organoids-as-model-for-infectious-diseases-culture-human-murine

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

University Medical Centre Utrecht

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.

Analysis of cell type-specific responses mediated by the type IV secretion system of Helicobacter pylori.

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor kappaB (NF-kappaB)-dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-kappaB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-gamma, IL-2, and tumor necrosis factor-alpha, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

Autoregulation of Th1-mediated inflammation by twist1.

Helicobacter pylori infection is a primary cause of peptic ulcers and is associated with gastric carcinogenesis. The H. pylori-induced pathophysiology may be linked to the deregulation of EGFR signalling. Elevated mucosal levels of EGF and the EGFR have been found in antral gastric biopsies of H. pylori-infected patients. A critical mechanism for regulating EGFR signalling is ligand-induced endocytosis. The internalized receptor recycles back to the plasma membrane for continued signalling or is targeted for degradation terminating receptor signalling. Here, we show that H. pylori blocks EGFR endocytosis and receptor degradation upon prolonged infection of gastric epithelial cells. Moreover, this inhibition occurs via a CagA-dependent, but CagA phosphorylation-independent activation of the non-receptor kinase c-Abl, which in turn phosphorylates the EGFR target site pY1173. This suggests a novel CagA-induced host cell response that is independent of CagA tyrosine phosphorylation. Our data indicate an intriguing strategy of H. pylori in host cell manipulations by altering selective receptor populations via a CagA-dependent endocytic mechanism. Furthermore, we identified a new role for c-Abl in phosphorylation of the EGFR target site pY1173 during H. pylori infection.

H. pylori selectively blocks EGFR endocytosis via the non-receptor kinase c-Abl and CagA.

The intracellular pathogen Legionella pneumophila activates the transcription factor NF-kappaB in macrophages and human epithelial cells, contributing to cytokine production and anti-apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF-kappaB by L. pneumophila in human epithelial cells, using a p65-GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin-deficient (DeltafliA and DeltaflaA) mutants could not induce this first, TLR5 and MyD88-dependent activation. The second p65 translocation event, however, is a long-term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65-GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IkappaBalpha and upregulation of anti-apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (DeltadotA; DeltaicmB/dotO), Dot/Icm effectors (DeltasdbA; DeltalubX) and two bacterial effector mutants (DeltaenhC; DeltaptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF-kappaB activation and intracellular replication and hints at an active interference of NF-kappaB signalling by L. pneumophila.

Temporal resolution of two-tracked NF-kappaB activation by Legionella pneumophila.

Post-translational modifications of core histones are important components of the epigenetic landscape. Recent investigations of bacterial or toxin-induced effects on histone phosphorylation and acetylation in host cells have linked the changes to transcriptional alterations of key cellular response pathways. However, these changes may have other reasons and functional consequences. Here, we show that infection of gastric epithelial cell lines with the carcinogenic bacterium Helicobacter pylori leads to changes in histone H3 phosphorylation: type IV secretion system (T4SS)-dependent decreases of H3 phosphorylation levels at serine 10 (pH3Ser10) and threonine 3 (pH3Thr3) were observed. Immunofluorescence experiments with pH3Ser10 and cyclin B1 revealed that a H. pylori-induced transient pre-mitotic arrest was responsible for the observed reduction. This causal link was substantiated further by showing that H. pylori causes a strong decrease of the cell division cycle 25 (CDC25C) phosphatase. As a consequence, mitotic histone H3 kinases such as vaccinia-related kinase 1 (VRK1) and Aurora B were not fully activated in infected cells. We show that VRK1 activity, measured using a kinase activity assay, was reduced after H. pylori infection by approximately 40%. Moreover, overexpression of VRK1, but not Aurora B, compensated for the H. pylori-induced decrease of pH3Ser10. Rephosphorylation of H3Ser10 was IkappaB kinase alpha (IKKalpha)-dependent and occurred at later time points of infection. Taken together, our work highlights the impact of bacterial pathogens on host cell chromatin; this modulation reflects the subversion of key cellular processes such as cell cycle progression.

Helicobacter pylori-induced modification of the histone H3 phosphorylation status in gastric epithelial cells reflects its impact on cell cycle regulation.

The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

The type III secretion effector NleE inhibits NF-kappaB activation.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-kappaB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-kappaB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-kappaB-induced genes is regulated by differential dynamics of single NF-kappaB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-kappaB activation have been observed in response to the cytokine tumor necrosis factor alpha (TNFalpha), little is known about the occurrence of oscillations in response to bacterial infections.

High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines.

&#x5E7D;&#x95E8;&#x87BA;&#x6746;&#x83CC;&#x4E0E;&#x80C3;&#x548C;&#x5341;&#x4E8C;&#x6307;&#x80A0;&#x6E83;&#x75A1;&#x4EE5;&#x53CA;&#x80C3;&#x764C;&#x76F8;&#x5173;&#x7684;&#x4EBA;&#x4F53;&#x80C3;&#x75C5;&#x83CC;&#x3002;&#x8D8A;&#x6765;&#x8D8A;&#x591A;&#x7684;&#x8BC1;&#x636E;&#x8868;&#x660E;&#x8FD9;&#x79CD;&#x75C5;&#x83CC;&#x7684;&#x52A8;&#x529B;&#x662F;&#x6210;&#x529F;&#x7684;&#x4EBA;&#x4F53;&#x80C3;&#x7EC4;&#x7EC7;&#x7684;&#x6B96;&#x6C11;&#x5316;&#x7684;&#x5148;&#x51B3;&#x6761;&#x4EF6;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x5B64;&#x7ACB; H.&#x87BA;&#x6746;&#x83CC; G27 HP0518 &#x7A81;&#x53D8;&#x4F53;&#x53C2;&#x5C55;&#x6539;&#x53D8;&#x7684;&#x52A8;&#x529B;&#x76F8;&#x6BD4;&#x5176;&#x7236;&#x6BCD;&#x7684;&#x5E94;&#x53D8;&#x3002;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x7A81;&#x53D8;&#x4F53;&#x8C03;&#x5236;&#x7684;&#x52A8;&#x529B;&#x76F8;&#x8FDE;&#x7684;&#x94FE;&#x63A5; O &#x7CD6;&#x57FA;&#x5316;&#x5BF9;&#x97AD;&#x6BDB;&#x86CB;&#x767D;&#x6C34;&#x5E73;&#x5347;&#x9AD8; (FlaA) &#x86CB;&#x767D;&#x3002;HP0518 &#x91CD;&#x7EC4;&#x86CB;&#x767D;&#x51CF;&#x5C11; H.&#x87BA;&#x6746;&#x83CC;&#x97AD;&#x6BDB;&#x86CB;&#x767D;&#x4F53;&#x5916;&#xFF0C;&#x6307;&#x793A; HP0518 &#x51FD;&#x6570;&#x4E2D;&#x86CB;&#x767D;&#x8D28;&#x53BB;&#x7CD6;&#x57FA;&#x7684; FlaA &#x86CB;&#x767D;&#x7684;&#x7CD6;&#x57FA;&#x5316;&#x6C34;&#x5E73;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x8D28;&#x8C31;&#x5206;&#x6790;&#x663E;&#x793A;&#x589E;&#x52A0;&#x7CD6;&#x57FA;&#x5316;&#x7684; HP0518 FlaA &#x662F;&#x56E0;&#x4E3A; pseudaminic FlaA &#xFF1B; &#x9178; (Pse) &#x6C34;&#x5E73;&#x7684;&#x53D8;&#x5316;HP0518 &#x7A81;&#x53D8;&#x4F53;&#x6D3E;&#x751F;&#x97AD;&#x6BDB;&#x86CB;&#x767D;&#x6240;&#x8F7D;&#x5927;&#x7EA6;&#x4E09;&#x500D;&#x66F4;&#x591A; Pse &#x6BD4;&#x7236;&#x6BCD;&#x7684;&#x538B;&#x529B;&#x3002;&#x8FDB;&#x4E00;&#x6B65;&#x8868;&#x578B;&#x548C;&#x5206;&#x5B50;&#x7279;&#x5F81;&#x8868;&#x660E;&#x8D85;&#x80FD;&#x52A8;&#x6027; HP0518 &#x7A81;&#x53D8;&#x5C55;&#x54C1;&#x4F18;&#x8D8A;&#x7684;&#x6B96;&#x6C11;&#x5316;&#x80FD;&#x529B;&#x548C;&#x968F;&#x540E;&#x89E6;&#x53D1;&#x589E;&#x5F3A;&#x7684; CagA &#x78F7;&#x9178;&#x548C; AGS &#x7EC6;&#x80DE; NF-&kappa; B &#x7684;&#x6FC0;&#x6D3B;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x663E;&#x793A; HP0518 &#x53C2;&#x4E0E;&#x86CB;&#x767D;&#x8D28;&#x53BB;&#x7CD6;&#x57FA;&#x7684;&#x97AD;&#x6BDB;&#x86CB;&#x767D;&#xFF0C;&#x4ECE;&#x800C;&#x8C03;&#x8282;&#x75C5;&#x539F;&#x52A8;&#x529B;&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8BC1;&#x5B9E; H.&#x87BA;&#x6746;&#x83CC;&#x97AD;&#x6BDB;&#x7684;&#x75C5;&#x539F;&#x5BBF;&#x4E3B;&#x7EC6;&#x80DE;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x4E2D;&#x7684;&#x7A81;&#x51FA;&#x4F5C;&#x7528;&#x548C;&#x8C03;&#x5236;&#x7684;&#x5BBF;&#x4E3B;&#x7EC6;&#x80DE;&#x53CD;&#x5E94;&#xFF0C;&#x53EF;&#x80FD;&#x4F1A;&#x5F71;&#x54CD;&#x53D1;&#x75C5;&#x8FC7;&#x7A0B;&#x3002;

&#x5E7D;&#x95E8;&#x87BA;&#x6746;&#x83CC; HP0518 &#x5F71;&#x54CD;&#x97AD;&#x6BDB;&#x86CB;&#x767D;&#x7CD6;&#x57FA;&#x5316;&#x6539;&#x53D8;&#x7EC6;&#x83CC;&#x7684;&#x52A8;&#x529B;&#x3002;

Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as gastric cancer. Mounting evidence suggests this pathogen's motility is prerequisite for successful colonization of human gastric tissues. Here, we isolated an H. pylori G27 HP0518 mutant exhibiting altered motility in comparison to its parental strain. We show that the mutant's modulated motility is linked to increased levels of O-linked glycosylation on flagellin A (FlaA) protein. Recombinant HP0518 protein decreased glycosylation levels of H. pylori flagellin in vitro, indicating that HP0518 functions in deglycosylation of FlaA protein. Furthermore, mass spectrometric analysis revealed increased glycosylation of HP0518 FlaA was due to a change in pseudaminic acid (Pse) levels on FlaA; HP0518 mutant-derived flagellin contained approximately threefold more Pse than the parental strain. Further phenotypic and molecular characterization demonstrated that the hyper-motile HP0518 mutant exhibits superior colonization capabilities and subsequently triggers enhanced CagA phosphorylation and NF-ÎºB activation in AGS cells. Our study shows that HP0518 is involved in the deglycosylation of flagellin, thereby regulating pathogen motility. These findings corroborate the prominent function of H. pylori flagella in pathogen-host cell interactions and modulation of host cell responses, likely influencing the pathogenesis process.

Helicobacter pylori HP0518 affects flagellin glycosylation to alter bacterial motility.

Helicobacter pylori est un pathog&egrave;ne humain gastrique, associ&eacute; &agrave; des ulc&egrave;res gastriques et duod&eacute;naux, mais aussi des cancers gastriques. Montage des preuves sugg&egrave;re la que motilit&eacute; de ce pathog&egrave;ne est la condition sine qua non &agrave; la colonisation r&eacute;ussie des tissus gastriques humaines. Ici, nous avons isol&eacute; une H. pylori G27 HP0518 mutante exposante alt&eacute;r&eacute;e motilit&eacute; en comparaison de sa souche parentale des. Nous montrons que la motilit&eacute; modul&eacute; de la mutante est li&eacute;e &agrave; des niveaux accrus de glycosylation O-li&eacute;e sur la flagelline une prot&eacute;ine (FlaA). Prot&eacute;ine recombinante de HP0518 abaiss&eacute; les taux de glycosylation de la flagelline de pylori H., in vitro, indiquant que HP0518 fonctionne de d&eacute;glycosylation de FlaA prot&eacute;ine. En outre, l&#39;analyse par spectrom&eacute;trie de masse a r&eacute;v&eacute;l&eacute; accrue de glycosylation de FlaA HP0518 &eacute;tait due &agrave; un changement dans pseudaminic acides (Pse) niveaux sur FlaA ; HP0518 d&eacute;riv&eacute;s de mutant flagelline contenait environ trois fois plus de Pse que la souche parentale. Nouvelles caract&eacute;risation ph&eacute;notypique et mol&eacute;culaire a d&eacute;montr&eacute; que le mutant HP0518 hyper-motile expose les fonctionnalit&eacute;s de colonisation sup&eacute;rieure et d&eacute;clenche par la suite am&eacute;lior&eacute;e CagA phosphorylation et activation de NF-&kappa;B dans les cellules de l&#39;AGS. Notre &eacute;tude montre que HP0518 est impliqu&eacute; dans la d&eacute;glycosylation de flagelline, donc r&eacute;glementer la motilit&eacute; pathog&egrave;ne. Ces r&eacute;sultats corroborent la fonction importante de flagelles pylori H. dans les interactions cellule h&ocirc;te-pathog&egrave;ne et la modulation des r&eacute;ponses de cellule h&ocirc;te, susceptibles d&#39;influencer le processus de la pathogen&egrave;se.

Helicobacter pylori HP0518 affecte la glycosylation de la flagelline d&#39;alt&eacute;rer la motilit&eacute; bact&eacute;rienne.

Helicobacter Pylori ist ein menschlicher Magen Erreger Magen und Zw&ouml;lffingerdarm-Geschw&uuml;re sowie Magenkrebs zugeordnet. Montage Beweis schl&auml;gt vor, diese Erreger Motilit&auml;t ist Voraussetzung f&uuml;r die erfolgreiche Besiedlung der menschliche Magen Gewebe. Hier isoliert wir eine H. Pylori G27 HP0518 mutant ausstellenden ver&auml;nderte Motilit&auml;t im Vergleich zu seiner elterlichen Belastung. Wir zeigen, dass der Mutant moduliert Motilit&auml;t erh&ouml;hte Konzentration von O-chromosomale Glycosylation auf Flagellin ein Protein (FlaA) verkn&uuml;pft ist. Rekombinante HP0518 verringert Protein Glycosylation Ebenen der H. Pylori Flagellin in vitro, dass HP0518 in Deglycosylation FlaA Protein fungiert. Dar&uuml;ber hinaus offenbart massenspektrometrischen Analyse war erh&ouml;hte Glykosylierung von HP0518 FlaA aufgrund einer &Auml;nderung in Pseudaminic S&auml;ure (Pse) Ebenen auf FlaA; HP0518 Mutant abgeleitete Flagellin enthielt etwa dreifache mehr Pse als die elterliche Belastung. Ph&auml;notypische und molekulare Charakterisierung zeigte ein weiteres Mal, dass der hyper-bewegliche HP0518-Mutant &uuml;berlegene Kolonisation Funktionen Exponate und anschlie&szlig;end erweiterte CagA-Phosphorylierung und NF-&kappa;B Aktivierung in AGS Zellen l&ouml;st. Unsere Studie zeigt, dass HP0518 die Deglycosylation der Flagellin, beteiligt ist und damit Regulierung Pathogen Motilit&auml;t. Diese Ergebnisse best&auml;tigen die herausragende Funktion des H. Pylori Flagellen in Pathogen-Host Zelle Interaktionen und Modulation der Host Zelle Antworten, wahrscheinlich den Pathogenese-Prozess zu beeinflussen.

Helicobacter Pylori HP0518 betrifft Flagellin Glycosylation, bakterielle Motilit&auml;t zu &auml;ndern.

L&#39;Helicobacter pylori &egrave; un patogeno umano gastrico associato a ulcera gastrica e duodenale, come pure il cancro gastrico. Prove di montaggio suggerisce che motilit&agrave; di questo agente patogeno &egrave; prerequisito per il successo colonizzazione dei tessuti umani gastrici. Qui, abbiamo isolato un H. pylori G27 HP0518 mutante espositrici alterata motilit&agrave; rispetto al suo ceppo parentale. Mostriamo che motilit&agrave; modulata del mutante &egrave; legata all&#39;aumento dei livelli di glicosilazione O-linked su flagellina una proteina (Kirschner). Proteina HP0518 ricombinante diminuzione dei livelli di glicosilazione di flagellina di pylori H. in vitro, che indica che HP0518 funzioni in deglycosylation della proteina Kirschner. Inoltre, analisi di spettrometria di massa ha rivelato glicosilazione aumentata di Kirschner HP0518 era dovuto ad un cambiamento nei livelli acidi pseudaminic (Pse) di Kirschner; HP0518 derivato mutante flagellina conteneva circa tre volte pi&ugrave; Pse rispetto al ceppo parentale. Ulteriore caratterizzazione fenotipica e molecolare hanno dimostrato che iper-motili mutante HP0518 esibisce le capacit&agrave; superiori di colonizzazione e innesca successivamente rafforzata CagA fosforilazione e l&#39;attivazione di NF-&kappa;B in cellule AGS. Il nostro studio mostra che HP0518 &egrave; coinvolto nel deglycosylation di flagellina, quindi regolazione della motilit&agrave; del patogeno. Questi risultati confermano la funzione prominente di flagelli di H. pylori nelle interazioni cellula ospite-agente patogeno e modulazione delle risposte delle cellule ospite, probabile che influenzano il processo di patogenesi.

Helicobacter pylori HP0518 colpisce flagellina glicosilazione di alterare la motilit&agrave; batterica.

&#x30D8;&#x30EA;&#x30B3;&#x30D0;&#x30AF;&#x30BF;&#x30FC;&#x30D4;&#x30ED;&#x30EA;&#x80C3;&#x30FB;&#x5341;&#x4E8C;&#x6307;&#x8178;&#x6F70;&#x760D;&#x3068;&#x3057;&#x3066;&#x80C3;&#x764C;&#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x4EBA;&#x9593;&#x306E;&#x80C3;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x3067;&#x3059;&#x3002;&#x8A3C;&#x62E0;&#x3092;&#x30DE;&#x30A6;&#x30F3;&#x30C8;&#x3059;&#x308B;&#x306B;&#x306F;&#x3053;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x306E;&#x904B;&#x52D5;&#x6027;&#x3072;&#x3068;&#x80C3;&#x764C;&#x7D44;&#x7E54;&#x306E;&#x6210;&#x529F;&#x690D;&#x6C11;&#x5730;&#x5316;&#x306E;&#x524D;&#x63D0;&#x6761;&#x4EF6;&#x3067;&#x3059;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x3001;H. &#x30D4;&#x30ED;&#x30EA;&#x83CC; G27 HP0518 &#x5909;&#x7570;&#x51FA;&#x5C55;&#x5909;&#x8CEA;&#x904B;&#x52D5;&#x305D;&#x306E;&#x89AA;&#x682A;&#x306B;&#x6BD4;&#x3079;&#x3066;&#x3092;&#x9694;&#x96E2;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x904B;&#x52D5;&#x6027;&#x5909;&#x7570;&#x4F53;&#x306E;&#x5909;&#x8ABF; (FlaA) &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30EC;&#x30D9;&#x30EB; o-&#x30B0;&#x30EA;&#x30B3;&#x30B7;&#x30EB; &#x30D5;&#x30E9;&#x30B8;&#x30A7;&#x30EA;&#x30F3;&#x4E0A;&#x306E;&#x5897;&#x52A0;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D44;&#x63DB;&#x3048; HP0518 &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x7CD6;&#x30EC;&#x30D9;&#x30EB; h. &#x30D4;&#x30ED;&#x30EA;&#x83CC;&#x306E;&#x30D5;&#x30E9;&#x30B8;&#x30A7;&#x30EA;&#x30F3; HP0518 FlaA &#x86CB;&#x767D;&#x8CEA;&#x306E; deglycosylation &#x3067;&#x6A5F;&#x80FD;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x4F53;&#x5916;&#x306E;&#x6E1B;&#x5C11;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x8CEA;&#x91CF;&#x5206;&#x6790;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B; HP0518 FlaA &#x306E;&#x5897;&#x52A0;&#x3057;&#x305F;&#x30B0;&#x30EA;&#x30B3;&#x30B7;&#x30EB;&#x5316; pseudaminic &#x9178; (Pse) &#x30EC;&#x30D9;&#x30EB; FlaA &#x4E0A;&#x3067;&#x306E;&#x5909;&#x66F4;&#x306B;&#x3088;&#x308A;&#x306A;&#x3063;&#x305F;;HP0518 &#x5909;&#x7570;&#x682A;&#x7531;&#x6765;&#x30D5;&#x30E9;&#x30B8;&#x30A7;&#x30EA;&#x30F3;&#x7D04; 3 &#x500D;&#x4EE5;&#x4E0A; Pse &#x3088;&#x308A;&#x89AA;&#x306E;&#x7DCA;&#x5F35;&#x3082;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x8868;&#x73FE;&#x578B;&#x304A;&#x3088;&#x3073;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x7684;&#x30CF;&#x30A4;&#x30D1;&#x30FC;&#x904B;&#x52D5;&#x6027;&#x306E; HP0518 &#x5909;&#x7570;&#x512A;&#x308C;&#x305F;&#x690D;&#x6C11;&#x5730;&#x5316;&#x306E;&#x80FD;&#x529B;&#x3092;&#x767A;&#x63EE;&#x3057;&#x3001;&#x305D;&#x306E;&#x5F8C;&#x5F37;&#x5316; CagA &#x30EA;&#x30F3;&#x9178;&#x5316;&#x3068; AGS &#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B; NF-&kappa; &#x6D3B;&#x6027;&#x5316;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x305D;&#x308C;&#x306B;&#x3088;&#x3063;&#x3066;&#x75C5;&#x539F;&#x4F53;&#x904B;&#x52D5;&#x306B;&#x95A2;&#x308F;&#x308B; HP0518 &#x306E;&#x30D5;&#x30E9;&#x30B8;&#x30A7;&#x30EA;&#x30F3;&#x3001;deglycosylation &#x3067;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x77E5;&#x898B;&#x306F;&#x3001;H. &#x30D4;&#x30ED;&#x30EA;&#x83CC;&#x3079;&#x3093;&#x6BDB;&#x75C5;&#x539F;&#x4F53;&#x30DB;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3067;&#x306E;&#x8457;&#x540D;&#x306A;&#x6A5F;&#x80FD;&#x3068;&#x75C5;&#x614B;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x53CA;&#x307C;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x30DB;&#x30B9;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x53CD;&#x5FDC;&#x306E;&#x5909;&#x8ABF;&#x3092;&#x88CF;&#x4ED8;&#x3051;&#x307E;&#x3059;&#x3002;

&#x30D8;&#x30EA;&#x30B3;&#x30D0;&#x30AF;&#x30BF;&#x30FC; &#x30FB; &#x30D4;&#x30ED;&#x30EA; HP0518 &#x7D30;&#x83CC;&#x306E;&#x904B;&#x52D5;&#x6027;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x30D5;&#x30E9;&#x30B8;&#x30A7;&#x30EA;&#x30F3;&#x7CD6;&#x9396;&#x4FEE;&#x98FE;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x307E;&#x3059;&#x3002;

&#xD5EC;&#xB9AC;&#xCF54;&#xBC15;&#xD130; &#xD30C;&#xC77C;&#xB85C;&#xB9AC;&#xB294; &#xC704;&#xC7A5; &#xBC0F; &#xC2ED;&#xC774;&#xC9C0; &#xC7A5; &#xADA4; &#xC591; &#xC704; &#xC554;&#xACFC; &#xAD00;&#xB828; &#xB41C; &#xC778;&#xAC04;&#xC758; &#xC704; &#xBCD1;&#xC6D0; &#xCCB4;. &#xC7A5;&#xCC29; &#xC99D;&#xAC70; &#xB098;&#xC654;&#xB2E4;&#xC774; &#xBCD1;&#xC6D0; &#xCCB4;&#xC758; &#xC6B4;&#xB3D9;&#xC774; &#xC131;&#xC744; &#xC778;&#xAC04;&#xC758; &#xC704; &#xC870;&#xC9C1;&#xC758; &#xC131;&#xACF5;&#xC801;&#xC778; &#xC2DD;&#xBBFC;&#xC9C0;&#xC5D0; &#xB300; &#xD55C; &#xC804;&#xC81C; &#xC870;&#xAC74;&#xC785;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xC6B4;&#xB3D9;&#xB97C; &#xD5E4; &#xD30C;&#xC77C;&#xB85C;&#xB9AC; G27 HP0518 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC804;&#xC2DC; &#xBCC0;&#xACBD; &#xB41C; &#xC131; &#xADF8; &#xBD80;&#xBAA8;&#xC758; &#xBD80;&#xB2F4;&#xC5D0; &#xBE44;&#xD574; &#xC808;&#xC5F0;. &#xC6B0;&#xB9AC;&#xB294; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xBCC0;&#xC870; &#xB41C; &#xC6B4;&#xB3D9; &#xC131; (FlaA) &#xB2E8;&#xBC31;&#xC9C8; flagellin&#xC5D0; O &#xC5F0;&#xACB0; glycosylation&#xC758; &#xC99D;&#xAC00; &#xC218;&#xC900;&#xC5D0; &#xC5F0;&#xACB0; &#xB41C; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. &#xC7AC;&#xC870;&#xD569; HP0518 &#xB2E8;&#xBC31;&#xC9C8; glycosylation &#xC218;&#xC900;&#xC758; H. pylori flagellin &#xC2DC;&#xD5D8;&#xAD00;, HP0518 FlaA &#xB2E8;&#xBC31;&#xC9C8;&#xC758; deglycosylation&#xC5D0;&#xC11C; &#xC791;&#xB3D9; &#xD558;&#xB294; &#xAC83;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xAC10;&#xC18C;. &#xB610;&#xD55C;, &#xB300;&#xB7C9; spectrometric &#xBD84;&#xC11D; &#xACF5;&#xAC1C; HP0518 FlaA &#xC99D;&#xAC00; glycosylation FlaA;&#xC5D0; pseudaminic &#xC0B0; (Pse) &#xB808;&#xBCA8; &#xBCC0;&#xACBD; &#xD588;&#xB2E4; HP0518 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD30C;&#xC0DD; flagellin &#xBD80;&#xBAA8;&#xC758; &#xBD80;&#xB2F4; &#xBCF4;&#xB2E4; &#xC57D; &#xC0BC; &#xBC30; &#xB354; &#xB9CE;&#xC740; Pse &#xD3EC;&#xD568;. &#xCD94;&#xAC00; phenotypic &#xBD84;&#xC790; &#xD2B9;&#xC131;&#xD654; &#xC2DC;&#xC5F0; &#xD558;&#xC774;&#xD37C; &#xBA54;&#xAD6C;&#xBBF8; HP0518 &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC6B0;&#xC218;&#xD55C; &#xC2DD;&#xBBFC; &#xAE30;&#xB2A5;&#xC744; &#xC804;&#xC2DC; &#xD558; &#xACE0; &#xC774;&#xD6C4;&#xC5D0; &#xD5A5;&#xC0C1; &#xB41C; CagA &#xC778; &#xC0B0;&#xD654; &#xBC0F; AGS &#xC138;&#xD3EC;&#xC5D0;&#xC11C; NF &kappa;B &#xD65C;&#xC131;&#xD654;&#xB97C; &#xD2B8;&#xB9AC;&#xAC70;&#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; HP0518 &#xD568;&#xC73C;&#xB85C;&#xC368; &#xBCD1;&#xC6D0; &#xCCB4;&#xC758; &#xC6B4;&#xB3D9; &#xC131; &#xC870;&#xC808; flagellin, deglycosylation&#xC5D0; &#xD3EC;&#xD568; &#xB41C;&#xC774; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. &#xC774; &#xBC1C;&#xACAC;&#xC740; &#xD655;&#xC99D; H. pylori flagella &#xBCD1;&#xC6D0; &#xCCB4;-&#xD638;&#xC2A4;&#xD2B8; &#xC140; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0;&#xC11C;&#xC758; &#xB208;&#xC5D0; &#xB744;&#xB294; &#xAE30;&#xB2A5; &#xBC0F; &#xBCC0;&#xC870; &#xAC00;&#xB2A5;&#xC131;&#xC774; pathogenesis &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xD638;&#xC2A4;&#xD2B8; &#xC140; &#xC751;&#xB2F5;&#xC758;.

&#xD5EC;&#xB9AC;&#xCF54;&#xBC15;&#xD130; &#xD30C;&#xC77C;&#xB85C;&#xB9AC; HP0518 flagellin glycosylation&#xC744; &#xC138;&#xADE0;&#xC758; &#xC6B4;&#xB3D9; &#xC131; &#xBCC0;&#xACBD;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC90D;&#xB2C8;&#xB2E4;.

Helicobacter pylori es un pat&oacute;geno humano de g&aacute;strico asociado con &uacute;lceras g&aacute;stricas y duodenales, as&iacute; como c&aacute;ncer g&aacute;strico. Creciente evidencia sugiere que la movilidad de este pat&oacute;geno es requisito previo para la colonizaci&oacute;n exitosa de tejidos g&aacute;stricos humanos. Aqu&iacute;, se aislaron una H. pylori G27 HP0518 mutante expositora alterada motilidad en comparaci&oacute;n con su cepa parental. Mostramos que motilidad modulado de la mutante est&aacute; ligada a los niveles crecientes de glicosilaci&oacute;n O vinculados en flagelina una prote&iacute;na (FlaA). La prote&iacute;na recombinante HP0518 disminuci&oacute;n en los niveles de glicosilaci&oacute;n de flagelina de H. pylori in vitro, lo que indica que HP0518 funciona en deglycosylation de prote&iacute;na FlaA. Adem&aacute;s, an&aacute;lisis de espectrometr&iacute;a de masa revelaron aumento de glicosilaci&oacute;n de HP0518 FlaA fue debido a un cambio en pseudaminic los niveles de &aacute;cido (Pse) en FlaA; HP0518 derivados del mutante flagelina conten&iacute;a aproximadamente triple Pse m&aacute;s que la cepa parental. Caracterizaci&oacute;n fenot&iacute;pica y molecular hab&iacute;a demostrado adem&aacute;s que el mutante HP0518 hyper-motile exhibe capacidades superiores de la colonizaci&oacute;n y posteriormente provoca mayor de fosforilaci&oacute;n de caga y activaci&oacute;n de NF-&kappa;B en c&eacute;lulas AGS. Nuestro estudio muestra que el HP0518 est&aacute; implicada en la deglycosylation de flagelina, tal modo regular la motilidad del pat&oacute;geno. Estos resultados corroboran la funci&oacute;n prominente de flagelos de H. pylori en las interacciones de c&eacute;lula hu&eacute;sped pat&oacute;geno y modulaci&oacute;n de respuestas de la c&eacute;lula del anfitri&oacute;n, probables que influyen en el proceso de patog&eacute;nesis.

Helicobacter pylori HP0518 afecta flagelina glicosilaci&oacute;n para alterar la motilidad bacteriana.

The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem cell biomarkers would accelerate our understanding of stem cell roles in tissue homeostasis and cancer. Membrane-expressed markers would also facilitate isolation of these adult stem cell populations for exploitation of their regenerative potential. Here, we review recent advances in adult stem cell biology, highlighting the promise and pitfalls of the candidate biomarkers of the various stem cell populations.

Tissue-resident adult stem cell populations of rapidly self-renewing organs.

The early stage of infection with Neisseria gonorrhoeae (Ngo), the causative agent of gonorrhoea, is marked by type IV pilus (Tfp)-mediated attachment and the formation of bacterial microcolonies on epithelial cells. Retraction of the Ngo Tfp generates substantial force on its substrate which can elicit host cell signalling. Here, we observed that this retraction force could also activate nuclear factor (NF)-κB, the central signalling cascade of innate immunity. Using a p65-GFP-expressing epithelial cell line, we show that piliated Ngo induce asynchronous NF-κB activation in infected cells, which is temporally associated with the formation of gonococcal microcolonies. A mutant lacking PilT, an ATPase necessary for Tfp retraction, induced markedly reduced NF-κB activation. This was accompanied by decreased NF-κB target gene transcription and cytokine release. The impaired ability of the pilT mutant to activate NF-κB was compensated by applying mechanical shear stress to the infected host cells, indicating that the mechanical forces generated by retractile pili are involved in the retraction-dependent activation of NF-κB elicited by gonococcal microcolonies. Thus, our work provides evidence for an intriguing relationship between microcolony growth, pilus retraction and host cell signalling, with likely implications with regard to the course of symptomatic versus asymptomatic gonococcal infections.

Activation of NF-κB by Neisseria gonorrhoeae is associated with microcolony formation and type IV pilus retraction.

Antimicrobial peptides are constituents of the first-line innate mucosal defense system that acts as a barrier to establishment of infection. The highly successful human gastric pathogen, Helicobacter pylori, is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including human β-defensin 3 (hBD3). We find that hBD3 is highly active against H. pylori in vitro and is rapidly induced during early infection via EGFR-dependent activation of MAP kinase and JAK/STAT signaling. However, during prolonged infection, hBD3 was subsequently downregulated by the H. pylori virulence determinant CagA. Upon translocation into host cells, CagA activated the cellular tyrosine phosphatase, SHP-2, terminating EGFR activation and downstream signaling and increasing bacterial viability. Chemical inhibition and knockdown of SHP-2 expression rescued hBD3 synthesis and bactericidal activity. Thus, we reveal how cagPAI-positive H. pylori strains use CagA to evade a key innate mucosal defense pathway to support the establishment of persistent infection.

The Helicobacter pylori virulence effector CagA abrogates human β-defensin 3 expression via inactivation of EGFR signaling.

Helicobacter pylori is a bacterial pathogen that colonizes the gastric niche of ∼ 50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H. pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ∼ 3000 H. pylori mutants revealed three non-cagPAI genes that affected NF-κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-κB, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded cofactor of T4SS function.

Helicobacter pylori outer membrane protein HopQ identified as a novel T4SS-associated virulence factor.

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."

Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium.

Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts. We show that short-chain fatty acids selectively increase the number of L cells, resulting in an elevation of GLP-1 release. This is accompanied by the upregulation of transcription factors associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L cells in mouse and human crypts as a potential basis for novel therapeutic strategies in patients with type 2 diabetes.

Generation of L cells in mouse and human small intestine organoids.

We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori.

In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection.

Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation.

A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.

Sina Bartfeld

Hubrecht Institute for Developmental Biology and Stem Cell Research

University Medical Centre Utrecht

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Sina Bartfeld

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Hubrecht Institute for Developmental Biology and Stem Cell Research

University Medical Centre Utrecht

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Hubrecht Institute for Developmental Biology and Stem Cell Research