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Type III secretion translocon assemblies that attenuate Yersinia virulence
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
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). (Hans Wolf-Watz)
Vise andre og tillknytning
2013 (engelsk)Inngår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, nr 7, s. 1088-1110Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD(A263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

sted, utgiver, år, opplag, sider
Wiley-Blackwell, 2013. Vol. 15, nr 7, s. 1088-1110
Emneord [en]
virulence, coiled-coil, effector delivery, pore formation, regulation, YopD
HSV kategori
Forskningsprogram
infektionssjukdomar; mikrobiologi; molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-67117DOI: 10.1111/cmi.12100ISI: 000320394800004OAI: oai:DiVA.org:umu-67117DiVA, id: diva2:610817
Forskningsfinansiär
Swedish Research Council
Merknad

This work, performed within the framework of the Umea Centre for Microbial Research-Linnaeus Program, was supported by grants from the Carl Tryggers Foundation for Scientific Research (M. S. F.), Swedish Research Council (H.W.-W., M. F., T. E., M. S. F.), Foundation for Medical Research at Umea University (M. S. F.) and J C Kempe Memorial Fund (T. R. C., A. A. A.).

Tilgjengelig fra: 2013-03-13 Laget: 2013-03-13 Sist oppdatert: 2019-11-12bibliografisk kontrollert
Inngår i avhandling
1. Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
Åpne denne publikasjonen i ny fane eller vindu >>Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Many Gram negative bacteria use type III secretion systems to cross-talk with eukaryotic cells. Type III secretion system assembly and function is tightly regulated. It initiates with assembly of a basal body-like structure, and is followed by a cytoplasmic-located substrate sorting and export platform that first engages with early substrates required for needle assembly. At the needle tip, a translocon is formed upon eukaryotic cell contact to allow the translocation of effector proteins to the host cell. The focus of this thesis is on understanding aspects of biogenesis, regulation and function of the translocon and its interaction with the host cell. Research questions are addressed in enteropathogenic Yersinia pseudotuberculosis model.

Prioritising the secretion of translocon components before effector proteins is a task given partly to the InvE/MxiC/HrpJ family of proteins. In Yersinia, homology to this protein family is partitioned over two proteins; YopN and TyeA. Certain Yersinia strains naturally produce a single YopN/TyeA polypeptide hybrid. To understand the implications of hybrid formation towards type III secretion control, a series of mutants were engineered to produce only a single hybrid peptide. Using in vitro assays revealed no difference in substrate secretion profiles between parent and mutants. Moreover, no obvious prioritisation of secretion between translocator and effector substrates was observed. Although these in vitro studies indicate that the YopN-TyeA single polypeptide is fully functionally competent, these mutants were attenuated in the mouse infection model. Hence, natural production of YopN and TyeA as a single polypeptide alone is unlikely to confer a fitness advantage to the infecting bacteria and is unlikely to orchestrate hierarchal substrate secretion.

The YopB and YopD translocon components form a pore in the host cell plasma membrane to deliver the effectors into the host cell. To better understand how YopD contributes to the biogenesis, function and regulation of the translocon pore, a series of mutants were constructed to disrupt two predicted α-helix motifs, one lying at the N-terminus and the other at the C-terminus. Based upon phenotypes associated with environmental control of Yop synthesis and secretion, effector translocation, evasion of phagocytosis, killing of immune cells and virulence in a mouse infection model, the mutants were grouped into three phenotypic classes. A particularly interesting mutant class maintained full T3SS function in vitro, but were attenuated for virulence in a murine oral-infection model. To better understand the molecular basis for these phenotypic differences, the effectiveness of RAW 264.7 cells to respond to infection by these mutants was scrutinised. Sixteen individual cytokines were profiled with mouse cytokine screen multiplex analysis. Signature cytokine profiles were observed that could again separate the different YopD mutants into distinct categories. The activation and supression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to modulate programmed cell death and antiphagocytosis pathways. Hence, the biogenesis of sub-optimal translocon pores alters host cell responsiveness and limits the ability of Yersinia to fortify against attack by both early and late arms of the host innate immune response.

The amount of bacteria now resistant to multiple antibiotics is alarming. By providing insights into a common virulence process, this work may ultimately facilitate the design of novel broad-acting inhibitors of type III secretion, and thereby be useful to treat an array of bacterial infections.

sted, utgiver, år, opplag, sider
Umeå: Umeå University, 2019. s. 100
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 2062
Emneord
Yersinia pseudotuberculosis, type III secretion system, translocation, translocon, regulation, bacteria-eukaryotic cell contact, cytokine profiling
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-165155 (URN)978-91-7855-157-6 (ISBN)
Disputas
2019-12-06, 933, Unod B 9, Norrlands universitetssjukhus, Umeå, 09:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-11-15 Laget: 2019-11-12 Sist oppdatert: 2019-11-14bibliografisk kontrollert

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