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Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis
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). (Matthew Francis)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). (Åke Forsberg)
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2013 (Engelska)Ingår i: PLOS ONE, 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. Vol. 8, nr 10, artikel-id e77767
Nyckelord [en]
secretion control, hierarchy, translocation, InvE family, ribosome slippage, virulence
Nationell ämneskategori
Mikrobiologi Biokemi och molekylärbiologi Mikrobiologi inom det medicinska området
Forskningsämne
mikrobiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-81379DOI: 10.1371/journal.pone.0077767ISI: 000325483600088PubMedID: 24098594Scopus ID: 2-s2.0-84884871476OAI: oai:DiVA.org:umu-81379DiVA, id: diva2:654855
Forskningsfinansiär
VetenskapsrådetTillgänglig från: 2013-10-08 Skapad: 2013-10-08 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Ingår i avhandling
1. Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
Öppna denna publikation i ny flik eller fönster >>Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Several pathogenic Gram-negative bacteria invest in sophisticated type III secretion systems (T3SS) to incapacitate their eukaryotic hosts. T3SSs can secrete protein cargo outside the bacterial cell and also target many of them into the eukaryotic cell interior. Internalized proteins promote bacterial colonization, survival and transmission, and can often cause severe disease. An example is the Ysc-Yop T3SS apparatus assembled by pathogenic Yersinia spp. A correctly assembled Ysc-Yop T3SS spans the Yersinia envelope and also protrudes from the bacterial surface. Upon host cell contact, this system is competent to secrete hydrophobic translocators that form a translocon pore in the host cell membrane to complete the delivery channel bridging both bacterial and host cells. Newly synthesized effector Yops may pass through this channel to gain entry into the host cell cytosol.As type III secretion (T3S) substrates function sequentially during infection, it is hypothesized that substrate export is temporally controlled to ensure that those required first are prioritized for secretion. On this basis three functional groups are classified as early (i.e. structural components), middle (i.e. translocators) and late (i.e. effectors). Factors considered to orchestrate the T3S of substrates are many, including the intrinsic substrate secretion signal sequences, customized chaperones, and recognition/sorting platforms at the base of the assembled T3SS. Investigating the interplay between these elements is critical for a better understanding of the molecular mechanisms governing export control during Yersinia T3S.To examine the composition of the N-terminal T3S signals of the YscX early substrate and the YopD middle substrate, these segments were altered by mutagenesis and the modified substrates analyzed for their T3S. Translational fusions between these signals and a signalless β-Lactamase were used to determine their optimal length required for efficient T3S. This revealed that YscX and YopD export is most efficiently supported by their first 15 N-terminal residues. At least for YopD, this is a peptide signal and not base upon information in the mRNA sequence. Moreover, features within and upstream of this segment contribute to their translational control. In parallel, bacteria were engineered to produce substrate chimeras where the N-terminal segments were exchanged between substrates of different classes in an effort to examine the temporal dynamics of T3S. In several cases, Yersinia producing chimeric substrates were defective in T3S activity, which could be a consequence of disturbing a pre-existing hierarchal secretion mechanism.YopN and TyeA regulatory molecules can be naturally produced as a 42 kDa YopN-TyeA hybrid, via a +1 frame shift event somewhere at the 5’-end of yopN. To study this event, Yersinia were engineered to artificially produce this hybrid, and these maintained in vitro T3S control of both middle and late substrates. However, modestly diminished directed targeting of effectors into eukaryotic cells correlated to virulence attenuation in vivo. Upon further investigation, a YopN C-terminal segment encompassing residues 278 to 287 was probably responsible, as this region is critical for YopN to control T3S, via enabling a specific interaction with TyeA.Investigated herein were molecular mechanisms to orchestrate substrate export by the T3SS of Yersinia. While N-terminal secretion signals may contribute to specific substrate order, the YopN and TyeA regulatory molecules do not appear to distinguish between the different substrate classes.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2013. s. 77
Serie
Doctoral thesis / Umeå University, Department of Molecular Biology
Nyckelord
Y. pseudotuberculosis, T3SS, YscX, YopD, assembly, translation control, temporal secretion.
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-70113 (URN)978-91-7459-566-6 (ISBN)
Disputation
2013-05-29, Norrlands universitetssjukhus, Biomedicinhuset, Byggnad 6L, Major Groove, Umeå Universitet, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2013-05-08 Skapad: 2013-05-05 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
2. Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
Öppna denna publikation i ny flik eller fönster >>Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
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
Yersinia pseudotuberculosis, type III secretion system, translocation, translocon, regulation, bacteria-eukaryotic cell contact, cytokine profiling
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-165155 (URN)978-91-7855-157-6 (ISBN)
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

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