cox ◽  
Boris Martinac
Keyword(s):  
Mechanical Properties ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Actin Filament ◽  
Force Spectroscopy ◽  
Shear Stresses ◽  
Cell Periphery ◽  
Static Conditions ◽  
Cells Cultured ◽  
Cell Cell

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm2). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells.

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