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Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Forsberg)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Francis)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Francis)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Forsberg)
Vise andre og tillknytning
2005 (engelsk)Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, nr 22, s. 7738-7752Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.

sted, utgiver, år, opplag, sider
American Society for Microbiology , 2005. Vol. 187, nr 22, s. 7738-7752
Emneord [en]
Amino acid sequence, bacterial proteins, binding sites, DNA; bacterial, HeLa cells, humans, molecular chaperones, molecular sequence data, protein interaction mapping, protein structure; tertiary, protein transport, sequence analysis; DNA, Yersinia pseudotuberculosis
HSV kategori
Identifikatorer
URN: urn:nbn:se:umu:diva-16672DOI: 10.1128/JB.187.22.7738-7752.2005ISI: 000233400200021PubMedID: 16267298OAI: oai:DiVA.org:umu-16672DiVA, id: diva2:156345
Tilgjengelig fra: 2007-10-08 Laget: 2007-10-08 Sist oppdatert: 2018-06-09bibliografisk kontrollert
Inngår i avhandling
1. Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis
Åpne denne publikasjonen i ny fane eller vindu >>Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis
2007 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.

The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.

Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.

Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

sted, utgiver, år, opplag, sider
Umeå: Molekylärbiologi (Teknat- och Medfak), 2007. s. 88
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1074
Emneord
Yersinia pseudotuberculosis, T3SS, YopD, translocation process, LcrH, class II chaperone, substrate secretion pathway specificity
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-985 (URN)91-7264-231-9 (ISBN)
Disputas
2007-02-16, Major Groove, 6 L NUS, Umeå Universitet, Umeå, 09:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2007-01-31 Laget: 2007-01-31 Sist oppdatert: 2019-01-23bibliografisk kontrollert

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