genetic-manipulation-ku80-strains-for-functional-genomic-analysis

Genetic Manipulation in <em>&Delta;ku80</em> Strains for Functional Genomic Analysis of <em>Toxoplasma gondii</em>

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

The Geisel School of Medicine at Dartmouth

We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). This method includes (i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques, (ii) in vivo cross-linking with formaldehyde, (iii) tandem affinity purification of bait proteins under fully denaturing conditions, and (iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions and permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of noncross-linked proteins to bait proteins. Two different negative controls were employed to eliminate the possibility of identifying background and nonspecific proteins as interacting partners, especially those caused by nonspecific binding to the stationary phase used for protein purification. In an initial demonstration of this approach, we tagged three Salmonella proteinsHimD, PduB and PhoPwith known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified for each bait protein, including the known binding partners such as HimA for HimD, as well as unexpected binding partners. Our results suggest that novel protein-protein interactions identified may be critical to pathogenesis by Salmonella.

A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis.

Nous avons modifi&eacute; avec succ&egrave;s une m&eacute;thode existante afin d&#39;&eacute;tudier les interactions entre les prot&eacute;ines de la bact&eacute;rie pathog&egrave;ne Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). Cette m&eacute;thode inclut (i) l&#39;ajout d&#39;une balise d&#39;histidine-biotine-histidine aux prot&eacute;ines app&acirc;t via des techniques d&#39;ADN recombinant, la r&eacute;ticulation (ii) in vivo avec le formald&eacute;hyde, la purification d&#39;affinit&eacute; en tandem (iii) des prot&eacute;ines d&#39;app&acirc;t dans des conditions enti&egrave;rement d&eacute;naturation et (iv) l&#39;identification des prot&eacute;ines r&eacute;ticul&eacute;es aux prot&eacute;ines app&acirc;t par chromatographie en phase liquide en conjonction avec la spectrom&eacute;trie de masse en tandem. R&eacute;ticulation in vivo stabilise les interactions entre prot&eacute;ines et autoris&eacute; la purification en deux &eacute;tapes ult&eacute;rieures men&eacute;e dans des conditions d&eacute;naturantes. La purification en deux &eacute;tapes a consid&eacute;rablement r&eacute;duit la liaison non sp&eacute;cifique des prot&eacute;ines bidimensionelle pour app&acirc;t des prot&eacute;ines. Deux contr&ocirc;les n&eacute;gatifs diff&eacute;rents ont &eacute;t&eacute; utilis&eacute;s pour &eacute;liminer la possibilit&eacute; d&#39;identifier des ant&eacute;c&eacute;dents et des prot&eacute;ines non sp&eacute;cifiques comme des partenaires qui interagissent, notamment ceux caus&eacute;s par une liaison non sp&eacute;cifique &agrave; la phase stationnaire, utilis&eacute;e pour la purification de la prot&eacute;ine. Dans une d&eacute;monstration initiale de cette approche, nous le tag trois Salmonella proteinsHimD, PduB et PhoPwith connus des partenaires de liaison qui variaient entre stable (par exemple, HimD) et transitoire (c.-&agrave;-d. PhoP). Des ensembles distincts de prot&eacute;ines qui interagissent ont &eacute;t&eacute; identifi&eacute;s pour chaque prot&eacute;ine app&acirc;t, y compris les partenaires de liaison connue comme HimA pour HimD, mais aussi des partenaires inattendus. Nos r&eacute;sultats sugg&egrave;rent que les interactions prot&eacute;ine-prot&eacute;ine nouvelle identifi&eacute;es peuvent &ecirc;tre critiques &agrave; la pathogen&egrave;se de Salmonella.

Une m&eacute;thode d&#39;&eacute;tude des interactions prot&eacute;ines-prot&eacute;ines li&eacute;es &agrave; la pathogen&egrave;se de salmonella typhimurium.

Abbiamo modificato con successo un metodo esistente per indagare sulle interazioni della proteina-proteina nel batterio patogeno Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). Questo metodo include (i) l&#39;aggiunta di un tag di istidina-biotina-istidina alle proteine esca tramite tecniche di DNA ricombinante, cross-linking (ii) in vivo con formaldeide, purificazione di affinit&agrave; (iii) tandem di proteine esca in condizioni completamente denaturazione e (iv) l&#39;identificazione delle proteine reticolato alle proteine esca dalla cromatografia liquido in combinazione con la spettrometria di massa tandem. Cross-linking in vivo stabilizzato le interazioni della proteina e consentito il passaggio di purificazione in due fasi successive condotto sotto condizioni di denaturazione. La purificazione di due passaggi notevolmente ridotto legame delle proteine noncross-linked per proteine l&#39;esca. Due diversi controlli negativi sono stati impiegati per eliminare la possibilit&agrave; di identificare proteine aspecifiche e sfondo come partner interagiscono tra loro, soprattutto quelle causate dal legame aspecifico con la fase stazionaria utilizzata per la purificazione della proteina. In una dimostrazione iniziale di questo approccio, abbiamo messo tre Salmonella proteinsHimD, PduB e PhoPwith noto partner di associazione che andavano da stabile (ad es., HimD) al transitorio (cio&egrave;, PhoP). Insiemi distinti di proteine interagenti sono stati identificati per ogni proteina esca, compreso il partner associazione conosciuta come HimA HimD, nonch&eacute; partner associazione inaspettata. I nostri risultati suggeriscono che le interazioni proteina-proteina romanzo identificate possono essere critiche a patogenesi di Salmonella.

Un metodo per indagare le interazioni della proteina-proteina correlate alla patogenesi di salmonella typhimurium.

Vaccinia virus infection can confer immunity to smallpox by inducing potent T cell and antibody responses. While the CD8 T cell response to vaccinia virus has been well characterized, less is known about factors required for priming and memory for the CD4 T cells. Focusing on two recently described epitopes, we show that after intranasal infection, both I1L and L4R epitopes are co-dominant during the acute response, but the I1L epitope dominates during memory. CD4 T cell priming was intact in the absence of CD80/86, however secondary responses were reduced. This contrasts with our previous data showing CD80/86-CD28 interaction is required for optimal primary and memory CD8 T cell responses. The absence of CD80/86 also changed the immunodominance hierarchy during memory, with the I1L and L4R responses becoming co-dominant in knockout mice. These data highlight different costimulatory requirements for primary CD4 and CD8 T cell responses to vaccinia virus.

Differential requirements for CD80/86-CD28 costimulation in primary and memory CD4 T cell responses to vaccinia virus.

Infection au virus de la vaccine peut immuniser &agrave; la variole en stimulant puissant des lymphocytes T et les r&eacute;ponses en anticorps. Alors que la r&eacute;ponse des lymphocytes T CD8 au virus de la vaccine a &eacute;t&eacute; bien caract&eacute;ris&eacute;e, on conna&icirc;t moins les facteurs n&eacute;cessaires &agrave; l&#39;amor&ccedil;age et la m&eacute;moire pour les cellules T CD4. En se concentrant sur deux &eacute;pitopes r&eacute;cemment d&eacute;crits, nous montrons que, apr&egrave;s l&#39;infection intranasale, I1L tant L4R &eacute;pitopes sont codominants au cours de la r&eacute;action aigu&euml;, mais l&#39;&eacute;pitope I1L domine au cours de la m&eacute;moire. Amor&ccedil;age de cellules T CD4 &eacute;tait intact en l&#39;absence de CD80/86, mais les r&eacute;ponses secondaires ont &eacute;t&eacute; r&eacute;duites. Ceci contraste avec nos donn&eacute;es ant&eacute;rieures montrant CD80/86-CD28 interaction est n&eacute;cessaire pour les r&eacute;ponses des cellules T CD8 optimales primaire et de la m&eacute;moire. L&#39;absence de CD80/86 a &eacute;galement modifi&eacute; la hi&eacute;rarchie de l&#39;immunodominance au cours de la m&eacute;moire, avec les r&eacute;ponses I1L et L4R devenant codominante dans les souris knock-out. Ces donn&eacute;es mettent en &eacute;vidence les diff&eacute;rentes exigences de costimulation pour primaires lymphocytes CD4 et CD8 T au virus de la vaccine.

Exigences diff&eacute;rentielles de costimulation CD80/86-CD28 dans les r&eacute;ponses des cellules T CD4 primaires et de la m&eacute;moire au virus de la vaccine.

Infezione da virus vaccinia pu&ograve; conferire l&#39;immunit&agrave; a vaiolo da indurre potenti delle cellule T e risposte anticorpali. Mentre la risposta delle cellule T CD8 virus vaccinia &egrave; stata ben caratterizzata, meno &egrave; conosciuto circa i fattori necessari per l&#39;innesco e la memoria per le cellule T CD4. Concentrandosi su due epitopi recentemente descritti, ci mostrano che dopo infezione intranasale, I1L sia L4R epitopi sono codominanti durante la risposta acuta, ma l&#39;epitopo I1L domina durante la memoria. Adescamento delle cellule T CD4 era intatto in assenza di CD80/86, tuttavia le risposte secondarie sono state ridotte. Questo contrasta con i nostri dati precedenti, mostrando CD80/86-CD28 interazione &egrave; necessaria per ottimale primaria e memoria risposte delle cellule T CD8. L&#39;assenza di CD80/86 ha cambiato anche la gerarchia immunodominance durante la memoria, con le risposte I1L e L4R diventando codominanti topi knockout. Questi dati evidenziano diversi requisiti di costimolazione per primarie risposte delle cellule CD4 e CD8 T al virus di vaccinia.

Differenziali requisiti per CD80/86-CD28 costimulation nella primaria e memoria risposte di cellule T CD4 a virus vaccinia.

Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-Î³-activated macrophages, conferring parasite survival inÂ vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.

Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence.

Les macrophages sont sp&eacute;cialis&eacute;s afin de d&eacute;tecter et de d&eacute;truire les microbes intracellulaires et encore un certain nombre de pathog&egrave;nes ont &eacute;volu&eacute; pour exploiter ce cr&eacute;neau hostile. Nous d&eacute;montrons que le parasite intracellulaire obligatoire Toxoplasma gondii d&eacute;sarme les m&eacute;canismes de clairance inn&eacute;e des macrophages en s&eacute;cr&eacute;tant une thr&eacute;onine kinase de s&eacute;rine appel&eacute;e ROP18, qui se lie &agrave; et phosphoryle GTPases li&eacute;es &agrave; l&#39;immunit&eacute; (IRGs). Substrat profilage de ROP18 a r&eacute;v&eacute;l&eacute; une pr&eacute;f&eacute;rence pour un motif conserv&eacute; au sein de la r&eacute;gion de commutateur j&#39;ai du domaine GTPase, une modification pr&eacute;vu de perturber la fonction de l&#39;IRG. Conform&eacute;ment &agrave; cela, expression de ROP18 &eacute;tait &agrave; la fois n&eacute;cessaire et suffisante pour bloquer le recrutement de Irgb6, qui devait &agrave; son tour pour la destruction du parasite. ROP18 la phosphorylation de IRGs emp&ecirc;ch&eacute; clairance au sein inflammatoires monocytes et macrophages activ&eacute;s par IFN-&gamma;, conf&eacute;rant de survie du parasite in vivo et la promotion de virulence. IRGs sont impliqu&eacute;es dans l&#39;autorisation d&#39;une vari&eacute;t&eacute; de pathog&egrave;nes intracellulaires, sugg&eacute;rant que les autres facteurs de virulence peuvent contrecarrer de m&ecirc;me ce m&eacute;canisme de d&eacute;fense cellulaire inn&eacute;.

Phosphorylation des GTPases immunit&eacute; li&eacute;es par une kinase de gondii-s&eacute;cr&eacute;t&eacute;e Toxoplasma favorise la virulence et la survie des macrophages.

I macrofagi sono specializzati per individuare e distruggere i microbi intracellulari e ancora un certo numero di agenti patogeni si &egrave; evoluti per sfruttare questa nicchia ostile. Qui dimostriamo che i parassiti intracellulari obbligati Toxoplasma gondii disarma meccanismi innati liquidazione macrofago secernendo una treonina chinasi di serina chiamata ROP18, che si lega a e fosforila l&#39;immunit&agrave;-correlati GTPases (IRG). Substrato profilatura di ROP18 ha rivelato una preferenza per un motivo conservata all&#39;interno della regione di interruttore di dominio GTPasi, una modifica che prevista a disturbare la funzione IRG. Coerentemente con questo, espressione di ROP18 era necessario sia sufficiente a bloccare l&#39;assunzione di Irgb6, che era a sua volta richiesto per la distruzione del parassita. ROP18 fosforilazione di IRG impedito liquidazione entro infiammatorie monociti e macrofagi IFN-&gamma;-attivato, che conferisce la sopravvivenza del parassita in vivo e promuovendo la virulenza. IRG sono implicati nella liquidazione di una variet&agrave; di agenti patogeni intracellulari, suggerendo che altri fattori di virulenza similmente possono contrastare questo meccanismo di difesa cellulare innato.

Fosforilazione di GTPases immunit&agrave;-correlati da una chinasi Toxoplasma gondii-secreto favorisce la virulenza e la sopravvivenza dei macrofagi.

Type II Toxoplasma gondii KU80 knockouts (Î”ku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Î”ku80 Î”hxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Î”ku80 Î”hxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Î”gra4 and Î”gra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Î”ku80 Î”hxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.

Type II Toxoplasma gondii KU80 d&eacute;bouchures (&Delta;ku80) d&eacute;ficientes en fin non-homologues rejoindre ont &eacute;t&eacute; d&eacute;velopp&eacute;s pour supprimer le trajet dominant m&eacute;diation int&eacute;gration al&eacute;atoire du ciblage des &eacute;pisomes. Ciblage de fr&eacute;quence dans la souche du type II &Delta;ku80 &Delta;hxgprt g&eacute;nique mesur&eacute;e &agrave; l&#39;orotate (FCT) et les loci de phosphoribosyltransf&eacute;rase uracile (UPRT) a &eacute;t&eacute; tr&egrave;s efficace. Pour &eacute;valuer le potentiel de la souche &Delta;hxgprt de type II &Delta;ku80 pour &eacute;tudier la fonction des g&egrave;nes affectant la biologie du kyste et stades latentes de l&#39;infection, nous avons cibl&eacute; la suppression de quatre g&egrave;nes antig&egrave;ne parasite (GRA4, GRA6, ROP7 et tgd057) qui codent caract&eacute;ris&eacute;es &eacute;pitopes T CD8 qui d&eacute;clenchent les populations correspondantes sp&eacute;cifiques de l&#39;antig&egrave;ne CD8 T cell associ&eacute;es au contr&ocirc;le de l&#39;infection. D&eacute;veloppement du kyste dans ces souches mutantes de type II n&#39;a pas &eacute;t&eacute; trouv&eacute; strictement d&eacute;pendante de l&#39;antig&egrave;ne-sp&eacute;cifiques CD8 T cell h6te. En revanche, un r&ocirc;le biologique important a &eacute;t&eacute; r&eacute;v&eacute;l&eacute; pour les prot&eacute;ines des granules denses GRA4 et GRA6 dans le d&eacute;veloppement de kystes puisque les charges de cerveau tissu kyste ont &eacute;t&eacute; r&eacute;duites drastiquement sp&eacute;cifiquement chez les souches mutantes avec GRA4 et/ou GRA6 supprim&eacute;. Compl&eacute;mentation des souches mutantes &Delta;gra4 et &Delta;gra6 &agrave; l&#39;aide d&#39;un all&egrave;le fonctionnel de la GRA supprim&eacute; codage r&eacute;gion plac&eacute;e sous le contr&ocirc;le du locus UPRT endog&egrave;ne s&#39;est av&eacute;r&eacute;e pour significativement restaurer fardeaux de kyste de cerveau. Ces r&eacute;sultats r&eacute;v&egrave;lent que les prot&eacute;ines GRA jouent un r&ocirc;le fonctionnel dans l&#39;&eacute;tablissement des charges de kyste et une infection latente. Collectivement, nos r&eacute;sultats sugg&egrave;rent qu&#39;un type de bagage g&eacute;n&eacute;tique de la &Delta;ku80 de &Delta;hxgprt II permet une analyse fonctionnelle des d&eacute;bits plus &eacute;lev&eacute;s du g&eacute;nome du parasite, de r&eacute;v&eacute;ler des aspects fondamentaux de la biologie de parasite contr&ocirc;lant la virulence, la pathogen&egrave;se et la transmission.

Souches de type II Toxoplasma gondii KU80 knockout permettent une analyse fonctionnelle des g&egrave;nes n&eacute;cessaires pour le d&eacute;veloppement de kystes et d&#39;une infection latente.

Tipo II Toxoplasma gondii KU80 knockouts (&Delta;ku80) carenti di unirsi nonhomologous fine sono stati sviluppati per eliminare la dominante via mediazione integrazione casuale di targeting episomes. Gene targeting per frequenza nel tipo II &Delta;ku80 &Delta;hxgprt ceppo misurata presso l&#39;orotato (= OPRT) e i loci di adenosin fosforibosiltrasferasi uracile (UPRT) era altamente efficiente. Per valutare il potenziale del ceppo tipo II &Delta;ku80 &Delta;hxgprt per esaminare la funzione del gene che interessa biologia cisti e stadi latente dell&#39;infezione, abbiamo mirato l&#39;eliminazione di quattro parassita antigene geni (GRA4, GRA6, ROP7 e tgd057) che codificano caratterizzati cellule T cell epitopi che suscitano corrispondente antigene-specifiche cellule T popolazioni di cellule associate al controllo dell&#39;infezione. Lo sviluppo di cisti in questi ceppi mutanti di tipo II non &egrave; stato trovato per essere strettamente dipendente da risposte antigene-specifiche cellule T cell host. Al contrario, un significativo ruolo biologico &egrave; stato rivelato per le proteine dei granuli densi GRA4 e GRA6 nello sviluppo di cisti poich&eacute; gli oneri di cisti tessuto cerebrale sono stati drasticamente ridotta in particolare ceppi mutanti con GRA4 o GRA6 cancellato. Complementazione dei ceppi mutanti &Delta;gra4 e &Delta;gra6 utilizzando un allele funzionale del GRA eliminato codifica regione posizionato sotto il controllo del locus UPRT endogeno &egrave; stato trovato per ripristinare in modo significativo gli oneri di cisti cerebrali. Questi risultati rivelano che le proteine GRA giocano un ruolo funzionale nella creazione di cisti oneri e infezione latente. Collettivamente, i nostri risultati suggeriscono che un tipo II &Delta;ku80 &Delta;hxgprt fondo genetico permette una velocit&agrave; superiore di analisi funzionale del genoma del parassita per rivelare gli aspetti fondamentali della biologia del parassita controllo virulenza, patogenesi e trasmissione.

Tipo II Toxoplasma gondii KU80 knockout ceppi consentono analisi funzionale dei geni necessari per lo sviluppo di cisti e di infezione latente.

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (Î”ROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in Î”ROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ÃŸ and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-Î³-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by Î”ROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. Î”ROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control.

La kinase ROP16 de Toxoplasma gondii est inject&eacute;e dans le cytosol de cellule h&ocirc;te o&ugrave; il active le signal transducteur et activateur de la transcription (STAT) -3 et STAT6. Ici, nous avons g&eacute;n&eacute;r&eacute; un mutant de d&eacute;l&eacute;tion ROP16 sur un Type je fond de souche parasite, comme un mutant de compl&eacute;mentation de contr&ocirc;le avec l&#39;expression de ROP16 restaur&eacute;e. Nous avons &eacute;tudi&eacute; le r&ocirc;le biologique de la mol&eacute;cule de ROP16 au cours de l&#39;infection par T. gondii. Infection des macrophages d&eacute;riv&eacute;s de la moelle osseuse de souris par des parasites rop16-supprim&eacute; (&Delta;ROP16) a entra&icirc;n&eacute; des quantit&eacute;s accrues de production de IL - 12p 40 par rapport &agrave; la souche parentale RH ROP16 positifs. Haut niveau production d&#39;IL - 12p 40 &Delta;ROP16 infection d&eacute;pendait de la mol&eacute;cule d&#39;adaptateur de la cellule h&ocirc;te MyD88, mais &eacute;tonnamment &eacute;tait ind&eacute;pendante de toute pr&eacute;c&eacute;demment reconnu T. gondii d&eacute;clench&eacute;e voie, reliant &agrave; MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1&szlig; et IL-18). En outre, ROP16 a &eacute;t&eacute; trouv&eacute; &agrave; la m&eacute;diation les effets suppresseurs de Toxoplasma sur la synth&egrave;se des cytokines induite par LPS dans les macrophages, ainsi que sur la production d&#39;oxyde nitrique induite par l&#39;IFN-&gamma; par les astrocytes et les cellules microgliales. En outre, ROP16 d&eacute;clench&eacute;e synth&egrave;se d&#39;h&ocirc;te cellule arginase-1 de mani&egrave;re STAT6-d&eacute;pendante. Dans les fibroblastes et les macrophages, induisent l&#39;arginase-1 en tachyzo&iuml;tes &Delta;ROP16 a dans la r&eacute;sistance &agrave; des conditions de famine de limiter l&#39;arginine, un acide amin&eacute; essentiel pour la r&eacute;plication et la virulence de ce parasite. &Delta;ROP16 tachyzo&iuml;tes qui n&#39;a provoqu&eacute; aucune cellule h&ocirc;te arginase 1 affiche une augmentation de la r&eacute;plication et la diffusion au cours de l&#39;infection in vivo. Nous concluons cette rencontre entre Toxoplasma ROP16 et de la cellule h&ocirc;te STAT signalisation cascade a des effets pl&eacute;iotropiques en aval qui agissent en multiple et complexe fa&ccedil;ons de diriger le cours de l&#39;infection.

Toxoplasma gondii rhoptry kinase ROP16 active STAT3 et STAT6 r&eacute;sultant dans le contr&ocirc;le de la croissance de l&#39;inhibition et d&eacute;pendante de l&#39;arginase-1 cytokine.

La chinasi ROP16 di Toxoplasma gondii viene iniettata nel citosol delle cellule ospite dove attiva trasduttore di segnale e attivatore di trascrizione (STAT) -3 e STAT6. Qui, abbiamo generato un mutante di delezione ROP16 su un tipo mi sfondo di ceppo del parassita, come pure un mutante di complementazione di controllo con espressione ROP16 restaurato. Abbiamo studiato il ruolo biologico della molecola ROP16 durante l&#39;infezione da T. gondii. Infezione di macrofagi di derivate dal midollo osseo del mouse con parassiti eliminati rop16 (&Delta;ROP16) ha portato in maggiore quantit&agrave; di produzione di IL - 12p 40 rispetto al ceppo parentale RH positivo ROP16. Alto livello produzione di IL - 12p 40 in &Delta;ROP16 infezione dipende dalla molecola adattatore host cell MyD88, ma sorprendentemente era indipendente da qualsiasi precedentemente riconosciuto T. gondii ha attivato percorso collegamento a MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1&szlig; e IL-18). Inoltre, ROP16 &egrave; stato trovato per mediare gli effetti soppressivi di Toxoplasma sulla sintesi di citochine indotta da LPS in macrofagi e sulla produzione di ossido nitrico indotta da IFN-&gamma; da astrociti e microglia. Inoltre, ROP16 innescato sintesi di host cell arginase-1 in modo STAT6-dipendente. In fibroblasti e macrofagi, fallimento per indurre arginase-1 da &Delta;ROP16 tachyzoites portato nella resistenza a condizioni di fame di limitare l&#39;arginina, un aminoacido essenziale per la replicazione e la virulenza di questo parassita. Tachyzoites &Delta;ROP16 che non &egrave; riuscito a indurre host cell arginase-1 visualizzato replica aumentata e diffusione durante l&#39;infezione in vivo. Possiamo concludere quell&#39;incontro tra Toxoplasma ROP16 e cellula ospite STAT segnalazione cascata ha gli effetti pleiotropici a valle che agiscono in multipli e complessa modi di dirigere il corso dell&#39;infezione.

Chinasi di Toxoplasma gondii rhoptry ROP16 attiva STAT3 e STAT6 con conseguente controllo di crescita di inibizione e arginase-1-dipendente di cytokine.

Article	Year
A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis. Journal of proteome research\| PubMed ID: 19206470	2009
Differential requirements for CD80/86-CD28 costimulation in primary and memory CD4 T cell responses to vaccinia virus. Cellular immunology\| PubMed ID: 21040905	2011
Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence. Cell host & microbe\| PubMed ID: 21147463	2010
Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryotic cell\| PubMed ID: 21531875	2011
Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control. PLoS pathogens\| PubMed ID: 21931552	2011

Leah M. Rommereim

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