Related Experiment Video
Updated: Aug 30, 2026
Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions
Published on: October 13, 2015
Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage
Gottfried Wilharm1, Wibke Neumayer, Jürgen Heesemann
1Department of Bacteriology, Max von Pettenkofer-Institut, Pettenkoferstr 9a, Munich D-80336, Germany. maier@m3401.mpk.med.uni-muenchen.de
Abstract:
Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) make use of a virulence plasmid-encoded type three secretion system (TTSS) to inject effector proteins into host cells. Y. enterocolitica YscM1 (LcrQ in Y. pestis and Y. pseudotuberculosis) and its homologue YscM2 are regulatory components of the TTSS that are also secreted by this transport apparatus. YscM1 and YscM2 share 57% identity and are believed to be functionally equivalent. We have recombinantly expressed and purified YscM1 and YscM2 in Escherichia coli. After expression as glutathione S-transferase (GST) fusions purification to near homogeneity was achieved by glutathione-Sepharose affinity chromatography followed by PreScission protease treatment to cleave off GST and gel filtration on a Superdex 75 column. Such recombinant YscM1 and YscM2 bound efficiently to the specific chaperone SycH, indicating proper folding of the purified proteins. Gel filtration analyses revealed that both YscM1 and YscM2 formed homodimers. The YscM1 and YscM2 homodimers could be dissociated at high ionic strength, indicating that salt bridges essentially contribute to the dimerization. We further demonstrated that YscM1 and YscM2 are susceptible to thrombin cleavage.
Insights
Pathogenic Yersinia bacteria use a type three secretion system (TTSS) to inject toxins. Researchers purified YscM1 and YscM2 proteins, finding they form dimers dependent on salt bridges.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- Pathogenic Yersinia species utilize a type three secretion system (TTSS) encoded on a virulence plasmid to deliver effector proteins into host cells.
- YscM1 (LcrQ) and YscM2 are TTSS regulatory proteins secreted via this apparatus, sharing 57% identity and presumed functional equivalence.
Purpose of the Study:
- To recombinantly express and purify YscM1 and YscM2 from Yersinia species.
- To characterize the biochemical properties, including folding, dimerization, and stability, of purified YscM1 and YscM2.
Main Methods:
- Recombinant expression of YscM1 and YscM2 as glutathione S-transferase (GST) fusions in Escherichia coli.
- Purification using glutathione-Sepharose affinity chromatography and PreScission protease cleavage.
- Gel filtration chromatography (Superdex 75) for size exclusion and dimerization analysis.
- Assays to assess binding to chaperone SycH and susceptibility to thrombin cleavage.
Main Results:
- Purified recombinant YscM1 and YscM2 proteins were obtained with high homogeneity.
- Both proteins demonstrated proper folding through efficient binding to the chaperone SycH.
- Gel filtration revealed that YscM1 and YscM2 exist as homodimers.
- Dimerization was shown to be dependent on salt bridges, as it could be disrupted by high ionic strength.
- YscM1 and YscM2 were found to be susceptible to thrombin cleavage.
Conclusions:
- YscM1 and YscM2 are stable, properly folded proteins capable of forming salt bridge-dependent homodimers.
- These findings provide biochemical insights into the regulation and function of TTSS components in pathogenic Yersinia.
- The characterization of YscM1 and YscM2 contributes to understanding the molecular mechanisms of Yersinia virulence.
