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YopD translocator function in Yersinia pseudotuberculosis type III secretion
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type III secretion systems (T3SS) are a common feature of Gram-negative bacteria, allowing them to inject anti-host effectors into the interior of infected eukaryotic cells. By this mechanism, these virulence factors help the bacteria to modulate eukaryotic cell function in its favor and subvert host innate immunity. This promotes a less hostile environment in which infecting bacteria can colonize and cause disease.

In pathogenic Yersinia, a crucial protein in this process is YopD. YopD is a T3S substrate that, together with YopB, forms a translocon pore in the host cell membrane through which the Yop effectors may gain access to the target-cell cytosol. The assembly of the translocator pore in plasma membranes is considered a fundamental feature of all T3SSs. How the pore is formed, what determines the correct size and ultimately the stoichiometry between YopD YopB, is still unknown. Portions of YopD are also observed inside HeLa cells. Moreover, YopD functions together with its T3S chaperone, LcrH, to control Yops synthesis in the bacterial cytoplasm. The multifunctional YopD may influence all these processes by compartmentalizing activities into discrete modular domains along the protein length. Therefore, understanding how particular domains and/or residues within these regions coordinate multiple functions of the protein will provide a platform to improve our knowledge of the molecular mechanisms behind translocation through T3SSs.

Comprehensive site-directed mutagenesis of the YopD C-terminal amphipathic α-helix domain, pinpointed hydrophobic residues as important for YopD function. Some YopD variants were defective in self-assembly and in the ability to interact with the needle tip protein, LcrV, which were required to facilitate bacterial T3S activity. A similar mutagenesis approach was used to understand the role of the two predicted coiled-coils located at the N-terminal and C-terminal region of YopD. The predicted N-terminal element that occurs solely in the Yersinia YopD translocator family is essential for optimal T3SS and full disease progression. The predicted YopD C-terminal coiled-coil shapes a functional translocon inserted into host cell membranes. This translocon was seen to be a dynamic structure facilitating at least two roles during effectors delivery into cells; one to guarantee translocon pore insertion into target cell membranes and the other to promote targeted activity of internalized effector toxins.

In Yersinia expression of yop genes and secretion of the corresponding polypeptides is tightly regulated at a transcriptional and post-transcriptional level. If T3S chaperones of the translocator class are known to influence transcriptional output of T3SS genes in other bacteria, we show that in Yersinia the class II T3S chaperone LcrH has no such effect on the LcrF transcriptional activator activity. We also demonstrate that there are possibly additional yop-regulatory roles for the LcrH chaperone besides forming a stable complex with YopD to impose post-transcriptional silencing on Yops synthesis. This mechanism that relies upon an active T3SS, might act independently of both YopD and the regulatory element LcrQ.

In conclusion, this work has sought to delineate the encrypted functions of the YopD translocator that contribute to Yersinia T3SS-dependent pathogenesis. Contributions of the YopD cognate chaperone LcrH in yop regulatory control are also presented.  

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2012. , 222 p.
Keyword [en]
Y. pseudotuberculosis, T3SS, translocon, YopD, coiled-coil, effector delivery, regulation, virulence
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-61544ISBN: 978-91-7459-483-6 (print)OAI: oai:DiVA.org:umu-61544DiVA: diva2:570477
Public defence
2012-12-14, Major Groove, Biomedicinhuset, Byggnad 6L, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-23 Created: 2012-11-19 Last updated: 2012-11-23Bibliographically approved
List of papers
1. YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis
Open this publication in new window or tab >>YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis
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2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, 25269-25284 p.Article in journal (Refereed) Published
Abstract [en]

YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2010
Keyword
Bacteria, Bacterial Genetics, Protein Cross-linking, Protein-Protein Interactions, Secretion, Yersinia, Amphipathic, Coiled-coil, Pore Formation, Regulation
National Category
Microbiology in the medical area
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-41848 (URN)10.1074/jbc.M110.144311 (DOI)000280682400020 ()20525687 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-01 Last updated: 2012-11-20Bibliographically approved
2. Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis
Open this publication in new window or tab >>Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis
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2012 (English)In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, no 8, 1729-1742 p.Article in journal (Refereed) Published
Abstract [en]

Pathogenic Yersinia all harbor a virulence plasmid-encoded Ysc–Yop T3SS. In this system, translocator function is performed by the hydrophobic proteins YopB and YopD. With the goal to better understand how YopD orchestrates yop-regulatory control, translocon pore formation and Yop effector translocation, we performed an in silico prediction of coiled-coil motifs in YopD and YopD-like sequences from other bacteria. Of interest was a predicted N-terminal coiled-coil that occurred solely in Yersinia YopD sequences. To investigate if this unique feature was biologically relevant, two in cis point mutations were generated with a view to disrupting this putative structure. Both mutants maintained full T3SS function in vitro in terms of environmental control of Yops synthesis and secretion, effector toxin translocation and evasion of phagocytosis and killing by cultured immune cells. However, these same mutants were attenuated for virulence in a murine oral-infection model. The cause of this tardy disease progression is unclear. However, these data indicate that any structural flaw in this element unique to the N-terminus will subtly compromise an aspect of YopD biology. Sub-optimal T3SSs are then formed that are unable to fortify Yersinia against attack by the host innate and adaptive immune response.

Place, publisher, year, edition, pages
Elsevier, 2012
Keyword
Pathogen-Host Interaction, Virulence Factor, Viruelnce, Bacteria
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-59466 (URN)10.1016/j.meegid.2012.07.016 (DOI)
Funder
Swedish Research Council, 2009-5628
Available from: 2012-09-19 Created: 2012-09-14 Last updated: 2017-12-07Bibliographically approved
3. Active type III translocon assemblies that attenuate Yersinia virulence
Open this publication in new window or tab >>Active type III translocon assemblies that attenuate Yersinia virulence
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(English)Manuscript (preprint) (Other academic)
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 a-helix motif at the C-terminus. Mutants YopDI262P and YopDK267P poorly localised 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 YopDA263P and YopDA270P 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. It is therefore probable that an active translocon makes a range of contributions during bacteria-host cell contact that extends beyond effector delivery per se.

National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-61539 (URN)
Available from: 2012-11-19 Created: 2012-11-19 Last updated: 2016-01-25
4. Influence of the LcrH chaperone on type III secretion system regulation in Yersinia pseudotuberculosis
Open this publication in new window or tab >>Influence of the LcrH chaperone on type III secretion system regulation in Yersinia pseudotuberculosis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Human pathogenic Yersiniae share a common virulence plasmid that encodes for the Ysc-Yop type III secretion system (T3SS). Control of yop expression involves several pathways in which their cross-talk is not completely understood. LcrF, an AraC-like transcriptional activator, is required for temperature-dependent yop-transcription. In contrast, a repressive effect of the T3S chaperone LcrH and the cognate translocator substrate YopD occurs through binding to yop mRNA and inhibiting translation; a process that is also thought to involve LcrQ. Several homologous members of the LcrH family of translocator-class of T3S chaperones can act as a cofactor to amplify the activity of transcriptional activators analogous to LcrF. However, we show here in Y. pseudotuberculosis that LcrH does not induce LcrF-dependent transcription of target genes. Moreover, a full length DlcrH null mutant in which YopB and YopD are rapidly degraded is totally de-repressed for Yop synthesis even though the anti-activator LcrQ is forced to accumulate in the cytoplasm through rendering the Ysc-Yop T3SS non-functional or ectopically producing LcrQ in trans. Typically, this mutant cannot grow at 37°C. Thus, in all respects, the DlcrH null mutant mirrors the regulatory defects established for Yersinia lacking the translocator and anti-activator YopD. On the other hand, Y. pseudotuberculosis producing the LcrHE30G point mutant that is defective for YscY chaperone binding exhibits a mild regulatory defect that permits some growth at 37°C, but is blind to the cytoplasmic accumulation of LcrQ. Critically however, this mutant still responds to repression caused by YopD accumulation, which is stably produced and efficiently secreted by this strain. Thus, our work with LcrHE30G indicates an additional regulatory function of this versatile T3S chaperone that is independent of the LcrF transcription factor and the YopD anti-activator.

 

National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-61540 (URN)
Available from: 2012-11-19 Created: 2012-11-19 Last updated: 2016-01-25

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Costa, Tiago R. D.

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