enteric-bacterial-invasion-intestinal-epithelial-cells-vitro-is

Enteric Bacterial Invasion Of Intestinal Epithelial Cells <em>In Vitro</em> Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

London School of Hygiene & Tropical Medicine

The Campylobacter jejuni pgl locus encodes an N-linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N-glycoprotein AcrA required for accepting an N-glycan. We found that the eukaryotic primary consensus sequence for N-glycosylation is N terminally extended to D/E-Y-N-X-S/T (Y, X not equalP) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N-glycosylated when they were either artificially introduced or when they were present in non-C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N-glycosylation sites and confirmed the requirement for a negatively charged side chain at position -2 in C. jejuni N-glycoproteins. N-glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the -2 position. Thus, bacterial N-glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.

Definition of the bacterial N-glycosylation site consensus sequence.

Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of "O-OTases" that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli.

Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation.

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought.

Delineation of the innate and adaptive T-cell immune outcome in the human host in response to Campylobacter jejuni infection.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide. Despite stringent microaerobic growth requirements, C. jejuni is ubiquitous in the aerobic environment and so must possess regulatory systems to sense and adapt to external stimuli, such as oxidative and aerobic (O(2)) stress. Reannotation of the C. jejuni NCTC11168 genome sequence identified Cj1556 (originally annotated as a hypothetical protein) as a MarR family transcriptional regulator, and further analysis indicated a potential role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic stress, decreased ability for intracellular survival in Caco-2 human intestinal epithelial cells and J774A.1 mouse macrophages, and a reduction in virulence in the Galleria mellonella infection model. Microarray analysis of gene expression changes in the Cj1556 mutant indicated negative autoregulation of Cj1556 expression and downregulation of genes associated with oxidative and aerobic stress responses, such as katA, perR, and hspR. Electrophoretic mobility shift assays confirmed the binding of recombinant Cj1556 to the promoter region upstream of the Cj1556 gene. cprS, which encodes a sensor kinase involved in regulation of biofilm formation, was also upregulated in the Cj1556 mutant, and subsequent studies showed that the mutant had a reduced ability to form biofilms. This study identified a novel C. jejuni transcriptional regulator, Cj1556, that is involved in oxidative and aerobic stress responses and is important for the survival of C. jejuni in the natural environment and in vivo.

The Campylobacter jejuni transcriptional regulator Cj1556 plays a role in the oxidative and aerobic stress response and is important for bacterial survival in vivo.

Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods.

Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis.

Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.

The discovery of the Campylobacter jejuni N-linked glycosylation system combined with its functional expression in Escherichia coli marked the dawn of a new era in glycoengineering. The process, termed protein glycan coupling technology (PGCT), has, in particular, been applied to the development of glycoconjugate vaccines. In this review, we highlight recent technical developments in this area, including the first structural determination of the coupling enzyme PglB, the use of glycotags for optimal glycan attachment and the possible applications of other glycosylation systems and how these may improve and extend PGCT.

Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design.

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Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world, however the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilise outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the co-ordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasmic-located, yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pre-treatment with proteinase K or polymixin B prior to co-incubation with IECs. Pre-treatment of IECs with methyl-beta-cyclodextrin partially blocks OMV induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

Campylobacter jejuni outer membrane vesicles play an important role in bacterial interactions with human intestinal epithelial cells.

Campylobacter jejuni est la cause pr&eacute;dominante de toxi-infection gastro-ent&eacute;rite dans les pays d&eacute;velopp&eacute;s, mais les bases mol&eacute;culaires de la pathog&eacute;nie n&#39;est pas clair. La s&eacute;cr&eacute;tion des facteurs de virulence est des principaux m&eacute;canismes par lesquels ent&eacute;ropathog&egrave;nes bact&eacute;riens interagissent avec les cellules h&ocirc;tes pour am&eacute;liorer la survie et/ou d&#39;endommager l&#39;h&ocirc;te. C. jejuni manque toutefois les syst&egrave;mes de s&eacute;cr&eacute;tion de virulence-associated poss&eacute;d&eacute;s par d&#39;autres pathog&egrave;nes ent&eacute;riques. Plusieurs bact&eacute;ries pathog&egrave;nes utilisent des v&eacute;sicules de la membrane externe (OMVs) pour l&#39;administration de facteurs de virulence dans les cellules h&ocirc;tes. En l&#39;absence de syst&egrave;mes de s&eacute;cr&eacute;tion de virulence-associated prototypique, OMVs pourraient &ecirc;tre une alternative importante pour la prestation coordonn&eacute;e de c. jejuni de prot&eacute;ines dans les cellules h&ocirc;tes. Analyse prot&eacute;omique de c. jejuni 11168H OMVs identifi&eacute; 151 prot&eacute;ines incluant des prot&eacute;ines p&eacute;riplasmiques et externes associ&eacute;e &agrave; la membrane, mais aussi de nombreux d&eacute;terminants, connues pour &ecirc;tre importante dans la survie et la pathogen&egrave;se, y compris les toxines distension toxine (CDT). C. jejuni OMVs contenait 16 glycoprot&eacute;ines N-li&eacute;es, indiquant un m&eacute;canisme d&#39;ex&eacute;cution par lequel ces p&eacute;riplasmique-situ&eacute;, pourtant les glycoprot&eacute;ines immunog&eacute;niques peuvent interagir avec les cellules h&ocirc;tes. C. jejuni OMVs poss&egrave;dent une activit&eacute; cytotoxique et induire une r&eacute;ponse immunitaire h&ocirc;te par les cellules &eacute;pith&eacute;liales intestinales T84 (IECs), qui n&#39;a pas &eacute;t&eacute; r&eacute;duite par pr&eacute;traitement OMV avec la prot&eacute;inase K ou polymixine B avant incubation avec Sava. Pr&eacute;traitement de l&#39;IECs avec m&eacute;thyl-b&ecirc;ta-cyclodextrine bloque partiellement l&#39;OMV induit immunitaire h6te, indiquant un r&ocirc;le pour les radeaux lipidiques dans les membranes plasmiques de cellules h&ocirc;tes lors des interactions avec c. jejuni OMVs. OMVs isol&eacute;s d&#39;un mutant de CFDC 11168H induite par interleukine-8 (IL-8) dans la m&ecirc;me mesure que sauvage OMVs, sugg&eacute;rant l&#39;OMV induction d&#39;IL-8 est ind&eacute;pendante de la CDT.

Campylobacter jejuni v&eacute;sicules de la membrane externe jouent un r&ocirc;le important dans les interactions bact&eacute;riennes avec les cellules &eacute;pith&eacute;liales intestinales humaines.

Campylobacter Jejuni ist die am weitesten verbreitete Ursache lebensmittelbedingter Magen-Darm in der entwickelten Welt, aber die molekulare Grundlage der Pathogenese unklar. Sekretion von Virulenzfaktoren ist ein wichtiger Mechanismus, mit dem enterischen bakterielle Krankheitserreger mit Wirtszellen zu &uuml;berleben zu verbessern und/oder zu einer Besch&auml;digung des Hosts interagieren. C. Jejuni fehlt jedoch die Virulenz-assoziierte Sekretion-Systeme von anderen enterischen Krankheitserregern besessen. Viele bakterielle Krankheitserreger nutzen Au&szlig;enmembran Bl&auml;schen (OMVs) f&uuml;r die Lieferung von Virulenzfaktoren in Wirtszellen. In Ermangelung der prototypische Virulenz-assoziierte Sekretion Systeme w&auml;re OMVs eine wichtige Alternative f&uuml;r die koordinierte Lieferung von C. Jejuni Proteine in Wirtszellen. Proteomic Analyse von C. Jejuni 11168H OMVs identifiziert 151 Proteine einschlie&szlig;lich periplasmatischen und &auml;u&szlig;ere Membran-assoziierte Proteine, aber auch viele Determinanten, die bekannterma&szlig;en im &Uuml;berleben und Pathogenese einschlie&szlig;lich der Cytolethal ausdehnend Toxin (CDT) wichtig. C. Jejuni OMVs enthaltenen 16 N gebundene Glykoproteine, angibt, eine &Uuml;bermittlungsmechanismus von denen diese periplasmatischen gelegen, noch immunogen Glykoproteine mit Wirtszellen interagieren k&ouml;nnen. C. Jejuni OMVs besitzen zytotoxische Aktivit&auml;t und induzieren eine Host Immunantwort von T84 intestinaler Epithelzellen (IECs), die nicht durch OMV Vorbehandlung mit Proteinase K oder Polymixin B vor Co-incubation mit IECs reduziert wurde. Vorbehandlung des IECs mit Methyl-Beta-Cyclodextrin blockiert teilweise OMV induzierte Host Immunantworten, angibt, eine Rolle f&uuml;r Lipid Rafts in Host Plasma Zellmembranen w&auml;hrend Interaktionen mit C. Jejuni 289.000. 289.000 Von 11168H Querschnitt Mutanten isoliert induzierte Interleukin-8 (IL-8) im gleichen Umfang wie Wildtyp 289.000, was darauf hindeutet, dass OMV Induktion von IL-8 unabh&auml;ngig von CDT ist.

Campylobacter Jejuni Au&szlig;enmembran Vesikeln spielen eine wichtige Rolle in bakterielle Interaktionen mit menschlichen intestinalen Epithelzellen.

Campylobacter jejuni &egrave; la pi&ugrave; diffusa causa di gastroenterite tossinfezioni alimentari nel mondo sviluppato, tuttavia la base molecolare della patogenesi &egrave; poco chiara. Secrezione di fattori di virulenza &egrave; un meccanismo chiave che patogeni enterici batterici interagiscono con cellule ospiti per migliorare la sopravvivenza e/o danneggiare l&#39;host. Tuttavia il c. jejuni manca i sistemi di secrezione di virulenza associati posseduti da altri patogeni enterici. Molti agenti patogeni batterici utilizzano vescicole di membrana esterna (OMVs) per la consegna dei fattori di virulenza nelle cellule ospiti. In assenza di sistemi di secrezione di virulenza associati prototipica, OMVs potrebbe essere un&#39;importante alternativa per la consegna coordinata di c. jejuni proteine nelle cellule ospiti. Analisi proteomic del c. jejuni 11168H OMVs identificate 151 proteine tra cui proteine di membrana associati periplasmica ed esterne, ma anche molti fattori determinanti noti per essere importante nella sopravvivenza e patogenesi compresa la cytolethal inducendo tossina (CDT). C. jejuni OMVs conteneva 16 glicoproteine N-linked, che indica un meccanismo di consegna da cui questi periplasmico-trova, ancora immunogene glicoproteine possono interagire con le cellule ospite. C. jejuni OMVs possiedono attivit&agrave; citotossica e indurre una risposta immunitaria da T84 cellule epiteliali intestinali (IECs), che non &egrave; stato ridotto di pretrattamento OMV con proteinasi K o polimixina B prima co-incubation con IECs. Pretrattamento dell&#39;IECs con metil-beta-ciclodestrina blocca parzialmente risposta immunitaria ospite OMV indotta, che indica un ruolo per le zattere lipidiche in host plasma membrane durante le interazioni con c. jejuni OMVs. OMVs isolato da un mutante cdtA 11168H indotta da interleukin-8 (IL-8) nella stessa misura come wild-type OMVs, suggerendo l&#39;induzione OMV di IL-8 &egrave; indipendente della CDT.

Campylobacter jejuni vescicole di membrana esterna svolgono un ruolo importante nelle interazioni batteriche con le cellule epiteliali intestinali umane.

&#x75C5;&#x614B;&#x306E;&#x5206;&#x5B50;&#x57FA;&#x76E4;&#x304C;&#x660E;&#x3089;&#x304B;&#x3067;&#x306F;&#x306A;&#x3044;&#x3057;&#x304B;&#x3057;&#x30AB;&#x30F3;&#x30D4;&#x30ED;&#x30D0;&#x30AF;&#x30BF;&#x30FC;&#x98DF;&#x54C1;&#x7531;&#x6765;&#x80C3;&#x8178;&#x708E;&#x3001;&#x5148;&#x9032;&#x56FD;&#x3067;&#x306E;&#x6700;&#x3082;&#x4E00;&#x822C;&#x7684;&#x306A;&#x539F;&#x56E0;&#x3067;&#x3059;&#x3002;&#x75C5;&#x539F;&#x6027;&#x56E0;&#x5B50;&#x306E;&#x5206;&#x6CCC;&#x8178;&#x5185;&#x7D30;&#x83CC;&#x6027;&#x75C5;&#x539F;&#x4F53;&#x304C;&#x751F;&#x5B58;&#x7387;&#x306E;&#x5411;&#x4E0A;&#x3084;&#x30DB;&#x30B9;&#x30C8;&#x306E;&#x640D;&#x50B7;&#x306B;&#x5BBF;&#x4E3B;&#x7D30;&#x80DE;&#x3068;&#x3092;&#x5BFE;&#x8A71;&#x3059;&#x308B;&#x4E3B;&#x8981;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3067;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057; C. jejuni &#x4ED6;&#x8178;&#x6EB6;&#x6027;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x306B;&#x3088;&#x308A;&#x4FDD;&#x6709;&#x306E;&#x75C5;&#x539F;&#x6027;&#x95A2;&#x9023;&#x5206;&#x6CCC;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x6B20;&#x3044;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x591A;&#x304F;&#x306E;&#x7D30;&#x83CC;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x5916;&#x819C;&#x5C0F;&#x80DE; (OMVs) &#x30DB;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x3078;&#x306E;&#x75C5;&#x539F;&#x56E0;&#x5B50;&#x306E;&#x914D;&#x4FE1;&#x306E;&#x305F;&#x3081;&#x306B;&#x6D3B;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x539F;&#x578B;&#x5206;&#x6CCC;&#x6BD2;&#x529B;&#x95A2;&#x9023;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x4E0D;&#x5728;&#x3067;&#x306F;&#x3001;OMVs &#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30CD;&#x30FC;&#x30C8;&#x4EA4;&#x4ED8; C. jejuni &#x86CB;&#x767D;&#x8CEA;&#x306E;&#x5BBF;&#x4E3B;&#x7D30;&#x80DE;&#x306B;&#x91CD;&#x8981;&#x306A;&#x9078;&#x629E;&#x80A2;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;C. jejuni 11168H OMVs &#x8B58;&#x5225;&#x3055;&#x308C;&#x305F; 151 &#x86CB;&#x767D;&#x8CEA;&#x30DA;&#x30EA;&#x30D7;&#x30E9;&#x30BA;&#x30E0;&#x3068;&#x5916;&#x5074;&#x306E;&#x819C;&#x6E96;&#x86CB;&#x767D;&#x8CEA;&#x3057;&#x307E;&#x3059;&#x304C;&#x3001;&#x751F;&#x5B58;&#x3068;&#x6BD2;&#x7D20; (CDT) distending &#x751F;&#x306A;&#x3069;&#x306E;&#x75C5;&#x56E0;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3082;&#x591A;&#x304F;&#x306E;&#x8981;&#x56E0;&#x3092;&#x542B;&#x3080;&#x306E;&#x30D7;&#x30ED;&#x30C6;&#x30AA;&#x30FC;&#x30E0;&#x89E3;&#x6790;&#x3002;C. jejuni OMVs &#x306B;&#x3088;&#x3063;&#x3066;&#x3053;&#x308C;&#x3089;&#x30DA;&#x30EA;&#x30D7;&#x30E9;&#x30BA;&#x30E0;&#x3042;&#x308B;&#x307E;&#x3060;&#x514D;&#x75AB;&#x539F;&#x6027;&#x7CD6;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30DB;&#x30B9;&#x30C8; &#x30BB;&#x30EB;&#x3068;&#x3084;&#x308A;&#x53D6;&#x308A;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x914D;&#x4FE1;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x793A;&#x3059;&#x3001;16 &#x306E; N &#x30EA;&#x30F3;&#x30AF;&#x7CD6;&#x304C;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;C. jejuni OMVs &#x7D30;&#x80DE;&#x50B7;&#x5BB3;&#x6D3B;&#x6027;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x308B;&#x3057;&#x3067;&#x306E; OMV &#x524D;&#x51E6;&#x7406;&#x30D7;&#x30ED;&#x30C6;&#x30A3;&#x30CA;&#x30FC;&#x30BC; K &#x307E;&#x305F;&#x306F;&#x30DD;&#x30EA;&#x30DF;&#x30AD;&#x30B7;&#x30F3; B &#x5C65;&#x4FEE;&#x3068;&#x305D;&#x3053;&#x3067;&#x306E;&#x524D;&#x306B;&#x4F4E;&#x4E0B;&#x3057;&#x306A;&#x3044;&#x30DB;&#x30B9;&#x30C8;&#x514D;&#x75AB;&#x5FDC;&#x7B54; T84 &#x8178;&#x4E0A;&#x76AE;&#x7D30;&#x80DE; &#xFF08;&#x5C65;&#x4FEE;) &#x304B;&#x3089;&#x3092;&#x8A98;&#x767A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30E1;&#x30C1;&#x30EB;-&beta;-&#x30B7;&#x30AF;&#x30ED;&#x30C7;&#x30AD;&#x30B9;&#x30C8;&#x30EA;&#x30F3;&#x306B;&#x3088;&#x308B;&#x5C65;&#x4FEE;&#x306E;&#x524D;&#x51E6;&#x7406;&#x306F;&#x90E8;&#x5206;&#x7684;&#x306B;&#x8A98;&#x8D77; OMV &#x5BBF;&#x4E3B;&#x514D;&#x75AB;&#x5FDC;&#x7B54;&#x306E;&#x30DB;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306B;&#x304A;&#x3051;&#x308B;&#x8102;&#x8CEA;&#x30E9;&#x30D5;&#x30C8;&#x306E;&#x5F79;&#x5272;&#x3092;&#x793A;&#x3059; C. jejuni OMVs &#x3068;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x4E2D;&#x306B;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3057;&#x307E;&#x3059;&#x3002;11168H cdtA &#x7A81;&#x7136;&#x5909;&#x7570;&#x4F53;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3055;&#x308C;&#x305F; OMVs &#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30ED;&#x30A4;&#x30AD;&#x30F3; 8 (IL-8) &#x91CE;&#x751F;&#x578B; OMVs&#x3001;IL-8 &#x306E; OMV &#x8A98;&#x5C0E; CDT &#x306E;&#x72EC;&#x7ACB;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3068;&#x540C;&#x3058;&#x7A0B;&#x5EA6;&#x306B;&#x8A98;&#x5C0E;&#x3002;

&#x30AB;&#x30F3;&#x30D4;&#x30ED;&#x30D0;&#x30AF;&#x30BF;&#x30FC;&#x8178;&#x708E;&#x5916;&#x819C;&#x5C0F;&#x80DE;&#x3072;&#x3068;&#x8178;&#x7BA1;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x3068;&#x7D30;&#x83CC;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3067;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x518D;&#x751F;&#x3057;&#x307E;&#x3059;&#x3002;

&#xADF8;&#xB7EC;&#xB098; Campylobacter jejuni pathogenesis&#xC758; &#xBD84;&#xC790; &#xAE30;&#xC900; &#xBA85;&#xD655; &#xD558;&#xC9C0; &#xC54A;&#xB2E4; &#xC120;&#xC9C4;&#xAD6D;, &#xC2DD;&#xD488;&#xC744; &#xB9E4;&#xAC1C;&#xB85C; &#xC704;&#xC7A5;&#xC5FC;&#xC758; &#xAC00;&#xC7A5; &#xB110;&#xB9AC; &#xD37C;&#xC9C4; &#xC6D0;&#xC778;&#xC785;&#xB2C8;&#xB2E4;. &#xB3C5;&#xC131; &#xC694;&#xC778;&#xC758; &#xBD84; &#xBE44;&#xB294; &#xC7A5;&#xC758; &#xC138;&#xADE0;&#xC131; &#xBCD1;&#xC6D0; &#xCCB4; &#xC0DD;&#xC874;&#xC744; &#xD5A5;&#xC0C1; &#xD558; &#xACE0; &#xD638;&#xC2A4;&#xD2B8;&#xB97C; &#xC190;&#xC0C1; &#xC2DC;&#xD0AC; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC640; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; &#xD575;&#xC2EC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xC774;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; C. jejuni &#xBD80;&#xC871; &#xC7A5;&#xC758; &#xB2E4;&#xB978; &#xBCD1;&#xC6D0; &#xADE0;&#xC5D0; &#xC758;&#xD574; &#xBCF4;&#xC720; &#xBD84; &#xBE44; &#xB3C5;&#xC131; &#xAD00;&#xB828; &#xC2DC;&#xC2A4;&#xD15C;. &#xB9CE;&#xC740; &#xC138;&#xADE0;&#xC131; &#xBCD1;&#xC6D0; &#xCCB4; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC5D0; &#xB3C5;&#xC131; &#xC694;&#xC778;&#xC758; &#xBC30;&#xB2EC;&#xC5D0; &#xB300; &#xD55C; &#xC678;&#xBD80; &#xB9C9; &#xC18C;&#xD3EC; (OMVs)&#xC744; &#xD65C;&#xC6A9;&#xD569;&#xB2C8;&#xB2E4;. &#xC6D0;&#xD615; &#xB3C5;&#xC131; &#xAD00;&#xB828; &#xBD84; &#xBE44; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xBD80;&#xC7AC;, OMVs &#xD638;&#xC2A4;&#xD2B8; &#xC140;&#xC73C;&#xB85C; C. jejuni &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xACF5;&#xB3D9;-ordinated &#xB0A9;&#xD488;&#xC744; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xB300;&#xC548;&#xC774; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. C. jejuni 11168H Omvs&#xC758; &#xBD84;&#xC11D; Proteomic 151 &#xB2E8;&#xBC31;&#xC9C8; periplasmic &#xBC0F; &#xC678;&#xBD80; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xAD00;&#xB828; &#xB2E8;&#xBC31;&#xC9C8; &#xD558;&#xC9C0;&#xB9CC; &#xB610;&#xD55C; &#xB9CE;&#xC740; &#xACB0;&#xC815; &#xC694;&#xC778; &#xC0DD;&#xC874;&#xACFC; pathogenesis &#xB3C5; &#xC18C; (&#xC11C;&#xBA38; &#xD0C0;&#xC784;) distending cytolethal&#xB97C; &#xD3EC;&#xD568; &#xD55C;&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC9C4; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC2DD;&#xBCC4; &#xB429;&#xB2C8;&#xB2E4;. C. jejuni OMVs immunogenic glycoproteins &#xC218; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC;&#xC640; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; &#xC544;&#xC9C1; periplasmic &#xC704;&#xCE58;&#xB294; &#xC774;&#xB4E4;&#xC5D0; &#xC758;&#xD574; &#xBC30;&#xB2EC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; 16 N &#xC5F0;&#xACB0; glycoproteins &#xD3EC;&#xD568;. C. jejuni OMVs cytotoxic &#xD65C;&#xB3D9;&#xC744; &#xBCF4;&#xC720; &#xD558; &#xACE0; &#xAC00;&#xC218;&#xBD84;&#xD574; K &#xB610;&#xB294; polymixin B co-incubation Iecs&#xC640; &#xC774;&#xC804; OMV &#xC804;&#xCC98;&#xB9AC;&#xC5D0; &#xC758;&#xD574; &#xAC10;&#xC18C; &#xD558;&#xC9C0;&#xB294; T84 &#xC7A5;&#xB0B4; &#xC0C1;&#xD53C; &#xC138;&#xD3EC; (IECs)&#xC5D0;&#xC11C; &#xD638;&#xC2A4;&#xD2B8; &#xBA74;&#xC5ED; &#xC751;&#xB2F5;&#xC744; &#xC720;&#xB3C4;. &#xBA54; &#xD2F8;-&#xBCA0;&#xD0C0;-cyclodextrin&#xACFC; Iecs&#xC758; &#xC804;&#xCC98;&#xB9AC;&#xB294; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; C. jejuni Omvs&#xC640; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xB9C9;&#xC5D0;&#xC11C; &#xC9C0;&#xC9C8; &#xB5CF;&#xBAA9;&#xC758; &#xC5ED;&#xD560;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; OMV &#xC720;&#xBC1C; &#xD638;&#xC2A4;&#xD2B8; &#xBA74;&#xC5ED; &#xBC18;&#xC751;&#xC744; &#xCC28;&#xB2E8; &#xD569;&#xB2C8;&#xB2E4;. OMVs 11168H cdtA &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xCCB4;&#xC5D0;&#xC11C; &#xBD84;&#xB9AC; &#xB41C; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; OMVs, IL-8&#xC758; OMV &#xC720;&#xB3C4; &#xC11C;&#xBA38; &#xD0C0;&#xC784;&#xC758; &#xB3C5;&#xB9BD;&#xC801;&#xC785;&#xB2C8;&#xB2E4; &#xC81C;&#xC548;&#xC73C;&#xB85C; &#xB3D9;&#xC77C;&#xD55C; &#xC815;&#xB3C4;&#xB85C; &#xC778;&#xD130; &#xB8E8; &#xD0A8;-8 (IL-8)&#xC744; &#xC720;&#xBC1C;.

Campylobacter jejuni &#xC678;&#xBD80; &#xB9C9; &#xC18C;&#xD3EC; &#xC778;&#xAC04;&#xC758; &#xCC3D; &#xC790; &#xC0C1;&#xD53C; &#xC138;&#xD3EC;&#xC640; &#xC138;&#xADE0; &#xC791;&#xC6A9;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;.

Campylobacter jejuni es la causa m&aacute;s frecuente de gastroenteritis transmitidas por los alimentos en el mundo desarrollado, sin embargo las bases moleculares de la patogenesia son confusa. Secreci&oacute;n de factores de virulencia es un mecanismo clave que bacterias pat&oacute;genas ent&eacute;ricas interact&uacute;an con las c&eacute;lulas del hu&eacute;sped para mejorar la supervivencia y/o da&ntilde;ar el anfitri&oacute;n. C. jejuni carece sin embargo de los sistemas de secreci&oacute;n de virulencia asociada pose&iacute;dos por otros pat&oacute;genos ent&eacute;ricos. Muchos pat&oacute;genos bacterianos utilizan ves&iacute;culas de membrana externa (OMVs) para la entrega de factores de virulencia en las c&eacute;lulas del hu&eacute;sped. En la ausencia de sistemas de secreci&oacute;n protot&iacute;pico de virulencia asociada, OMVs podr&iacute;an ser una alternativa importante para la prestaci&oacute;n coordinada de C. jejuni prote&iacute;nas en las c&eacute;lulas del hu&eacute;sped. An&aacute;lisis prote&oacute;mico de C. jejuni 11168H OMVs identifican 151 prote&iacute;nas incluyendo prote&iacute;nas asociadas a membrana periplasmic y externas, pero tambi&eacute;n muchos determinantes sabe que es importante para la supervivencia y la patogenesia incluyendo el cytolethal distensi&oacute;n toxina (CDT). C. jejuni OMVs conten&iacute;a 16 glicoprote&iacute;nas N-ligados, que indica un mecanismo de entrega por que &eacute;stos periplasmic ubicados, pero inmunog&eacute;nicas glicoprote&iacute;nas pueden interactuar con las c&eacute;lulas del hu&eacute;sped. C. jejuni OMVs poseen actividad citot&oacute;xica e inducir una respuesta inmune de anfitri&oacute;n de T84 c&eacute;lulas epiteliales intestinales (IECs), que no se redujo por el tratamiento previo de la OMV con proteinasa K o polimixina B antes de nexos con IECs. Tratamiento previo de CA con metil-beta-ciclodextrina bloquea parcialmente las respuestas inmunitarias de la OMV inducida por host, indicando un papel para las balsas de l&iacute;pidos en las membranas de plasma de la c&eacute;lula anfitri&oacute;n durante interacciones con C. jejuni OMVs. OMVs aislados de un mutante de cdtA de 11168H inducida por Interleucina-8 (IL-8) en la misma medida que el salvaje-tipo OMVs, sugiriendo OMV inducci&oacute;n de IL-8 es independiente del CDT.

Campylobacter jejuni ves&iacute;culas de membrana externa juegan un papel importante en las interacciones bacterianas con las c&eacute;lulas epiteliales intestinales humanas.

Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l(-1) of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.

Article	Year
Definition of the bacterial N-glycosylation site consensus sequence. The EMBO journal\| PubMed ID: 16619027	2006
Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation. Journal of bacteriology\| PubMed ID: 17890310	2007
Delineation of the innate and adaptive T-cell immune outcome in the human host in response to Campylobacter jejuni infection. PloS one\| PubMed ID: 21085698	2010
The Campylobacter jejuni transcriptional regulator Cj1556 plays a role in the oxidative and aerobic stress response and is important for bacterial survival in vivo. Journal of bacteriology\| PubMed ID: 21642451	2011
Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis. Microbial cell factories\| PubMed ID: 22276812	2012
Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment. Infection and immunity\| PubMed ID: 22354027	2012
Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design. Journal of medical microbiology\| PubMed ID: 22516134	2012
Campylobacter jejuni outer membrane vesicles play an important role in bacterial interactions with human intestinal epithelial cells. Infection and immunity\| PubMed ID: 22966047	2012
Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis. Open biology\| PubMed ID: 23697804	2013

Dominic C. Mills

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