Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

University of Virginia Health Sciences Center

&#x7535;&#x538B;&#x95E8;&#x63A7;&#x9499;&#x79BB;&#x5B50;&#x901A;&#x9053;&#x8C03;&#x8282; Ca2 + &#x6D3B;&#x52A8;&#x4ECB;&#x5BFC;&#x7684;&#x795E;&#x7ECF;&#x53D1;&#x80B2;&#xFF0C;&#x5305;&#x62EC;&#x795E;&#x7ECF;&#x611F;&#x5E94;&#x3001; &#x795E;&#x7ECF;&#x9012;&#x8D28;&#x8868;&#x578B;&#x89C4;&#x8303;&#x548C;&#x7A81;&#x8D77;&#x7684;&#x8BB8;&#x591A;&#x65B9;&#x9762;&#x5177;&#x6709;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x975E;&#x6D32;&#x722A;&#x87FE;&#x80DA;&#x80CE;&#xFF0C;&#x5728;&#x5B9A;&#x4E49;&#x7684;&#x901A;&#x9053;&#x7684;&#x52A8;&#x529B;&#x5B66;&#x5C5E;&#x6027; 10 &#x7684;&#x5B54; 1 &#x53D7;&#x4F53;&#x53CA;&#x5176;&#x4E9A;&#x5F00;&#x53D1;&#x8FC7;&#x7A0B;&#x4E2D;&#x63CF;&#x8FF0;&#x7684;&#x65F6;&#x7A7A;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x3002;&#x539F;&#x4F4D;&#x6742;&#x4EA4;&#x6280;&#x672F;&#x6307;&#x793A; CaV1.2&#x3001; CaV2.1&#x3001; CaV2.2&#x3001; CaV3.2 &#x8868;&#x793A;&#x5728;&#x6574;&#x4E2A;&#x795E;&#x7ECF;&#x7BA1; neurula &#x9636;&#x6BB5;&#x3002;&#x8FD9;&#x4E9B; CaV1.3 &#x548C; CaV2.3&#xFF0C;&#x540E;&#x671F;&#x5C3E;&#x82BD;&#xFF0C;&#x5F00;&#x59CB;&#x5728; CaV3.1 &#x548C;&#x65E9;&#x671F;&#x5C3E;&#x82BD;&#x9636;&#x6BB5;&#xFF0C;&#x5728;&#x590D;&#x6742;&#x7684;&#x6A21;&#x5F0F;&#xFF0C;&#x5728;&#x8111;&#x548C;&#x810A;&#x9AD3;&#x901A;&#x8FC7;&#x6E38;&#x6CF3;&#x874C;&#x86AA;&#x9636;&#x6BB5;&#x5185;&#x68C0;&#x6D4B;&#x5230;&#x3002;&#x4E9A;&#x57FA; 1 &#x53D7;&#x4F53;&#x53CA;&#x5176;&#x5404;&#x79CD;&#x9644;&#x52A0;&#x8868;&#x8FBE;&#x5F0F;&#x89C2;&#x5BDF;&#x9885;&#x795E;&#x7ECF;&#x8282;&#x3001; &#x89C6;&#x7F51;&#x819C;&#x3001; &#x55C5;&#x4E0A;&#x76AE;&#x3001; &#x677E;&#x679C;&#x817A;&#x548C;&#x5FC3;&#x5230;&#x3002;&#x4E0D;&#x540C; 1 &#x53D7;&#x4F53;&#x53CA;&#x5176;&#x4E9A;&#x57FA;&#x7684;&#x72EC;&#x7279;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x8868;&#x660E;&#x4ED6;&#x4EEC;&#x6B63;&#x5728;&#x7CBE;&#x786E;&#x65F6;&#x7A7A;&#x8C03;&#x63A7;&#xFF0C;&#x5728;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x671F;&#x95F4;&#x670D;&#x52A1;&#x7684;&#x5177;&#x4F53;&#x804C;&#x80FD;&#x3002;

&#x514B;&#x9686;&#x53CA;&#x7535;&#x538B;&#x95E8;&#x63A7;&#x6027;&#x9499;&#x901A;&#x9053;&#x5728;&#x5F00;&#x53D1;&#x8FC7;&#x7A0B;&#x4E2D;&#x7684;&#x722A;&#x87FE; 1 &#x53D7;&#x4F53;&#x53CA;&#x5176;&#x4E9A;&#x5355;&#x4F4D;&#x3002;

Voltage-gated calcium channels play a critical role in regulating the Ca2+ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore-forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that CaV1.2, CaV2.1, CaV2.2, and CaV3.2 are expressed during neurula stages throughout the neural tube. These, along with CaV1.3 and CaV2.3, beginning at early tail bud stages, and CaV3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development.

Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during development.

Les canaux calciques voltage-d&eacute;pendants jouent un r&ocirc;le essentiel dans la r&eacute;gulation de l&#39;activit&eacute; de Ca2 + qui intervient dans nombreux aspects du d&eacute;veloppement neuronal, y compris l&#39;induction neurale, sp&eacute;cification du ph&eacute;notype neurotransmetteur et la croissance des neurites. En utilisant les embryons de Xenopus laevis, nous d&eacute;crivons les patrons d&#39;expression spatiale et temporelle au cours du d&eacute;veloppement des 10 pore-forming alpha1 sous-unit&eacute;s qui d&eacute;finissent les propri&eacute;t&eacute;s cin&eacute;tiques des canaux. Hybridation in situ indique que CaV1.2, CaV2.1, CaV2.2 et CaV3.2 sont exprim&eacute;es au cours des stades neurula tout au long du tube neural. Ceux-ci, ainsi que CaV1.3 et CaV2.3, d&egrave;s les premiers stades de bourgeon de queue et CaV3.1 aux derniers stades de bourgeon de queue, sont d&eacute;tect&eacute;s dans des motifs complexes dans le cerveau et la moelle &eacute;pini&egrave;re par &eacute;tapes de t&ecirc;tard de natation. Expression suppl&eacute;mentaires de diverses sous-unit&eacute;s alpha 1 a &eacute;t&eacute; observ&eacute;e dans les ganglions cr&acirc;niens, r&eacute;tine, &eacute;pith&eacute;lium olfactif, glande pin&eacute;ale et le coeur. Les patrons d&#39;expression unique pour les sous-unit&eacute;s diff&eacute;rentes alpha1 sugg&egrave;re qu&#39;ils sont sous r&eacute;glementation spatiale et temporelle pr&eacute;cise et sont au service des fonctions sp&eacute;cifiques au cours du d&eacute;veloppement embryonnaire.

Clonage et caract&eacute;risation de voltage-d&eacute;pendants de calcium channel alpha1 sous-unit&eacute;s de Xenopus laevis au cours du d&eacute;veloppement.

Spannung-Gating Kalzium-Kan&auml;le spielen eine entscheidende Rolle bei der Regulierung der Ca2 +-Aktivit&auml;t, die viele Aspekte der neuronalen Entwicklung, einschlie&szlig;lich der neuronalen Induktion, Neurotransmitter Ph&auml;notyp Spezifikation und Neurit Auswuchs vermittelt. Xenopus Laevis Embryonen verwenden, beschreiben wir die r&auml;umliche und zeitliche Muster w&auml;hrend der Entwicklung der 10 Pore-bildenden alpha1-Untereinheiten, die der Kan&auml;le kinetischen Eigenschaften definieren. In situ Hybridisierung zeigt, dass CaV1.2, CaV2.1, CaV2.2 und CaV3.2 w&auml;hrend Neurula-B&uuml;hnen das Neuralrohr ausgedr&uuml;ckt werden. Diese, zusammen mit CaV1.3 und CaV2.3, beginnend bei Schwanz Knospe Anfangsphase und CaV3.1 am Schwanz Knospe Sp&auml;tstadium, werden in komplexen Mustern in das Gehirn und das R&uuml;ckenmark durch Schwimmen Kaulquappe Stadien erkannt. Zus&auml;tzlicher Ausdruck der verschiedenen alpha1-Untereinheiten wurde in die kraniale Ganglien, Netzhaut, olfaktorischen Epithel, Zirbeldr&uuml;se und Herzen beobachtet. Das einzigartige Muster f&uuml;r die verschiedenen alpha1-Untereinheiten schl&auml;gt vor, sie sind unter pr&auml;zisen r&auml;umlichen und zeitlichen Regelungen und dienen bestimmte Funktionen w&auml;hrend der Embryonalentwicklung.

Klonierung und Charakterisierung von Spannung-Gating Kalzium-Kanal alpha1-Untereinheiten in Xenopus Laevis w&auml;hrend der Entwicklung.

Canali del calcio voltaggio-dipendente giocano un ruolo critico nella regolazione dell&#39;attivit&agrave; Ca2 + che media molti aspetti dello sviluppo neurale, tra cui induzione neurale, specifica del fenotipo di neurotrasmettitore e crescita dei neuriti. Utilizzando embrioni di Xenopus laevis, descriviamo i pattern di espressione spaziale e temporale durante lo sviluppo del 10 subunit&agrave; alfa1 formando dei pori che definiscono propriet&agrave; cinetica canali. Ibridazione in situ indica che CaV1.2, CaV2.1, CaV2.2 e CaV3.2 sono espresse nelle fasi di neurula in tutto il tubo neurale. Questi, insieme a CaV1.3 e CaV2.3, inizio alle prime fasi di germoglio di coda e CaV3.1 alle ultime fasi di gemma di coda, sono rilevati in modelli complessi all&#39;interno del cervello e del midollo spinale attraverso fasi girino di nuoto. Ulteriore espressione di diverse subunit&agrave; alfa1 &egrave; stata osservata nel gangli cranici, retina, epitelio olfattivo, ghiandola pineale e nel cuore. I modelli di espressione univoco per le subunit&agrave; alfa1 differenti suggerisce sono sotto regolazione spaziale e temporale precisa e servono specifiche funzioni durante lo sviluppo embrionale.

Clonazione e caratterizzazione del calcio voltaggio-dipendenti del canale subunit&agrave; alfa1 di Xenopus laevis durante lo sviluppo.

&#x96FB;&#x5727;&#x96FB;&#x4F4D;&#x4F9D;&#x5B58;&#x6027;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x795E;&#x7D4C;&#x8A98;&#x5C0E;&#x795E;&#x7D4C;&#x7A81;&#x8D77;&#x4F38;&#x9577;&#x3001;&#x795E;&#x7D4C;&#x4F1D;&#x9054;&#x7269;&#x8CEA;&#x8868;&#x73FE;&#x578B;&#x4ED5;&#x69D8;&#x306A;&#x3069;&#x3092;&#x542B;&#x3080;&#x3001;&#x795E;&#x7D4C;&#x306E;&#x958B;&#x767A;&#x306E;&#x591A;&#x304F;&#x306E;&#x5074;&#x9762;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B; ca 2 + &#x6D3B;&#x6027;&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x80DA;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6642;&#x7A7A;&#x306E;&#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x304C;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306E;&#x52D5;&#x529B;&#x5B66;&#x7684;&#x7279;&#x6027;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x3001;10 &#x7D30;&#x5B54;&#x5F62;&#x6210; &alpha; 1 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x958B;&#x767A;&#x4E2D;&#x306B;&#x8A18;&#x8FF0;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x307E;&#x307E;&#x306E;&#x4EA4;&#x914D;&#x306F;&#x3001;CaV1.2&#x3001;CaV2.1&#x3001;CaV2.2&#x3001;&#x304A;&#x3088;&#x3073; CaV3.2 neurula &#x6BB5;&#x968E;&#x795E;&#x7D4C;&#x7BA1;&#x3092;&#x901A;&#x3057;&#x3066;&#x8868;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306F;&#x3001;CaV1.3 &#x3068; CaV2.3&#x3001;&#x521D;&#x671F;&#x5C3E;&#x82BD;&#x306E;&#x6BB5;&#x968E;&#x3001;&#x5F8C;&#x671F;&#x30C6;&#x30FC;&#x30EB;&#x82BD;&#x6BB5;&#x968E;&#x3067;&#x306F;&#x3001;CaV3.1 &#x3067;&#x59CB;&#x3081;&#x3066;&#x3001;&#x8133;&#x3068;&#x810A;&#x9AC4;&#x30AA;&#x30BF;&#x30DE;&#x30B8;&#x30E3;&#x30AF;&#x30B7;&#x6BB5;&#x968E;&#x6C34;&#x6CF3;&#x3092;&#x901A;&#x3058;&#x3066;&#x5185;&#x306E;&#x8907;&#x96D1;&#x306A;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3067;&#x691C;&#x51FA;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x8FFD;&#x52A0;&#x306E;&#x5F0F;&#x5404;&#x7A2E; &alpha; 1 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x982D;&#x84CB;&#x795E;&#x7D4C;&#x7BC0;, &#x7DB2;&#x819C;, &#x55C5;&#x4E0A;&#x76AE;&#x3001;&#x677E;&#x679C;&#x4F53;&#x3068;&#x5FC3;&#x3067;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306E;&#x7570;&#x306A;&#x308B; &alpha; 1 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3067;&#x306F;&#x6642;&#x7A7A;&#x306E;&#x7CBE;&#x5BC6;&#x306A;&#x898F;&#x5247;&#x306E;&#x4E0B;&#x3067;&#x3001;&#x840C;&#x82BD;&#x671F;&#x306E;&#x958B;&#x767A;&#x4E2D;&#x306B;&#x7279;&#x5B9A;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x308B;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x96FB;&#x4F4D;&#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3068;&#x30AD;&#x30E3;&#x30E9;&#x30AF;&#x30BF;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3; &alpha; 1 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8; &#x30A2;&#x30D5;&#x30EA;&#x30AB;&#x30C4;&#x30E1;&#x30AC;&#x30A8;&#x30EB;&#x306E;&#x958B;&#x767A;&#x4E2D;&#x306B;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC804;&#xC555; &#xAC1C;&#xD3D0; &#xCE7C;&#xC298; &#xCC44;&#xB110; &#xC870;&#xC808; &#xC2E0;&#xACBD; &#xC720;&#xB3C4;, &#xC2E0;&#xACBD; &#xC804;&#xB2EC; &#xBB3C;&#xC9C8; &#xD615; &#xC0AC;&#xC591; &#xBC0F; neurite &#xD30C;&#xC0DD;&#xBB3C;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#xC2E0;&#xACBD; &#xAC1C;&#xBC1C;&#xC758; &#xB9CE;&#xC740; &#xCE21;&#xBA74;&#xC744; &#xC911;&#xC7AC; &#xD558;&#xB294; Ca2 + &#xD65C;&#xB3D9;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. Xenopus laevis &#xBC30;&#xC544;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xCC44;&#xB110; &#xD0A4;&#xB124;&#xD2F1; &#xC18D;&#xC131;&#xC744; &#xC815;&#xC758; &#xD558;&#xB294; 10 &#xAE30; &#xACF5; &#xD615;&#xC131; alpha1 subunits&#xC758; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xACF5;&#xAC04; &#xBC0F; &#xC2DC;&#xAC04; &#xC2DD; &#xD328;&#xD134; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xC81C;&#xC790;&#xB9AC; &#xAD50; &#xC7A1; CaV1.2, CaV2.1, CaV2.2, &#xBC0F; CaV3.2 &#xC2E0;&#xACBD; &#xD29C;&#xBE0C;&#xC5D0; &#xAC78;&#xCCD0; neurula &#xB2E8;&#xACC4; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. CaV1.3 &#xBC0F; CaV2.3, &#xB9D0; &#xAF2C;&#xB9AC; &#xAF43; &#xBD09; &#xC624;&#xB9AC; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xCD08;&#xAE30; &#xAF2C;&#xB9AC; &#xAF43; &#xBD09; &#xC624;&#xB9AC; &#xB2E8;&#xACC4; &#xBC0F; Cav3.1&#xC5D0;&#xC11C; &#xC2DC;&#xC791;&#xC774; &#xB450;&#xB1CC;&#xC640; &#xCC99;&#xC218; &#xC218;&#xC601; &#xC62C;&#xCC59;&#xC774; &#xB2E8;&#xACC4;&#xB97C; &#xD1B5;&#xD574; &#xB0B4;&#xC5D0;&#xC11C; &#xBCF5;&#xC7A1; &#xD55C; &#xD328;&#xD134;&#xC73C;&#xB85C; &#xAC80;&#xC0C9; &#xB429;&#xB2C8;&#xB2E4;. &#xB2E4;&#xC591; &#xD55C; alpha1 subunits&#xC758; &#xCD94;&#xAC00; &#xD45C;&#xD604; &#xB450;&#xAC1C;&#xACE8; &#xC2E0;&#xACBD;, &#xB9DD;&#xB9C9;, &#xD6C4; &#xAC01; &#xC0C1;&#xD53C;, &#xB1CC;&#xC758; &#xC1A1;&#xACFC;&#xC120;, &#xBC0F; &#xC2EC;&#xC7A5;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4;. &#xB2E4;&#xB978; alpha1 subunits&#xC758; &#xB3C5;&#xD2B9;&#xD55C; &#xC2DD; &#xD328;&#xD134; &#xADF8;&#xB4E4;&#xC740; &#xC815;&#xD655; &#xD558; &#xAC8C; &#xACF5;&#xAC04; &#xBC0F; &#xC2DC;&#xAC04; &#xADDC;&#xC815;&#xC740; &#xBC30;&#xC544; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD2B9;&#xC815; &#xAE30;&#xB2A5;&#xC744; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xAC74;&#xC758; &#xD55C;&#xB2E4;.

&#xBCF5;&#xC81C; &#xBC0F; &#xC804;&#xC555; &#xAC1C;&#xD3D0; &#xCE7C;&#xC298;&#xC758; &#xD2B9;&#xC131;&#xD654; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; alpha1 subunits Xenopus laevis&#xC758; &#xCC44;&#xB110;.

Canales voltaje-bloqueados del calcio juegan un papel cr&iacute;tico en la regulaci&oacute;n de la actividad Ca2 + que interviene en muchos aspectos del desarrollo neurol&oacute;gico, incluyendo la inducci&oacute;n neural, especificaci&oacute;n del fenotipo de neurotransmisor y consecuencia neurita. Utilizando embriones de Xenopus laevis, describimos los patrones espaciales y temporales de expresi&oacute;n durante el desarrollo de las 10 formaci&oacute;n de poro alpha1 subunidades que definen las propiedades cin&eacute;ticas de los canales. Hibridaci&oacute;n in situ indica que CaV1.2, CaV2.1, modulates y CaV3.2 se expresan en N&eacute;urula etapas a lo largo del tubo neural. Estos, junto con CaV1.3 y CaV2.3, a partir de las primeras fases de brote de la cola y CaV3.1 en etapas tard&iacute;as del brote de la cola, se detectan en patrones complejos dentro del cerebro y la m&eacute;dula espinal a trav&eacute;s de etapas de renacuajo de nataci&oacute;n. Expresi&oacute;n adicional de varias subunidades alpha1 fue observada en los ganglios craneales, retina, epitelio olfativo, gl&aacute;ndula pineal y coraz&oacute;n. Los patrones de expresi&oacute;n &uacute;nica para las subunidades diferentes alpha1 sugieren tal reglamento precisa espacial y temporal y est&aacute;n cumpliendo funciones espec&iacute;ficas durante el desarrollo embrionario.

Clonaci&oacute;n y caracterizaci&oacute;n de calcio voltaje-bloqueados del canal alpha1 subunidades en Xenopus laevis durante el desarrollo.

Infection with the human-specific bacterial pathogen Neisseria gonorrhoeae triggers a potent, local inflammatory response driven by polymorphonuclear leukocytes (neutrophils or PMNs). PMNs are terminally differentiated phagocytic cells that are a vital component of the host innate immune response and are the first responders to bacterial and fungal infections. PMNs possess a diverse arsenal of components to combat microorganisms, including the production of reactive oxygen species and release of degradative enzymes and antimicrobial peptides. Despite numerous PMNs at the site of gonococcal infection, N. gonorrhoeae can be cultured from the PMN-rich exudates of individuals with acute gonorrhea, indicating that some bacteria resist killing by neutrophils. The contribution of PMNs to gonorrheal pathogenesis has been modeled in vivo by human male urethral challenge and murine female genital inoculation and in vitro using isolated primary PMNs or PMN-derived cell lines. These systems reveal that some gonococci survive and replicate within PMNs and suggest that gonococci defend themselves against PMNs in two ways: they express virulence factors that defend against PMNs' oxidative and non-oxidative antimicrobial components, and they modulate the ability of PMNs to phagocytose gonococci and to release antimicrobial components. In this review, we will highlight the varied and complementary approaches used by N. gonorrhoeae to resist clearance by human PMNs, with an emphasis on gonococcal gene products that modulate bacterial-PMN interactions. Understanding how some gonococci survive exposure to PMNs will help guide future initiatives for combating gonorrheal disease.

Resistance of Neisseria gonorrhoeae to neutrophils.

Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome.

M. Brittany Johnson

Department of Microbiology,
Immunology,
and Cancer Biology,
Department of Microbiology, Immunology, and Cancer Biology

University of Virginia Health Sciences Center

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

M. Brittany Johnson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology,Immunology,and Cancer Biology,Department of Microbiology, Immunology, and Cancer Biology

University of Virginia Health Sciences Center

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Department of Microbiology,
Immunology,
and Cancer Biology,
Department of Microbiology, Immunology, and Cancer Biology