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Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion
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)
Department of Medical Countermeasures, Swedish Defense Research Agency, Division of NBC12 Defense, Umeå, Sweden.
Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Åke Forsberg)
Vise andre og tillknytning
2011 (engelsk)Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, s. 6683-6700Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

sted, utgiver, år, opplag, sider
American Society for Microbiology , 2011. Vol. 193, nr 23, s. 6683-6700
Emneord [en]
mRNA, amphipathic, effector, hierarchy, translation, chaperone
HSV kategori
Forskningsprogram
infektionssjukdomar; mikrobiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-49681DOI: 10.1128/JB.00210-11OAI: oai:DiVA.org:umu-49681DiVA, id: diva2:456511
Forskningsfinansiär
Swedish Research Council, 2009-5628Tilgjengelig fra: 2011-11-16 Laget: 2011-11-15 Sist oppdatert: 2018-06-08bibliografisk kontrollert
Inngår i avhandling
1. Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
Åpne denne publikasjonen i ny fane eller vindu >>Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
2013 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Umeå: Umeå universitet, 2013. s. 77
Serie
Doctoral thesis / Umeå University, Department of Molecular Biology
Emneord
Y. pseudotuberculosis, T3SS, YscX, YopD, assembly, translation control, temporal secretion.
HSV kategori
Forskningsprogram
mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-70113 (URN)978-91-7459-566-6 (ISBN)
Disputas
2013-05-29, Norrlands universitetssjukhus, Biomedicinhuset, Byggnad 6L, Major Groove, Umeå Universitet, Umeå, 09:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2013-05-08 Laget: 2013-05-05 Sist oppdatert: 2018-06-08bibliografisk kontrollert

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