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Regulation of Yersinia Yop-effector delivery by translocated YopE
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Wolf-Watz)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Wolf-Watz)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Fällman)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). (Wolf-Watz)
Visa övriga samt affilieringar
2008 (Engelska)Ingår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, nr 3-4, s. 183-192Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

Ort, förlag, år, upplaga, sidor
Elsevier , 2008. Vol. 298, nr 3-4, s. 183-192
Nyckelord [en]
YopE; GAP activity; ExoS; ExoT, feedback inhibition, translocation, type III secretion
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Identifikatorer
URN: urn:nbn:se:umu:diva-20424DOI: 10.1016/j.ijmm.2007.04.007PubMedID: 17597003OAI: oai:DiVA.org:umu-20424DiVA, id: diva2:208670
Tillgänglig från: 2009-03-19 Skapad: 2009-03-19 Senast uppdaterad: 2018-06-09
Ingår i avhandling
1. Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence
Öppna denna publikation i ny flik eller fönster >>Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence
2005 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In order to establish an extra-cellular infection the gram-negative bacteria Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to translocate a set of anti-host effectors into eukaryotic cells. The toxins disrupt signalling pathways important for phagocytosis, cytokine production and cell survival. Secretion and translocation via this T3SS is strictly regulated on several levels. In this context, the function of YopE and LcrH during Yersinia infections has been analysed.

YopE is an essential translocated effector that disrupts the actin cytoskeleton of infected eukaryotic cells, by inactivating small GTPases through its GTPase activating protein (GAP) activity. However, cytotoxicity can be uncoupled from in vitro GAP activity towards the RhoA, Rac1 and Cdc42 GTPases. Furthermore, in vivo studies of the YopE GAP activity revealed that only RhoA and Rac1 are targeted, but this is not a pre-requisite for Yersinia virulence. Hence, YopE must target one or more additional GTPases to cause disease in mice.

YopE was the only Yersinia effector that blocks LDH release from infected cells. Moreover, translocated YopE could regulate the level of subsequent effector translocation by a mechanism that involved the YopE GAP function and another T3S component, YopK. Loss of translocation control elevated total T3S gene expression in the presence of eukaryotic cells. This indicated the existence of a regulatory loop for feedback control of T3S gene expression in the bacteria that originates from the interior of the eukaryotic cell after effector translocation is completed. This might represent the true virulence function of YopE.

Exoenzyme S (ExoS) of Pseudomonas aeruginosa has a YopE-like GAP domain with similar activity towards RhoA, Rac1 and Cdc42. However, ExoS is unable to complement hyper-translocation resulting from loss of YopE. This indicates a unique function for YopE in translocation control in Yersinia that might be dependent on correct intracellular localisation. It follows that the Membrane Localisation Domain in YopE was important for translocation control, but dispensable for cytotoxicity and blockage of LDH release.

YopD and its cognate chaperone LcrH are negative regulatory elements of the T3S regulon and together with YopB, are involved in the effector translocation process. Randomly generated point mutants in LcrH specifically effected stability and secretion of both the YopB and YopD substrates in vitro and prevented their apparent insertion as translocon pores in the membranes of infected cells. Yet, these mutants still produced stable substrates in the presence of eukaryotic cells and most could mediate at least partial effector translocation. Thus, only minimal amounts of the YopB and YopD translocator proteins are needed for translocation and the LcrH chaperone may regulate this process from inside the bacteria.

Ort, förlag, år, upplaga, sidor
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2005. s. 53
Nyckelord
Yersinia pseudotuberculosis, bacterial pathogenesis, YopE, LcrH, virulence, effector translocation, type III secretion, regulation
Nationell ämneskategori
Odontologi
Identifikatorer
urn:nbn:se:umu:diva-646 (URN)91-7305-977-3 (ISBN)
Disputation
2005-12-16, 13:00 (Engelska)
Handledare
Tillgänglig från: 2005-11-24 Skapad: 2005-11-24 Senast uppdaterad: 2019-01-24Bibliografiskt granskad
2. The multifunctional GAP protein YopE of Yersinia is involved in effector translocation control and virulence
Öppna denna publikation i ny flik eller fönster >>The multifunctional GAP protein YopE of Yersinia is involved in effector translocation control and virulence
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[sv]
Det multifunktionella GAP proteinet YopE från Yersinia är involverat i kontroll av effektortranslokering och virulens
Abstract [en]

The Gram-negative bacterium Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to establish infections. The T3SS translocates a diverse set of effector proteins directly into the host cells. The coordinate action of the translocated effectors blocks the innate immune system of the host and ensures extracellular proliferation of the bacterium. YopE is an essential effector that disrupts the actin cytoskeleton of infected host cells. This cytotoxicity is caused by the inactivation of RhoGTPases by the GTPase Activating Protein (GAP) activity of YopE. YopE was demonstrated to inactivate the RhoGTPases Rac1 and RhoA in vivo. However, Rac1 and RhoA inactivation was not a prerequisite for cytotoxicity or virulence. Thus, YopE must have additional targets during infection. Surprisingly, avirulent yopE mutants had lost the control of Yop expression in the presence of target cells and they all overtranslocated effectors. It appeared as if translocated YopE was able to control Yop expression and effector translocation via a feedback inhibition mechanism. This feedback inhibition was dependent on functional GAP activity. Translocation control could also be mediated by exogenous GAP activity, suggesting that effector translocation control might be a general property of all bacterial GAP proteins. Besides YopE, the regulatory protein YopK was also found to be involved in the effector translocation control process. Clearly, as demonstrated in virulence, the roles for YopE and YopK are intimately related.                       Further, YopE possesses a membrane localization domain (MLD) required for proper localization. A yopE∆MLD mutant had lost the feedback inhibition of YopE expression and was avirulent. Hence, the effector translocation control of YopE requires both proper localization as well as functional GAP activity.                                           In addition, fish keratocytes were established as a novel model system for Y. pseudotuberculosis infections. YopE was found to be the sole effector responsible for cytotoxicity towards the keratocytes. Further, induction of cytotoxicity required fully native YopE protein which indicated that the keratocytes would be useful as a sensitive model system for further studies of YopE mediated phenotypes.

In summary, this thesis work has sought to unravel the multiple functions of translocated YopE. A novel role was elucidated where Yersinia utilizes translocated YopE to control the process of effector translocation into host cells. This regulatory control was connected to virulence in the mouse model of disease. Thus, perhaps YopE should be considered also as a regulatory protein besides being a classical effector.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2010. s. 56
Nyckelord
Yersinia, T3SS, YopE, GAP activity, translocation control, virulence
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-37960 (URN)978-91-7459-100-2 (ISBN)
Disputation
2010-12-13, N320, Naturvetarhuset, Umeå universitet, Umeå, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2010-11-22 Skapad: 2010-11-19 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
3. Role of the Yersinia protein YopK in microbe-host interactions
Öppna denna publikation i ny flik eller fönster >>Role of the Yersinia protein YopK in microbe-host interactions
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

There are three human pathogenic species of the genus Yersiniae: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. To cause disease, these strains inhibit several key innate defense mechanisms, including phagocytosis, the critical process for bacterial clearance. The ability of Yersinia to evade the immune defense is dependent on delivery of virulence effectors, Yersinia outer proteins (Yops), into the interacting cell by a mechanism involving the type III secretion machinery. We have shown that the virulence protein YopK plays an important role in the control of Yop effector translocation via a feedback mechanism involving another virulence protein, YopE. We also found that YopK participated in regulation of Yop effector translocation by modulating level and ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, using a yeast two-hybrid screen with YopK as a bait, the eukaryotic protein RACK1 was identified as a target for this virulence protein. We found that RACK1 was engaged upon Y. pseudotuberculosis-mediated β1-integrin activation, where it was recruited to phagocytic cups. Downregulation of RACK1 by RNAi resulted in a reduced ability of Y. pseudotuberculosis to block phagocytosis, indicating that RACK1 is required for efficient Yersinia-mediated antiphagocytosis. Based on our data, we suggest a model where Yersinia, via YopK, targets RACK1 to ensure a directed delivery of the Yop effectors to the “right place” where they bind to and inactivate their targets, resulting in efficient inhibition of phagocytosis.  

A yopK mutant strain over-delivers Yop effectors, but is still avirulent in mice, indicating that YopK is important for the fine-tuning of effector protein delivery during infection. To analyse this, we investigated the importance of YopK during in vivo infection. We found that a yopK mutant colonized Peyer’s patches and the mesenteric lymph node more rapidly compared to wild-type Y. pseudotuberculosis, but was unable to spread systemically to liver and spleen and cause full disease in mice. Further, we showed that a yopK mutant was able to colonize liver and spleen and cause full disease in mice lacking the main phagocytes, polymorphonuclear leukocytes (PMNs). We also showed that YopK was important for Yersinia-mediated silencing of the PMN response.

To summarize, we suggest that YopK is important for Yersinia to evade the PMN defense and thereby spread systemically and cause disease. YopK is proposed to do this by allowing a controlled, directed Yop effector delivery that is just sufficient to inhibit host immune defense mechanisms. The controlled and precise delivery of virulence effectors avoids inappropriate triggering of PMNs and thereby an enhanced immune response favoring the host.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2012. s. 58
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1492
Nyckelord
Yersinia, YopK, T3SS, antiphagocytosis, neutrophil, translocation, virulence
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-53585 (URN)978-91-7459-408-9 (ISBN)
Disputation
2012-04-27, Major Groove, Building 6L, Umeå University, Umeå, 09:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Vetenskapsrådet, K2008-58X-11222-14-3
Tillgänglig från: 2012-04-04 Skapad: 2012-04-03 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet)Institutionen för molekylärbiologi (Medicinska fakulteten)Umeå Centre for Microbial Research (UCMR)Molekylär Infektionsmedicin, Sverige (MIMS)
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