Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage

Wilharm, Gottfried; Wibke, Neumayer; Heesemann, Jürgen

doi:10.1016/s1046-5928(03)00183-9

Cited by 12 publications

(12 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, P. aeruginosa has evolved multiple signaling pathways to fine-tune the regulation of the type III secretion system in response to the environmental changes. Similarly, Yersinia has been reported to have several regulators, such as an activator, VirF, and repressor molecules, LcrQ, YscM1, YscM2, and YmoA, that are involved in the control of yop gene transcription (7,48,51). Current efforts are focused on the elucidation of the molecular mechanism by which PtrB mediates suppression of the TTSS.…”

Section: Discussionmentioning

confidence: 99%

PtrB of Pseudomonas aeruginosa Suppresses the Type III Secretion System under the Stress of DNA Damage

Jin

2005

J Bacteriol

View full text Add to dashboard Cite

In a search for regulatory genes of the type III secretion system (TTSS) in Pseudomonas aeruginosa, transposon (Tn5) insertional mutants of the prtR gene were found defective in the TTSS. PrtR is an inhibitor of prtN, which encodes a transcriptional activator for pyocin synthesis genes. In P. aeruginosa, pyocin synthesis is activated when PrtR is degraded during the SOS response. Treatment of a wild-type P. aeruginosa strain with mitomycin C, a DNA-damaging agent, resulted in the inhibition of TTSS activation. A prtR/prtN double mutant had the same TTSS defect as the prtR mutant, and complementation by a prtR gene but not by a prtN gene restored the TTSS function. Also, overexpression of the prtN gene in wild-type PAK had no effect on the TTSS; thus, PrtN is not involved in the repression of the TTSS. To identify the PrtR-regulated TTSS repressor, another round of Tn mutagenesis was carried out in the background of a prtR/prtN double mutant. Insertion in a small gene, designated ptrB, restored the normal TTSS activity. Expression of ptrB is specifically repressed by PrtR, and mitomycin C-mediated suppression of the TTSS is also abolished in a ptrB mutant strain. Therefore, PtrB is a new TTSS repressor that coordinates TTSS repression and pyocin synthesis under the stress of DNA damage.

show abstract

Section: Discussionmentioning

confidence: 99%

PtrB of Pseudomonas aeruginosa Suppresses the Type III Secretion System under the Stress of DNA Damage

Jin

2005

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…In this model, stable complexes of YscM1-SycH are more numerous than YscM1-SycE complexes in turn favoring transfer rates of YopH Ͼ YopE. An exchange of SycH with YscM1, which exists as a homodimer (47), drives YopH release into the mammalian host cell, and depletion of SycH-YopH complexes initiates the transfer of YopE by a similar mechanism. Further our data indicate that SycE has a propensity to interact with YscM1 and the Y. enterocolitica protein YscM2 but not YopH.…”

Section: Discussionmentioning

confidence: 99%

Novel Protein-Protein Interactions of the Yersinia pestis Type III Secretion System Elucidated with a Matrix Analysis by Surface Plasmon Resonance and Mass Spectrometry

Świętnicki

O’Brien

Holman

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Binary complexes formed by components of theYopB and YopD, in conjunction with LcrV, are thought to form a pore in the membrane of mammalian cells through which the Yops (3, 4) are delivered. The cellular targets of some Yops are known. For example, YopH is a potent tyrosine phosphatase that dephosphorylates macrophage p130Cas and focal adhesion kinase proteins and disrupts focal adhesions (5-7), YopE is a GTPase-activating protein that causes actin cytoskeleton depolymerization (8), and YpkA is a Ser/Thr kinase that interferes with Rho-mediated cellular signaling (9). Recent data suggest that YopM targets the cellular kinases protein kinase C-like 2 and ribosomal S6 protein kinase 1 (10). Transport of YopB, YopD, YopE, YopH, and YopT through the injectosome (11-13) is facilitated by formation of a complex between the secreted protein and a specific Yop chaperone (Syc) also encoded by pCD1. The three other effectors, YopJ, YopM, and YopO (YpkA), do not appear to require cognate secretion chaperones for transport (13). Comparatively little is known about the transient interactions involved in the assembly of the injectosome, the precise order of assembly and delivery of the bacterial proteins into mammalian cells, and the energy source for the transport. To add to the complexity of the problem, protein-RNA and protein-DNA interactions are postulated for some components of the TTSS (14, 15). Deleting any single component of the TTSS attenuates bacterial virulence, suggesting that the Yops, Sycs, and other accessory proteins assemble into a large multisubunit complex. As an essential component of the coordinately regulated low Ca 2ϩ response stimulon (LCR) of pCD1, LcrF stimulates maximum expression of LcrV and Yops at 37°C, and this activation step requires host cell contact or Ca 2ϩ depletion (16,17).As an approach to study the assembly of the Y. pestis virulence machinery, we used a two-step screening process to identify direct interactions between pairs of TTSS proteins. Protein interactions were first detected by surface plasmon resonance (SPR) and then classified according to the strength of interaction at equilibrium. Positive interactions indicated by SPR were next confirmed by MALDI-TOF mass spectrometry. Our results suggest that the combination of SPR and MALDI-TOF mass spectrometry is a powerful method for rapidly identifying protein-protein interactions involved in complex macromolecular assemblies such as the type III secretion system. MATERIALS AND METHODS Protein Expression and Purification-The open reading frames encoding Y. pestis YopK, LcrG, LcrH, LcrQ, and YmoA were amplified from genomic DNA from Y. pestis biovar Orientalis, strain 195/P, kindly

show abstract

“…For purification of recombinant Yersinia PEPC, a GST fusion construct based on pGEX-4T3 (Amersham Biosciences) was used. Plasmids for expression of GST-YscM1 and GST-YscM2 fusions have been described (38). The ppc mutant was generated as described (15) by replacement of the ppc gene with a kanamycin resistance cassette mediated by homologous recombination of a transformed PCR product.…”

Section: Methodsmentioning

confidence: 99%

“…GST fusions based on pGEX-4T3, which harbor a thrombin cleavage site, were used for initial PEPC binding studies (affinity purification and native gel electrophoresis). For production of recombinant YscM1/ YscM2 without GST, pGEX-4T3-based constructs cannot be used, since YscM1/YscM2 harbor an intrinsic thrombin cleavage site (38). Therefore, recombinant YscM1 and YscM2 were produced as described using pGEX-6P3 as vector (38).…”

Section: Methodsmentioning

confidence: 99%

Cross-talk between Type Three Secretion System and Metabolism in Yersinia

Schmid

Wibke

Trülzsch

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage

Cited by 12 publications

References 22 publications

PtrB of Pseudomonas aeruginosa Suppresses the Type III Secretion System under the Stress of DNA Damage

PtrB of Pseudomonas aeruginosa Suppresses the Type III Secretion System under the Stress of DNA Damage

Novel Protein-Protein Interactions of the Yersinia pestis Type III Secretion System Elucidated with a Matrix Analysis by Surface Plasmon Resonance and Mass Spectrometry

Cross-talk between Type Three Secretion System and Metabolism in Yersinia

Contact Info

Product

Resources

About