expression-exogenous-antigens-mycobacterium-bovis-bcg-vaccine-via-non

Expression of Exogenous Antigens in the <em>Mycobacterium bovis</em> BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

University of British Columbia

The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study of mycobacterial genetics, which culminated in the publication of the full genome sequence of many mycobacterial strains. Since then, many genes and open reading frames of unknown function have been described and the expression of their encoded proteins is critical toward understanding the pathogenesis of TB and developing therapeutic and preventive strategies. Therefore there is an increased need for highly efficient methods for cloning of mycobacterial genes, as the limited cloning flexibility of current Escherichia coli-mycobacteria shuttle vectors remains a frequent impediment in genetic manipulation of mycobacteria. In order to overcome this limitation, we have converted representative extrachromosomal and integrative vectors into multiple destination mycobacterial vectors for one-step and restriction enzyme-free recombination cloning methodology that uses in vitro site-specific recombination. We provide several examples that highlight the potential of recombination cloning for gene expression in slow and fast-growing mycobacteria. Thus, a gene of interest can be transferred by simple recombination into our mycobacterial destination vectors, which serve a multitude of functional genomic studies.

A broad-range of recombination cloning vectors in mycobacteria.

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Mycobacterial nucleoside diphosphate kinase blocks phagosome maturation in murine RAW 264.7 macrophages.

Pannexins &#x662F;&#x7531;&#x5176;&#x5BF9;&#x6606;&#x866B;&#x7684;&#x7F1D;&#x9699;&#x8FDE;&#x63A5;&#x86CB;&#x767D;&#x540C;&#x6E90;&#x6807;&#x8BC6;&#x7684;&#x4E91;&#x5C71;&#x901A;&#x9053;&#x86CB;&#x767D;&#x7684;&#x4E00;&#x7C7B;&#x3002;Pannexin &#x5BB6;&#x5EAD;&#x7531;&#x4E09;&#x4E2A;&#x6210;&#x5458;&#x3001; Panx1&#x3001; Panx2 &#x548C; Panx3&#xFF0C;&#x7EC4;&#x6210;&#xFF0C;&#x6BCF;&#x4E2A;&#x8FD9;&#x4E9B;&#x86CB;&#x767D;&#x8D28;&#x5728;&#x7EC6;&#x80DE;&#x8FC7;&#x7A0B;&#x4E2D;&#x53D1;&#x6325;&#x7684;&#x4F5C;&#x7528;&#x662F;&#x4ECD;&#x5728;&#x8C03;&#x67E5;&#x4E2D;&#x3002;Panx3 &#x8868;&#x8FBE;&#x5F0F;&#x7684;&#x524D;&#x51E0;&#x4EFD;&#x62A5;&#x544A;&#x8868;&#x660E;&#x9AA8;&#x9ABC;&#x808C;&#x7EC4;&#x7EC7;&#x4E2D;&#x5BCC;&#x96C6;&#x7684;&#x6240;&#x4EE5;&#x6211;&#x4EEC;&#x6709;&#x8FDB;&#x4E00;&#x6B65;&#x8C03;&#x67E5;&#x8FD9;&#x79CD;&#x5206;&#x5E03;&#x4E0E;&#x514D;&#x75AB;&#x8367;&#x5149;&#x6CD5;&#x6D4B;&#x91CF;&#x53D1;&#x5C55;&#x7684;&#x5C0F;&#x9F20;&#x80DA;&#x80CE;&#x3002;&#x9AD8;&#x6C34;&#x5E73;&#x7684; Panx3 intramembranous &#x9885;&#x9762;&#x6241;&#x9AA8;&#xFF0C;&#x4EE5;&#x53CA;&#x957F;&#x9AA8;&#x7684;&#x9611;&#x5C3E;&#x548C;&#x8F74;&#x5411;&#x9AA8;&#x67B6;&#x4E2D;&#x53D1;&#x73B0;&#x3002;&#x8FD9;&#x79CD;&#x5206;&#x5E03;&#x662F;&#x8868;&#x8FBE;&#x5F0F;&#x7684;&#x6210;&#x9AA8;&#x7EC6;&#x80DE;&#x548C;&#x80A5;&#x5927;&#x8F6F;&#x9AA8;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x7ED3;&#x679C;&#x3002;&#x6B64;&#x5916;&#xFF0C;Panx3 &#x542F;&#x52A8;&#x5B50;&#x5305;&#x542B;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x53C2;&#x4E0E;&#x9AA8;&#x7684;&#x5F62;&#x6210;&#xFF0C;&#x5047;&#x5B9A;&#x7ED1;&#x5B9A;&#x7AD9;&#x70B9;&#x548C;&#x6211;&#x4EEC;&#x5C55;&#x793A;&#x57FA;&#x5730;-275 &#x548C;-283 &#x4E4B;&#x95F4;&#x7684;&#x5E8F;&#x5217;&#x662F;&#x987A;&#x5E94; Runx2 &#x6FC0;&#x6D3B;&#x3002;&#x4E24;&#x8005;&#x5408;&#x8BA1;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x8868;&#x660E; Panx3 &#x53EF;&#x80FD;&#x4F1A;&#x8D77;&#x5230;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#x5728;&#x9AA8;&#x9ABC;&#x53D1;&#x80B2;&#x548C; Runx2 &#x4F9D;&#x8D56;&#x7684;&#x65B0;&#x9776;&#x6807;&#x7684;&#x4FE1;&#x53F7;&#x3002;

Pannexin 3 &#x662F; Runx2&#xFF0C;&#x6210;&#x9AA8;&#x7EC6;&#x80DE;&#x548C;&#x6210;&#x719F;&#x7684;&#x751F;&#x957F;&#x677F;&#x8F6F;&#x9AA8;&#x7EC6;&#x80DE;&#x6240;&#x8868;&#x8FBE;&#x7684;&#x65B0;&#x9776;&#x6807;&#x3002;

Pannexins are a class of chordate channel proteins identified by their homology to insect gap junction proteins. The pannexin family consists of three members, Panx1, Panx2, and Panx3, and the role each of these proteins plays in cellular processes is still under investigation. Previous reports of Panx3 expression indicate enrichment in skeletal tissues, so we have further investigated this distribution by surveying the developing mouse embryo with immunofluorescence. High levels of Panx3 were detected in intramembranous craniofacial flat bones, as well as long bones of the appendicular and axial skeleton. This distribution is the result of expression in both osteoblasts and hypertrophic chondrocytes. Furthermore, the Panx3 promoter contains putative binding sites for transcription factors involved in bone formation, and we show that the sequence between bases -275 and -283 is responsive to Runx2 activation. Taken together, our data suggests that Panx3 may serve an important role in bone development, and is a novel target for Runx2-dependent signaling.

Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes.

Pannexins sont une classe de prot&#xE9;ines canaux chord&#xE9;s identifi&#xE9;s par leur homologie avec les insectes prot&#xE9;ines des jonctions lacunaires. La famille se compose pannexine de trois membres, Panx1, les Panx2 et Panx3, et le r&#xF4;le de chacune de ces prot&#xE9;ines joue dans les processus cellulaires est toujours sous enqu&#xEA;te. Les rapports pr&#xE9;c&#xE9;dents de Panx3 expression indiquent un enrichissement des tissus squelettiques, de sorte que nous avons examin&#xE9; plus avant cette distribution par l&#x26;#39;arpentage l&#x26;#39;embryon de souris en d&#xE9;veloppement avec immunofluorescence. Des niveaux &#xE9;lev&#xE9;s de Panx3 ont &#xE9;t&#xE9; d&#xE9;tect&#xE9;s dans intramembranaires craniofaciales os plats, ainsi que les os longs de l&#x26;#39;appendiculaire et squelette axial. Cette r&#xE9;partition est le r&#xE9;sultat de l&#x26;#39;expression dans les deux ost&#xE9;oblastes et les chondrocytes hypertrophiques. En outre, le promoteur contient Panx3 sites de liaison putatifs pour les facteurs de transcription impliqu&#xE9;s dans la formation osseuse, et nous montrons que la s&#xE9;quence entre les bases -275 -283 et est sensible &#xE0; l&#x26;#39;activation Runx2. Pris dans leur ensemble, nos donn&#xE9;es sugg&#xE8;rent que Panx3 peut jouer un r&#xF4;le important dans le d&#xE9;veloppement des os, et est une nouvelle cible pour Runx2 signalisation d&#xE9;pendante.

Pannexine 3 est une nouvelle cible pour Runx2, exprim&#xE9;e par les ost&#xE9;oblastes matures et chondrocytes de la plaque de croissance

Pannexins sind eine Klasse der Chordatiere-Kanal-Proteine, die durch ihre Homologie Insekt L&uuml;cke Kreuzung Proteine identifiziert. Die Pannexin-Familie besteht aus drei Mitgliedern, Panx1, Panx2 und Panx3, und die Rolle, die jede dieser Proteine in zellul&auml;re Prozesse spielt wird noch untersucht. Fr&uuml;here Berichte des Panx3 Ausdrucks geben Anreicherung in Skelett-Gewebe, damit wir diese Verteilung weiter untersucht haben, durch die Erhebung des entwickelnden Maus-Embryos mit Immunfluoreszenz. Hohes Ma&szlig; an Panx3 wurden bei intramembranous kraniofaziale flachen Knochen, sowie die langen Knochen des Skeletts appendicular und axiale festgestellt. Diese Verteilung ist das Ergebnis des Ausdrucks in Osteoblasten und hypertropher Chondrozyten. Dar&uuml;ber hinaus Panx3 Projekttr&auml;ger enth&auml;lt vermeintliche Bindungsstellen f&uuml;r Transkriptionsfaktoren, die Knochenbildung beteiligt, und wir zeigen, dass die Sequenz zwischen Basen-275 und-283 Runx2 Aktivierung tr&auml;gt. Zusammengenommen vermuten unsere Daten Panx3 kann eine wichtige Rolle in der Knochenentwicklung dienen, und ist ein neuartiges Ziel f&uuml;r Runx2-abh&auml;ngige signalisieren.

Pannexin 3 ist ein neues Ziel f&uuml;r Runx2, ausgedr&uuml;ckt durch die Osteoblasten und Reife Wachstum Platte Chondrozyten.

Pannexins sono una classe di proteine canale infundibolo identificati dal loro omologia delle proteine di giunzione divario dell&#39;insetto. La famiglia pannexin &egrave; costituito da tre membri, Panx1, Panx2 e Panx3, e il ruolo che ciascuna di queste proteine svolge nei processi cellulari &egrave; ancora sotto inchiesta. Precedenti relazioni di Panx3 espressione indicano arricchimento nei tessuti scheletrici, quindi abbiamo studiato ulteriormente questa distribuzione di misurazione dell&#39;embrione di topo in via di sviluppo con immunofluorescenza. Alti livelli di Panx3 sono stati individuati intramembranosa craniofacciale ossa piatte, come pure le lunghe ossa dello scheletro assiale e appendicolare. Questa distribuzione &egrave; il risultato dell&#39;espressione in osteoblasti e condrociti ipertrofica. Inoltre, il promotore Panx3 contiene siti di legame putativo per fattori di trascrizione coinvolti nella formazione del tessuto osseo, e ci mostra che la sequenza tra basi-275 e-283 &egrave; sensibile alle Runx2 attivazione. Presi insieme, i nostri dati suggeriscono Panx3 pu&ograve; servire un ruolo importante nello sviluppo osseo, che &egrave; un romanzo bersaglio per Runx2-dipendente di segnalazione.

Pannexin 3 &egrave; un romanzo bersaglio per Runx2, espressa da osteoblasti e condrociti placca matura crescita.

Pannexins&#x6606;&#x866B;&#x306E;&#x30AE;&#x30E3;&#x30C3;&#x30D7;&#x7D50;&#x5408;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306B;&#x305D;&#x308C;&#x3089;&#x306E;&#x76F8;&#x540C;&#x6027;&#x306B;&#x3088;&#x3063;&#x3066;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F;&#x810A;&#x7D22;&#x52D5;&#x7269;&#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30AF;&#x30E9;&#x30B9;&#x3067;&#x3059;&#x3002; pannexin&#x306E;&#x5BB6;&#x65CF;&#x306F;3&#x4EBA;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3001;Panx1&#x3001;Panx2&#x3001;&#x3068;Panx3&#x3067;&#x69CB;&#x6210;&#x3055;&#x308C;&#x3066;&#x304A;&#x308A;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x5404;&#x3005;&#x306F;&#x7D30;&#x80DE;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3067;&#x679C;&#x305F;&#x3059;&#x5F79;&#x5272;&#x306F;&#x3001;&#x73FE;&#x5728;&#x8ABF;&#x67FB;&#x4E2D;&#x3067;&#x3059;&#x3002; Panx3&#x5F0F;&#x306E;&#x524D;&#x306E;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x3067;&#x306F;&#x3001;&#x9AA8;&#x683C;&#x7D44;&#x7E54;&#x3067;&#x6FC3;&#x7E2E;&#x3092;&#x793A;&#x3059;&#x306E;&#x3067;&#x3001;&#x6211;&#x3005;&#x306F;&#x3055;&#x3089;&#x306B;&#x514D;&#x75AB;&#x767A;&#x5C55;&#x9014;&#x4E0A;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x80DA;&#x3092;&#x8ABF;&#x67FB;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x3053;&#x306E;&#x30C7;&#x30A3;&#x30B9;&#x30C8;&#x30EA;&#x30D3;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x305F;&#x3002; Panx3&#x9AD8;&#x30EC;&#x30D9;&#x30EB;&#x306E;&#x819C;&#x5185;&#x9854;&#x9762;&#x5E73;&#x5766;&#x306A;&#x9AA8;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3001;&#x56DB;&#x80A2;&#x3068;&#x8EF8;&#x9AA8;&#x683C;&#x306E;&#x9577;&#x9AA8;&#x304C;&#x691C;&#x51FA;&#x3055;&#x308C;&#x305F;&#x3002;&#x3053;&#x306E;&#x30C7;&#x30A3;&#x30B9;&#x30C8;&#x30EA;&#x30D3;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;&#x9AA8;&#x82BD;&#x7D30;&#x80DE;&#x3068;&#x80A5;&#x5927;&#x8EDF;&#x9AA8;&#x7D30;&#x80DE;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x5F0F;&#x306E;&#x7D50;&#x679C;&#x3067;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;Panx3&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306F;&#x3001;&#x9AA8;&#x5F62;&#x6210;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306E;&#x63A8;&#x5B9A;&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x304C;&#x542B;&#x307E;&#x308C;&#x3066;&#x304A;&#x308A;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x57FA;&#x5730;-275&#x3068;-283&#x306E;&#x9593;&#x306E;&#x914D;&#x5217;&#x306F;&#x3001;Runx2&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x5FDC;&#x7B54;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x3001;&#x6211;&#x3005;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;Panx3&#x306F;&#x3001;&#x9AA8;&#x306E;&#x767A;&#x9054;&#x306B;&#x304A;&#x3044;&#x3066;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3001;&#x3068;Runx2&#x4F9D;&#x5B58;&#x6027;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x305F;&#x306A;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3067;&#x3059;&#x3002;

Pannexin 3&#x306F;&#x3001;&#x9AA8;&#x82BD;&#x7D30;&#x80DE;&#x3068;&#x6210;&#x719F;&#x6210;&#x9577;&#x677F;&#x8EDF;&#x9AA8;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x308B;&#x767A;&#x73FE;&#x3001;Runx2&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3067;&#x3042;&#x308B;

Pannexins&#xB294; &#xCC99; &#xCC44;&#xB110; &#xB2E8;&#xBC31;&#xC9C8; &#xACE4;&#xCDA9; &#xAC2D; &#xC815;&#xC158; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xADF8;&#xB4E4;&#xC758; homology&#xB85C; &#xC2DD;&#xBCC4; &#xB41C; &#xD074;&#xB798;&#xC2A4;. Pannexin &#xD328;&#xBC00;&#xB9AC; &#xC138; &#xD68C;&#xC6D0;, Panx1, Panx2, &#xBC0F; Panx3, &#xAD6C;&#xC131; &#xB418;&#xC5B4; &#xC788;&#xC73C;&#xBA70; &#xAC01;&#xAC01;&#xC774; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC138;&#xD3EC; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0;&#xC11C; &#xC218;&#xD589; &#xD558;&#xB294; &#xC5ED;&#xD560;&#xC740; &#xC544;&#xC9C1; &#xC870;&#xC0AC;&#xB97C; &#xBC1B;&#xACE0;. &#xADF8;&#xB798;&#xC11C; &#xC6B0;&#xB9AC; &#xCD94;&#xAC00; &#xBA74;&#xC5ED; &#xD615;&#xAD11; &#xAC80;&#xC0AC;&#xC640; &#xAC1C;&#xBC1C; &#xB9C8;&#xC6B0;&#xC2A4; &#xBC30;&#xC544;&#xB97C; &#xC870;&#xC0AC; &#xD558; &#xC5EC;&#xC774; &#xBC30;&#xD3EC; &#xC870;&#xC0AC;&#xB294; Panx3 &#xC2DD;&#xC758; &#xC774;&#xC804; &#xBCF4;&#xACE0;&#xC11C; &#xACE8;&#xACA9; &#xC870;&#xC9C1;&#xC5D0; &#xB18D;&#xCD95;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. Panx3 &#xB192;&#xC740; &#xC218;&#xC900;&#xC758; intramembranous craniofacial &#xD3B8;&#xD3C9;&#xD55C; &#xBF08;&#xC5D0;&#xC11C; appendicular &#xBC0F; &#xCD95; &#xACE8;&#xACA9;&#xC758; &#xAE34; &#xBF08;&#xAC00; &#xBC1C;&#xACAC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xBD84;&#xD3EC;&#xB294; osteoblasts &#xBC0F; hypertrophic chondrocytes&#xC5D0; &#xC2DD;&#xC758; &#xACB0;&#xACFC;&#xC785;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, Panx3 &#xBAA8;&#xD130; &#xD3EC;&#xD568; putative &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xB300; &#xD55C; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC694;&#xC778; &#xBF08; &#xD615;&#xC131;&#xC5D0; &#xAD00;&#xC5EC; &#xD558; &#xACE0; &#xC6B0;&#xB9AC; &#xAE30;&#xC9C0;-275&#xC640;-283 &#xC0AC;&#xC774;&#xC758; &#xC21C;&#xC11C;&#xB294; Runx2 &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xBC18;&#xC751; &#xBCF4;&#xC5EC;. &#xD568;&#xAED8; &#xCC0D;&#xC740;, &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130;&#xB294; Panx3 &#xBF08; &#xAC1C;&#xBC1C;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xACF5;&#xD560; &#xC218; &#xBC0F; Runx2 &#xC885;&#xC18D;&#xC5D0; &#xB300; &#xD55C; &#xC18C;&#xC124; &#xB300;&#xC0C1; &#xC2E0;&#xD638;&#xB294; &#xB098;&#xC654;&#xB2E4;.

Pannexin 3 Runx2, osteoblasts &#xBC0F; &#xC131;&#xC219; &#xC131;&#xC7A5; &#xD50C;&#xB808;&#xC774;&#xD2B8; chondrocytes &#xD45C;&#xD604;&#xC5D0; &#xB300; &#xD55C; &#xC18C;&#xC124; &#xB300;&#xC0C1; &#xC774;&#xB2E4;.

Pannexins son una clase de prote&#xED;nas de los canales cordados identificadas por su homolog&#xED;a con las prote&#xED;nas de las uniones de insectos. La familia pannexin consta de tres miembros, y Panx1 Panx2 y Panx3, y el papel de cada una de estas prote&#xED;nas juega en los procesos celulares est&#xE1; a&#xFA;n bajo investigaci&#xF3;n. En informes anteriores de la expresi&#xF3;n Panx3 indican un enriquecimiento en los tejidos &#xF3;seos, por lo que han investigado esta distribuci&#xF3;n mediante una encuesta el embri&#xF3;n de rat&#xF3;n en desarrollo con inmunofluorescencia. Los altos niveles de Panx3 se detectaron en intramembranosas huesos planos craneofaciales, as&#xED; como huesos largos de la apendicular y esqueleto axial. Esta distribuci&#xF3;n es el resultado de la expresi&#xF3;n, tanto en los osteoblastos y los condrocitos hipertr&#xF3;ficos. Por otra parte, el promotor Panx3 contiene supuestos sitios de uni&#xF3;n para factores de transcripci&#xF3;n implicados en la formaci&#xF3;n &#xF3;sea, y nos muestran que la secuencia entre -275 y -283 bases responde a Runx2 activaci&#xF3;n. En conjunto, nuestros datos sugieren que Panx3 puede servir un papel importante en el desarrollo &#xF3;seo, y es un nuevo objetivo para Runx2 dependiente de la se&#xF1;alizaci&#xF3;n.

Pannexin 3 es un nuevo objetivo para Runx2, expresada por los osteoblastos y condrocitos maduros placa de crecimiento

Genes encoding vanillin dehydrogenase (vdh) and vanillate O-demethylase (vanAB) were identified in Rhodococcus jostii RHA1 using gene disruption and enzyme activities. During growth on vanillin or vanillate, vanA was highly upregulated while vdh was not. This study contributes to our understanding of lignin degradation by RHA1 and other actinomycetes.

Vanillin catabolism in Rhodococcus jostii RHA1.

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

BCG vaccine, introduced almost 100years ago, is the only option to prevent TB disease. It effectively protects newborns from meningeal TB but fails to prevent adult pulmonary TB. TB kills 1.3million people annually in areas where BCG vaccination is widely practiced. Thus, more effective TB vaccines are urgently needed. Others and we have shown that BCG mimics features of virulent M. tuberculosis, in particular attenuation of essential macrophage functions such as phagosome maturation and antigen presentation. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the cysteine protease Cathepsin S (CatS), which prevents MHC II molecule maturation and proper antigen peptide loading. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to (i) contain and process ingested bacteria and (ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells.

Alice Lau

Division of Infectious Diseases,
Department of Medicine and Vancouver Costal Health Research Institute,
Division of Infectious Diseases, Department of Medicine and Vancouver Costal Health Research Institute

University of British Columbia

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Alice Lau

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University of British Columbia

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Division of Infectious Diseases,
Department of Medicine and Vancouver Costal Health Research Institute,
Division of Infectious Diseases, Department of Medicine and Vancouver Costal Health Research Institute