remote-magnetic-actuation-micrometric-probes-for-situ-3d-mapping

Remote Magnetic Actuation of Micrometric Probes for <em>in situ</em> 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Sorbonne Universités, UPMC

We report here the formation of adhesive conjugates between living cells and properly tailored colloidal liquid droplets bearing a cationic surfactant. We show that the droplets could wet cell surface with a well-defined contact angle, allowing direct determination of the energy of adhesion. We also describe the effect of cationic surfactant concentration on adhesion efficiency. This provides new tools to probe living cell surface properties and find practical laws for cell adhesion on well-defined surfaces.

Wetting of liquid droplets on living cells.

The establishment of specific molecular bonds between a cell and a facing surface is involved in many physiological and technological situations. Using micrometric magnetic particles, we have explored the formation of specific molecular bonds between the cell and surfaces bearing complementary ligands under passive conditions. Streptavidin-coated particles were targeted to the cell surface of a B-cell line through a specific biotinylated antibody against the CD19 receptor. Flow cytometry, optical microscopy, and micropipette experimental techniques have been used. Main findings have been that cell surface receptor density acted like a switch for particle capture with a threshold value found here equal to 1.6 x 10(3) receptor/ microm(2). This led to exclusion from binding of the cells of lowest receptor density. The density threshold was modulated by the length of the binding link and the physics of the cell/particle collision. We suggest that the shear stress is one of the main determinants of the characteristics of binding. We also show that several thousand receptors were involved in the cell particle contact at the end of the binding process, although only eight bonds are required for the initial capture of a particle. A passive binding inhibition process due to link concentration by the initial contact was proposed to account for the small number of particles per cell.

Specific recognition of macroscopic objects by the cell surface: evidence for a receptor density threshold revealed by micrometric particle binding characteristics.

Specific molecular bonds between apposing surfaces play a central role in many biological structures and functions. They display a widely varying anchoring to the cell surface, and they are subject to forces that affect their binding characteristics due to their hydrodynamic environments. Here, we examine both anchoring and shearing aspects using simplified model systems aimed at gaining insight into the formation of a 2D bond collection under stress using two different surface anchors. The highly specific streptavidin-biotin molecular bond was chosen as the model receptor-ligand pair, and grafted colloids were used as model surfaces. To explore the role of the surface anchor, we grafted biotin onto the particle surface following two different approaches: first, the grafting was performed directly on the particle amine functions; second, a 35-nm-long PEG spacer was used. Hybrid particle classes were brought into contact in a homogeneous shear (between 200 s(-)(1) and 1200 s(-)(1)) using a cone plate geometry. The bond association and dissociation kinetics were given by the time course assemblage of hybrid particles into doublets. We observed saturating kinetics profiles that we interpreted as a linkage-breakage equilibrium, which yielded the on and off rates. We found that the biotin-PEG spacer was needed in order to observe significant binding at any shear rate. We also showed that only the number of collisions per unit time, generated by the shear, affected the on rate of the binding. Neither the exerted forces nor the collision lifetime had any effect. The off rate decreased with shear, possibly because of the shortening of the force duration, which results from the increasing shear rate.

Molecular bond formation between surfaces: anchoring and shearing effects.

The development of surface-attached biofilm bacterial communities is considered an important source of nosocomial infections. Recently, bacterial interference via signaling molecules and surface active compounds was shown to antagonize biofilm formation, suggesting that nonantibiotic molecules produced during competitive interactions between bacteria could be used for biofilm reduction. Hence, a better understanding of commensal/pathogen interactions within bacterial community could lead to an improved control of exogenous pathogens. To reveal adhesion or growth-related bacterial interference, we investigated interactions between uropathogenic and commensal Escherichia coli in mixed in vitro biofilms. We demonstrate here that the uropathogenic strain CFT073 and all E. coli expressing group II capsules release into their environment a soluble polysaccharide that induces physicochemical surface alterations, which prevent biofilm formation by a wide range of Gram-positive and Gram-negative bacteria. We show that the treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens. These findings identify capsular polymers as antiadhesion bacterial interference molecules, which may prove to be of significance in the design of new strategies to limit biofilm formation on medical in dwelling devices.

Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide.

The development of bacteria on abiotic surfaces has important public health and sanitary consequences. However, despite several decades of study of bacterial adhesion to inert surfaces, the biophysical mechanisms governing this process remain poorly understood, due, in particular, to the lack of methodologies covering the appropriate time scale. Using micrometric colloidal surface particles and flow cytometry analysis, we developed a rapid multiparametric approach to studying early events in adhesion of the bacterium Escherichia coli. This approach simultaneously describes the kinetics and amplitude of early steps in adhesion, changes in physicochemical surface properties within the first few seconds of adhesion, and the self-association state of attached and free-floating cells. Examination of the role of three well-characterized E. coli surface adhesion factors upon attachment to colloidal surfaces--curli fimbriae, F-conjugative pilus, and Ag43 adhesin--showed clear-cut differences in the very initial phases of surface colonization for cell-bearing surface structures, all known to promote biofilm development. Our multiparametric analysis revealed a correlation in the adhesion phase with cell-to-cell aggregation properties and demonstrated that this phenomenon amplified surface colonization once initial cell-surface attachment was achieved. Monitoring of real-time physico-chemical particle surface properties showed that surface-active molecules of bacterial origin quickly modified surface properties, providing new insight into the intricate relations connecting abiotic surface physicochemical properties and bacterial adhesion. Hence, the biophysical analytical method described here provides a new and relevant approach to quantitatively and kinetically investigating bacterial adhesion and biofilm development.

A short-time scale colloidal system reveals early bacterial adhesion dynamics.

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Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow-generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.

Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress.

Infections bact&eacute;riennes ciblant le sang conduisent &agrave; un large &eacute;ventail de maladies d&eacute;vastatrices comme le choc septique et la m&eacute;ningite. Pour &eacute;tudier ce type crucial d&#39;infection, son environnement sp&eacute;cifique doit &ecirc;tre pris en compte, en particulier les forces m&eacute;caniques g&eacute;n&eacute;r&eacute;es par la circulation sanguine. Dans une &eacute;tude ant&eacute;rieure, &agrave; l&#39;aide de Neisseria meningitidis comme mod&egrave;le, nous avons observ&eacute; que des microcolonies de bact&eacute;ries formant &agrave; la surface des cellules endoth&eacute;liales dans la lumi&egrave;re du vaisseau sont remarquablement r&eacute;sistants aux contraintes m&eacute;caniques. La pr&eacute;sente &eacute;tude vise &agrave; identifier les bases mol&eacute;culaires de cette r&eacute;sistance. Formes de N. meningitidis agr&eacute;gats ind&eacute;pendamment des cellules de l&#39;h&ocirc;te, mais nous montrons ici que coh&eacute;sifs forces impliqu&eacute;es dans ces agr&eacute;gats bact&eacute;riens ne suffisent pas &agrave; expliquer la stabilit&eacute; des colonies sur les surfaces de la cellule. R&eacute;sultats impliquent que les attributs de cellules h&ocirc;tes renforcer la coh&eacute;sion de microcolonie. Microcolonies sur la surface des cellules induisent une r&eacute;ponse cellulaire comprenant de nombreuses protub&eacute;rances cellulaires similaires &agrave; filopodes qui viennent en contact &eacute;troit avec toutes les bact&eacute;ries dans les microcolonies. Conform&eacute;ment &agrave; un r&ocirc;le de cette r&eacute;action cellulaire, h&ocirc;te cellule lipidique microdomaine perturbation simultan&eacute;ment inhibe cette r&eacute;ponse et rendu des microcolonies sensibles aux forces de tra&icirc;n&eacute;e g&eacute;n&eacute;r&eacute; par &eacute;coulement de sang. Nous puis identifi&eacute;e par une approche g&eacute;n&eacute;tique, le composant de pili type IV PilV comme un facteur d&eacute;clenchant de la restructuration de la membrane plasmique et toujours trouv&eacute; que les microcolonies form&eacute;s par un mutant pilV sont tr&egrave;s sensibles &agrave; la tension de cisaillement. Notre &eacute;tude montre que les bact&eacute;ries manipulent les fonctions des cellules h&ocirc;tes pour r&eacute;organiser la surface de la cellule h&ocirc;te pour former des structures de type filopodes qui am&eacute;liorent la coh&eacute;sion des microcolonies et donc la colonisation des vaisseaux sanguins dans les conditions s&eacute;v&egrave;res de la circulation sanguine.

Pathog&egrave;ne bact&eacute;rien extracellulaire induit host cell surface r&eacute;organisation &agrave; r&eacute;sister &agrave; la contrainte de cisaillement.

Bakterielle Infektionen, die gezielt des Blutkreislauf f&uuml;hren zu einer Vielzahl von verheerenden Krankheiten wie septischer Schock und Meningitis. Um diese entscheidende Art der Infektion zu studieren, muss seine spezifische Umgebung, vor allem die mechanischen Kr&auml;fte, die durch den Blutfluss generiert ber&uuml;cksichtigt werden. In einer fr&uuml;heren Studie mit Neisseria Meningitidis als Modell beobachteten wir, dass bakterielle Microcolonies bilden auf der Oberfl&auml;che der Endothelzellen in das Lumen des Schiffes bemerkenswert widerstandsf&auml;hig gegen&uuml;ber mechanischer Beanspruchung sind. Der vorliegenden Studie zielt darauf ab, die molekulare Basis dieser Resistenz zu identifizieren. N. Meningitidis Formen fasst unabh&auml;ngig von Wirtszellen, aber wir zeigen hier, dass diese bakterielle Aggregate beteiligt verbindenden Kr&auml;fte nicht ausreichen, um die Stabilit&auml;t der Kolonien auf Zelle Oberfl&auml;chen zu erkl&auml;ren sind. Ergebnisse implizieren, dass Host Zelle Attribute Microcolony Zusammenhalt st&auml;rken. Microcolonies auf der Zelloberfl&auml;che induzieren eine zellul&auml;re Antwort bestehend aus zahlreichen zellul&auml;ren Vorspr&uuml;nge &auml;hnlich wie Filopodium, die in engem Kontakt mit alle Bakterien in die Microcolony kommen. In &Uuml;bereinstimmung mit eine Rolle dieser zellul&auml;re Reaktion, Host Zellaufschluss Lipid Microdomain gleichzeitig gehemmt diese Antwort und Microcolonies empfindlich auf Blut flie&szlig;en generierte Widerstandskr&auml;fte gerendert. Wir dann durch einen genetischen Ansatz, die Typ-IV-Pili-Komponente PilV als ausl&ouml;senden Faktor der Plasmamembran Reorganisation, identifiziert und durchweg festgestellt, dass Microcolonies gebildet von einem PilV Mutant sehr empfindlich auf Stress zu scheren. Unsere Studie zeigt, dass Bakterien manipulieren Host-Zelle-Funktionen der Host-Zellenoberfl&auml;che zur Form Filopodium-Strukturen neu zu organisieren, die den Zusammenhalt der Microcolonies und daher Blutgef&auml;&szlig; Kolonisation unter den harten Bedingungen der Blutbahn zu st&auml;rken.

Extrazellul&auml;re bakteriellen Erreger induziert Host Zelle Oberfl&auml;che Reorganisation Schubspannung zu widerstehen.

Infezioni batteriche nel flusso sanguigno di targeting conducono a una vasta gamma di devastanti malattie quali lo shock settico e meningite. Per studiare questo fondamentale tipo di infezione, suo ambiente specifico deve essere preso in considerazione, in particolare le forze meccaniche generate dal flusso sanguigno. In un precedente studio utilizzando Neisseria meningitidis come un modello, abbiamo osservato che la microcolonies batterica formando sulla superficie delle cellule endoteliali nel lume del vaso sono straordinariamente resistente alle sollecitazioni meccaniche. Il presente studio si propone di identificare le basi molecolari di questa resistenza. Forme di N. meningitidis aggrega indipendentemente dalle cellule ospiti, eppure qui dimostriamo che le forze coesive coinvolte in questi aggregati batterici non sono sufficienti a spiegare la stabilit&agrave; delle colonie sulle superfici delle cellule. Risultati implicano che gli attributi cella host migliorano la coesione microcolony. Microcolonies sulla superficie delle cellule inducono una risposta cellulare costituito da numerosi cellulari sporgenze simili a filopodia che vengono a stretto contatto con tutti i batteri nella microcolony. Coerente con il ruolo di questa risposta cellulare, distruzione microdomain host cellulare lipidico contemporaneamente inibito questa risposta e reso microcolonies sensibili alle forze di trascinamento generato dal flusso di sangue. Abbiamo quindi identificato, da un approccio genetico, il componente di pili di tipo IV PilV come un fattore scatenante della riorganizzazione della membrana plasmatica e costantemente trovato che microcolonies formata da un mutante di pilV sono altamente sensibili alle sollecitazioni di taglio. Il nostro studio dimostra che i batteri manipolare le funzioni della cellula ospite per riorganizzare la superficie della cellula ospite per formare filopodia-come strutture che aumentano la coesione del microcolonies e pertanto colonizzazione vaso sanguigno sotto le dure condizioni di flusso sanguigno.

Extracellulare batterico patogeno induce ospite delle cellule superficiali riorganizzazione per resistere a sollecitazioni di taglio.

&#x7D30;&#x83CC;&#x611F;&#x67D3;&#x75C7;&#x306F;&#x8840;&#x6D41;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306F;&#x58CA;&#x6EC5;&#x7684;&#x306A;&#x6557;&#x8840;&#x75C7;&#x3084;&#x9AC4;&#x819C;&#x708E;&#x306A;&#x3069;&#x306E;&#x75BE;&#x60A3;&#x306E;&#x5E83;&#x3044;&#x914D;&#x5217;&#x306B; &#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x91CD;&#x8981;&#x306A;&#x7A2E;&#x985E;&#x306E;&#x611F;&#x67D3;&#x75C7;&#x3092;&#x7814;&#x7A76;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x305D;&#x306E;&#x7279;&#x5B9A;&#x306E;&#x74B0;&#x5883;&#x3001;&#x7279;&#x306B;&#x8840;&#x6D41;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x308B;&#x6A5F;&#x68B0;&#x529B;&#x8003;&#x616E;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x9AC4;&#x819C;&#x708E;&#x83CC;&#x3068;&#x3057;&#x3066;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x4EE5;&#x524D;&#x306E;&#x7814;&#x7A76;&#x3067;&#x8840;&#x7BA1;&#x5185;&#x8154;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x5F62;&#x6210;&#x7D30;&#x83CC;&#x306E; microcolonies &#x304C;&#x6A5F;&#x68B0;&#x7684;&#x30B9;&#x30C8;&#x30EC;&#x30B9;&#x306B;&#x975E;&#x5E38;&#x306B;&#x8010;&#x6027;&#x304C;&#x3042;&#x308B;&#x3092;&#x89B3;&#x5BDF;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x672C;&#x7814;&#x7A76;&#x306F;&#x3053;&#x306E;&#x62B5;&#x6297;&#x6027;&#x306E;&#x5206;&#x5B50;&#x57FA;&#x76E4;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x76EE;&#x7684;&#x3068;.(&#x540D;) &#x9AC4;&#x819C;&#x708E;&#x83CC;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x79C1;&#x305F;&#x3061;&#x3053;&#x3053;&#x3067;&#x3053;&#x308C;&#x3089;&#x7D30;&#x83CC;&#x306E;&#x51DD;&#x96C6;&#x4F53;&#x306E;&#x95A2;&#x4E0E;&#x306E;&#x51DD;&#x96C6;&#x529B;&#x304C;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306B;&#x690D;&#x6C11;&#x5730;&#x306E;&#x5B89;&#x5B9A;&#x6027;&#x3092;&#x8AAC;&#x660E;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5341;&#x5206;&#x306A;&#x3055;&#x308C;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x3001;&#x307E;&#x3060;&#x5BBF;&#x4E3B;&#x7D30;&#x80DE;&#x3068;&#x306F;&#x7121;&#x95A2;&#x4FC2;&#x3092;&#x96C6;&#x8A08;&#x3057;&#x307E;&#x3059;&#x3002;&#x30DB;&#x30B9;&#x30C8; &#x30BB;&#x30EB;&#x306E;&#x5C5E;&#x6027;&#x304C;&#x30B3;&#x30ED;&#x30CB;&#x30FC;&#x306E;&#x7D50;&#x675F;&#x3092;&#x5F37;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x307E;&#x3059;&#x3002;Microcolonies &#x7D30;&#x80DE;&#x8868;&#x9762;&#x306B;&#x7D30;&#x83CC;&#x306E;&#x30B3;&#x30ED;&#x30CB;&#x30FC;&#x306B;&#x7DCA;&#x5BC6;&#x306B;&#x9023;&#x7D61;&#x3092;&#x4ED8;&#x5C5E;&#x591A;&#x6570;&#x7D30;&#x80DE;&#x7A81;&#x8D77;&#x30D5;&#x30A3;&#x30ED;&#x30DD;&#x30C7;&#x30A3;&#x30A2;&#x306B;&#x4F3C;&#x3066;&#x304B;&#x3089;&#x6210;&#x308B;&#x7D30;&#x80DE;&#x5FDC;&#x7B54;&#x3092;&#x8A98;&#x767A;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x3042;&#x308B;&#x3053;&#x306E;&#x7D30;&#x80DE;&#x5FDC;&#x7B54;&#x306E;&#x5F79;&#x5272;&#x306F;&#x3001;&#x30DB;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x8102;&#x8CEA;&#x306E;&#x5275;&#x88FD;&#x505C;&#x6B62;&#x306F;&#x540C;&#x6642;&#x306B;&#x3053;&#x306E;&#x5FDC;&#x7B54;&#x3092;&#x6291;&#x5236;&#x3057;&#x3001;microcolonies &#x304C;&#x8840;&#x6D41;&#x751F;&#x6210;&#x6297;&#x529B;&#x306B;&#x654F;&#x611F;&#x306A;&#x30EC;&#x30F3;&#x30C0;&#x30EA;&#x30F3;&#x30B0;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x8B58;&#x5225;&#x3001;&#x907A;&#x4F1D;&#x7684;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3067;&#x306F;&#x3001;&#x30BF;&#x30A4;&#x30D7; IV &#x30D4;&#x30EA; pili &#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8; PilV &#x306E;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306E;&#x518D;&#x7DE8;&#x3001;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x56E0;&#x5B50;&#x3068;&#x3057;&#x3066;&#x3001;&#x4E00;&#x8CAB;&#x3057;&#x3066; microcolonies pilV &#x5909;&#x7570;&#x4F53;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F62;&#x6210;&#x3055;&#x308C;&#x308B;&#x305B;&#x3093;&#x65AD;&#x5FDC;&#x529B;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x6975;&#x3081;&#x3066;&#x654F;&#x611F;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306E;&#x7814;&#x7A76;&#x306F;&#x7D30;&#x83CC; microcolonies &#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x690D;&#x6C11;&#x5730;&#x8840;&#x7BA1;&#x8840;&#x6D41;&#x306E;&#x904E;&#x9177;&#x306A;&#x6761;&#x4EF6;&#x4E0B;&#x3067;&#x306E;&#x7D50;&#x675F;&#x3092;&#x5F37;&#x5316;&#x3059;&#x308B;&#x7CF8;&#x72B6;&#x4EEE;&#x8DB3;&#x69D8;&#x69CB;&#x9020;&#x3092;&#x5F62;&#x6210;&#x3059;&#x308B;&#x30DB;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x3092;&#x518D;&#x7DE8;&#x6210;&#x3059;&#x308B;&#x5BBF;&#x4E3B;&#x7D30;&#x80DE;&#x306E;&#x4F5C;&#x7528;&#x3092;&#x64CD;&#x4F5C;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x7D30;&#x80DE;&#x5916;&#x306E;&#x7D30;&#x83CC;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x305B;&#x3093;&#x65AD;&#x5FDC;&#x529B;&#x306B;&#x62B5;&#x6297;&#x3059;&#x308B;&#x30DB;&#x30B9;&#x30C8; &#x30BB;&#x30EB;&#x306E;&#x8868;&#x9762;&#x518D;&#x7DE8;&#x3092;&#x8A98;&#x5C0E;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC138;&#xADE0;&#xC131; &#xAC10;&#xC5FC;&#xC774; &#xD608; &#xB958;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xB2E4;&#xC591; &#xD55C; &#xCE58;&#xBA85;&#xC801;&#xC778; &#xBD80;&#xD328; &#xC2DC;&#xD0A4;&#xB294; &#xCDA9;&#xACA9;, &#xB1CC; &#xC218; &#xB9C9; &#xC5FC; &#xB4F1; &#xC9C8;&#xBCD1;&#xC73C;&#xB85C; &#xC774;&#xC5B4;&#xC9C8;. &#xC774; &#xC911;&#xC694; &#xD55C; &#xC885;&#xB958;&#xC758; &#xAC10;&#xC5FC; &#xC5F0;&#xAD6C;, &#xD2B9;&#xC815; &#xD658;&#xACBD; &#xACE0;&#xB824;, &#xD2B9;&#xD788;&#xC5D0;&#xC11C; &#xD608;&#xC561;&#xC758; &#xD750;&#xB984;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC131; &#xB41C; &#xAE30;&#xACC4;&#xC801;&#xC778; &#xD798;&#xC744; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. Neisseria meningitidis &#xBAA8;&#xB378;&#xB85C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC774;&#xC804; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C;&#xB294; &#xC120;&#xBC15; &#xB8E8;&#xBA58;&#xC5D0;&#xC11C; &#xB0B4; &#xD53C; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xD615;&#xC131; &#xD558;&#xB294; &#xC138;&#xADE0; microcolonies&#xB294; &#xAE30;&#xACC4;&#xC801; &#xC2A4;&#xD2B8;&#xB808;&#xC2A4;&#xC5D0; &#xC800;&#xD56D;&#xB825;&#xC774; &#xD604;&#xC800; &#xD558; &#xAC8C; &#xAD00;&#xCC30; &#xD569;&#xB2C8;&#xB2E4;. &#xD604;&#xC7AC;&#xAC00; &#xC800;&#xD56D; &#xBD84;&#xC790; &#xB2E8;&#xC704;&#xB85C; &#xD30C;&#xC545; &#xD558;&#xB294; &#xAC83;&#xC744; &#xBAA9;&#xD45C;&#xB85C; &#xACF5;&#xBD80; &#xD569;&#xB2C8;&#xB2E4;. &#xBA85;. meningitidis &#xC591;&#xC2DD;&#xC744; &#xC6B0;&#xB9AC;&#xAC00; &#xC5EC;&#xAE30; &#xACB0;&#xC18D;&#xB825; &#xC788;&#xB294; &#xC138;&#xB825; &#xC774;&#xB7EC;&#xD55C; &#xC138;&#xADE0; &#xC9D1;&#xACC4;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xC2DD;&#xBBFC;&#xC9C0;&#xC758; &#xC548;&#xC815;&#xC131;&#xC744; &#xC124;&#xBA85; &#xD558;&#xAE30;&#xC5D0; &#xCDA9;&#xBD84; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC544;&#xC9C1; &#xD638;&#xC2A4;&#xD2B8; &#xC140; &#xBCC4;&#xB3C4;&#xB85C; &#xC9D1;&#xACC4; &#xD569;&#xB2C8;&#xB2E4;. &#xACB0;&#xACFC; &#xD638;&#xC2A4;&#xD2B8; &#xC140; &#xD2B9;&#xC131; microcolony &#xC751;&#xC9D1;&#xB825;&#xC744; &#xAC15;&#xD654; &#xC758;&#xBBF8;. Microcolonies &#xC140; &#xD45C;&#xBA74;&#xC5D0; &#xC218;&#xB9CE;&#xC740; &#xC140;&#xB8F0;&#xB7EC; &#xB3CC;&#xCD9C; &#xBE44;&#xC2B7;&#xD55C; filopodia&#xB97C; microcolony&#xC5D0;&#xC11C; &#xBAA8;&#xB4E0; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC640; &#xAE34;&#xBC00; &#xD55C; &#xC811;&#xCD09;&#xC5D0;&#xC640; &#xC11C; &#xC774;&#xB8E8;&#xC5B4;&#xC9C4; &#xC138;&#xD3EC; &#xC751;&#xB2F5;&#xC744; &#xC720;&#xB3C4;. &#xC77C;&#xCE58;&#xC774; &#xC138;&#xD3EC; &#xBC18;&#xC751;&#xC758; &#xC5ED;&#xD560;, &#xD638;&#xC2A4;&#xD2B8; &#xC140; &#xC9C0;&#xC9C8; microdomain &#xC911;&#xB2E8; &#xB3D9;&#xC2DC;&#xC5D0;&#xC774; &#xC751;&#xB2F5;&#xC744; &#xC800;&#xD574; &#xBC0F; &#xD608;&#xC561; &#xD750;&#xB984; &#xC0DD;&#xC131; &#xB4DC;&#xB798;&#xADF8; &#xC138;&#xB825;&#xC5D0; &#xBBFC;&#xAC10;&#xD55C; microcolonies &#xB80C;&#xB354;&#xB9C1;. &#xC6B0;&#xB9AC;&#xB294; &#xB2E4;&#xC74C; &#xC2DD;&#xBCC4;, &#xC720;&#xC804;&#xC790; &#xC811;&#xADFC; &#xC720;&#xD615; IV pili &#xAD6C;&#xC131; &#xC694;&#xC18C; PilV &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xAC1C;&#xD3B8;&#xC758; &#xD2B8;&#xB9AC;&#xAC70; &#xC694;&#xC18C;&#xB85C; &#xD558; &#xACE0; &#xC9C0;&#xC18D;&#xC801;&#xC73C;&#xB85C; pilV &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC758;&#xD574; &#xD615;&#xC131; &#xB41C; microcolonies&#xB294; &#xC804;&#xB2E8; &#xC2A4;&#xD2B8;&#xB808;&#xC2A4;&#xC5D0; &#xB9E4;&#xC6B0; &#xBBFC;&#xAC10;&#xD55C; &#xBC1C;&#xACAC;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C;&#xB294; &#xBC15;&#xD14C;&#xB9AC;&#xC544; &#xC870;&#xC791; &#xD638;&#xC2A4;&#xD2B8; &#xC140; &#xD568;&#xC218; microcolonies&#xC640; &#xB530;&#xB77C;&#xC11C; &#xD608; &#xB958; &#xB7C9;&#xC758; &#xAC00;&#xD639;&#xD55C; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xD608;&#xAD00; &#xC2DD;&#xBBFC;&#xC9C0;&#xC758; &#xC751;&#xC9D1;&#xB825;&#xC744; &#xAC15;&#xD654; &#xD558;&#xB294; &#xD615;&#xD0DC;&#xB85C; filopodia &#xAC19;&#xC740; &#xAD6C;&#xC870;&#xC5D0; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xC7AC;&#xAD6C;&#xC131;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;.

&#xC138;&#xD3EC; &#xC678; &#xC138;&#xADE0;&#xC131; &#xBCD1;&#xC6D0; &#xCCB4; &#xC804;&#xB2E8; &#xC751;&#xB825;&#xC5D0; &#xC800;&#xD56D; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xC870;&#xC9C1; &#xAC1C;&#xD3B8;&#xC744; &#xC720;&#xB3C4; &#xD569;&#xB2C8;&#xB2E4;.

Infecciones bacterianas a la circulaci&oacute;n sangu&iacute;nea conducen a una amplia gama de enfermedades tales como choque s&eacute;ptico y meningitis devastadoras. Para estudiar este tipo crucial de infecci&oacute;n, su entorno espec&iacute;fico debe tenerse en cuenta, en particular las fuerzas mec&aacute;nicas generadas por el flujo de sangre. En un estudio anterior usando Neisseria meningitidis como modelo, observamos que microcolonias bacterianas formando en la superficie de la c&eacute;lula endotelial en el lumen del vaso son extremadamente resistentes a la tensi&oacute;n mec&aacute;nica. El presente estudio pretende identificar la base molecular de esta resistencia. Formas de N. meningitidis agregados independientemente de las c&eacute;lulas del hu&eacute;sped, pero aqu&iacute; demostramos que cohesivo las fuerzas involucradas en estos agregados bacterianos no son suficientes para explicar la estabilidad de las colonias en las superficies de la c&eacute;lula. Resultados implican que atributos de c&eacute;lula anfitri&oacute;n mejoran cohesi&oacute;n microcolony. Microcolonias en la superficie celular inducen una respuesta celular compuesta por numerosas protuberancias celulares similares a filopodios que vienen en contacto con todas las bacterias en el microcolony. Consistente con un papel de esta respuesta celular, anfitri&oacute;n c&eacute;lula l&iacute;pidos microdomain perturbaci&oacute;n simult&aacute;neamente inhibe esta respuesta y prestados microcolonias sensibles a las fuerzas de arrastre generados por el flujo de sangre. Luego identificamos, por un enfoque gen&eacute;tico, el componente de pili de tipo IV PilV como un factor desencadenante de la reorganizaci&oacute;n de la membrana plasm&aacute;tica y encontradas consistentemente que microcolonias formados por un mutante de pilV son muy sensibles a la tensi&oacute;n de esquileo. Nuestro estudio muestra que las bacterias manipulan funciones de la c&eacute;lula hu&eacute;sped para reorganizar la superficie de la c&eacute;lula hu&eacute;sped para formar filopodios-como las estructuras que mejoran la cohesi&oacute;n de las microcolonias y por lo tanto la colonizaci&oacute;n del vaso sangu&iacute;neo bajo las duras condiciones de la circulaci&oacute;n sangu&iacute;nea.

Pat&oacute;geno bacteriano extracelular induce reorganizaci&oacute;n superficial de c&eacute;lula de anfitri&oacute;n para resistir la tensi&oacute;n de esquileo.

T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.

T-cell artificial focal triggering tools: linking surface interactions with cell response.

The T-cell is one of the main players in the mammalian immune response. It ensures antigen recognition at the surface of antigen-presenting cells in a complex and highly sensitive and specific process, in which the encounter of the T-cell receptor with the agonist peptide associated with the major histocompatibility complex triggers T-cell activation. While signaling pathways have been elucidated in increasing detail, the mechanism of TCR triggering remains highly controversial despite active research published in the past 10 years. In this paper, we present a short overview of pending questions on critical initial events associated with T-cell triggering. In particular, we examine biophysical approaches already in use, as well as future directions. We suggest that the most recent advances in fluorescence super-resolution imaging, coupled with the new classes of genetic fluorescent probes, will play an important role in elucidation of the T-cell triggering mechanism. Beyond this aspect, we predict that exploration of mechanical cues in the triggering process will provide new clues leading to clarification of the entire mechanism.

Early T-cell activation biophysics.

In natural conditions many bacterial populations are found as surface-attached communities exhibiting features distinct from those of planktonic cells. We focus here on the question of initial adhesion, the mechanisms of which are still far from being fully understood. Recently, the frontier between microbiologists and physicists has become increasingly permeable, boosting implementation of new methodological approaches for better elucidating the intricate aspects of initial bacterial adhesion. After discussing briefly the main sources of complexity that confuse the understanding of the early steps of cell-surface attachment, we present a selection of physical methods enabling real-time measurement of early adhesion kinetics in live cells. We also discuss the limitations and pitfalls that might appear when applying such methodologies - initially designed for studying physically ideal systems - to analysis of these, more complex, living systems. We address mainly on the use of dispersed-surfaces flow cytometry (DS-FCM), quartz microbalance (QCM) and surface plasmon resonance (SPR) approaches, and give a brief survey of new perspectives in optical microscopy. We conclude that the use of combined and multiparametric technical approaches will lead to significant advances in providing a comprehensive understanding of the early events in bacterial adhesion.

Short time-scale bacterial adhesion dynamics.

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored simultaneously to verify the cell commitment in the activation process. [Ca(2+)](i) increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process.

Force generation upon T cell receptor engagement.

Liquid colloids, in the form of droplets grafted with specific biomolecules, are emerging as potential biomimetic systems. Here we show for the first time the possibility of forming hybrid conjugates between an advanced living cell model, the T-cell of the Jurkat cell line, and a specifically grafted droplet. Using T-cells expressing a fluorescent chimeric protein associated with the TCR/CD3 complex and fluorescent ligand-grafted droplets, we demonstrate formation of an interfacial contact concentrated in linking molecules, the morphology and dynamics of which strongly depend on the targeted receptor. The sequence of events ranges from the initial concentration of molecules following an unbound molecule gradient to active actin-driven spreading and fragmentation of the contact, ending with droplet internalization. We observed synchronized colocalization of receptors and ligands driven by cell dynamics and closely mirrored by the droplet interface. Using intracellular calcium probe Fura-2, we also showed that the cell/droplet interaction can trigger the T-cell signaling cascade. By examining molecular dynamics using FRAP measurements, we observed a nearly frozen cell droplet joining interface. Taken together, our results point to liquid colloids as promising new tools both for probing cell surface interactions and receptor dynamics and for manipulating biological cell functions.

Biomimetic droplets for artificial engagement of living cell surface receptors: the specific case of the T-cell.

Most bacteria live in the form of adherent communities forming three-dimensional material anchored to artificial or biological surfaces, with profound impact on many human activities. Biofilms are recognized as complex systems but their physical properties have been mainly studied from a macroscopic perspective. To determine biofilm local mechanical properties, reveal their potential heterogeneity, and investigate their relation to molecular traits, we have developed a seemingly new microrheology approach based on magnetic particle infiltration in growing biofilms. Using magnetic tweezers, we achieved what was, to our knowledge, the first three-dimensional mapping of the viscoelastic parameters on biofilms formed by the bacterium Escherichia coli. We demonstrate that its mechanical profile may exhibit elastic compliance values spread over three orders of magnitude in a given biofilm. We also prove that heterogeneity strongly depends on external conditions such as growth shear stress. Using strains genetically engineered to produce well-characterized cell surface adhesins, we show that the mechanical profile of biofilm is exquisitely sensitive to the expression of different surface appendages such as F pilus or curli. These results provide a quantitative view of local mechanical properties within intact biofilms and open up an additional avenue for elucidating the emergence and fate of the different microenvironments within these living materials.

Article	Year
Wetting of liquid droplets on living cells. Journal of colloid and interface science\| PubMed ID: 12505074	2002
Specific recognition of macroscopic objects by the cell surface: evidence for a receptor density threshold revealed by micrometric particle binding characteristics. Biophysical journal\| PubMed ID: 15111442	2004
Molecular bond formation between surfaces: anchoring and shearing effects. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 16378410	2006
Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16894146	2006
A short-time scale colloidal system reveals early bacterial adhesion dynamics. PLoS biology\| PubMed ID: 18613749	2008
Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress. PLoS pathogens\| PubMed ID: 19247442	2009
T-cell artificial focal triggering tools: linking surface interactions with cell response. PloS one\| PubMed ID: 19274104	2009
Early T-cell activation biophysics. HFSP journal\| PubMed ID: 20514131	2009
Short time-scale bacterial adhesion dynamics. Advances in experimental medicine and biology\| PubMed ID: 21557073	2011
Force generation upon T cell receptor engagement. PloS one\| PubMed ID: 21572959	2011
Biomimetic droplets for artificial engagement of living cell surface receptors: the specific case of the T-cell. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 22414256	2012
Mapping of bacterial biofilm local mechanics by magnetic microparticle actuation. Biophysical journal\| PubMed ID: 22995513	2012

Nelly Henry

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JoVE Journal Publications

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