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YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
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, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
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2010 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 33, s. 25269-25284Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
American Society for Biochemistry and Molecular Biology , 2010. Vol. 285, nr 33, s. 25269-25284
Nyckelord [en]
Bacteria, Bacterial Genetics, Protein Cross-linking, Protein-Protein Interactions, Secretion, Yersinia, Amphipathic, Coiled-coil, Pore Formation, Regulation
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Forskningsämne
infektionssjukdomar
Identifikatorer
URN: urn:nbn:se:umu:diva-41848DOI: 10.1074/jbc.M110.144311ISI: 000280682400020PubMedID: 20525687OAI: oai:DiVA.org:umu-41848DiVA, id: diva2:407950
Tillgänglig från: 2011-04-11 Skapad: 2011-04-01 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Ingår i avhandling
1. YopD translocator function in Yersinia pseudotuberculosis type III secretion
Öppna denna publikation i ny flik eller fönster >>YopD translocator function in Yersinia pseudotuberculosis type III secretion
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.  

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2012. s. 222
Nyckelord
Y. pseudotuberculosis, T3SS, translocon, YopD, coiled-coil, effector delivery, regulation, virulence
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-61544 (URN)978-91-7459-483-6 (ISBN)
Disputation
2012-12-14, Major Groove, Biomedicinhuset, Byggnad 6L, Umeå University, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2012-11-23 Skapad: 2012-11-19 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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Costa, Tiago R DBröms, Jeanette EÅhlund, Monika KForsberg, ÅkeFrancis, Matthew S

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Costa, Tiago R DBröms, Jeanette EÅhlund, Monika KForsberg, ÅkeFrancis, Matthew S
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Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet)Umeå Centre for Microbial Research (UCMR)Klinisk bakteriologiMolekylär Infektionsmedicin, Sverige (MIMS)
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