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Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå University. (Matthew Francis)ORCID-id: 0000-0002-7286-1104
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University , 2019. , s. 100
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 2062
Nyckelord [en]
Yersinia pseudotuberculosis, type III secretion system, translocation, translocon, regulation, bacteria-eukaryotic cell contact, cytokine profiling
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-165155ISBN: 978-91-7855-157-6 (tryckt)OAI: oai:DiVA.org:umu-165155DiVA, id: diva2:1369537
Disputation
2019-12-06, 933, Unod B 9, Norrlands universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-11-15 Skapad: 2019-11-12 Senast uppdaterad: 2019-11-14Bibliografiskt granskad
Delarbeten
1. Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis
Öppna denna publikation i ny flik eller fönster >>Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis
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2013 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 10, artikel-id e77767Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

Ort, förlag, år, upplaga, sidor
San Francisco: Public Library of Science, 2013
Nyckelord
secretion control, hierarchy, translocation, InvE family, ribosome slippage, virulence
Nationell ämneskategori
Mikrobiologi Biokemi och molekylärbiologi Mikrobiologi inom det medicinska området
Forskningsämne
mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-81379 (URN)10.1371/journal.pone.0077767 (DOI)000325483600088 ()24098594 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet
Tillgänglig från: 2013-10-08 Skapad: 2013-10-08 Senast uppdaterad: 2019-11-12Bibliografiskt granskad
2. Type III secretion translocon assemblies that attenuate Yersinia virulence
Öppna denna publikation i ny flik eller fönster >>Type III secretion translocon assemblies that attenuate Yersinia virulence
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2013 (Engelska)Ingår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, nr 7, s. 1088-1110Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Wiley-Blackwell, 2013
Nyckelord
virulence, coiled-coil, effector delivery, pore formation, regulation, YopD
Nationell ämneskategori
Mikrobiologi Biokemi och molekylärbiologi Mikrobiologi inom det medicinska området
Forskningsämne
infektionssjukdomar; mikrobiologi; molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-67117 (URN)10.1111/cmi.12100 (DOI)000320394800004 ()
Forskningsfinansiär
Vetenskapsrådet
Anmärkning

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.).

Tillgänglig från: 2013-03-13 Skapad: 2013-03-13 Senast uppdaterad: 2019-11-12Bibliografiskt granskad
3. Impact of Defective Translocon Assemblies on Hierarchal Yop Effector Translocation by Yersinia pseudotuberculosis
Öppna denna publikation i ny flik eller fönster >>Impact of Defective Translocon Assemblies on Hierarchal Yop Effector Translocation by Yersinia pseudotuberculosis
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Identifikatorer
urn:nbn:se:umu:diva-165173 (URN)
Tillgänglig från: 2019-11-13 Skapad: 2019-11-13 Senast uppdaterad: 2019-11-14

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