Part:BBa_K2871000

Map20, T3SS export signal peptide from Map gene

Map20 is a signal sequence encoding a 20 amino acid long signal peptide derived from the map gene from the Enteropathogenic E. coli strain E22. It is used as an N-terminal tag for a protein to be translocated by the E. coli type-3-secretion system (T3SS). This T3SS signal tag is described by Charpentier & Oswald (2004) to be sufficient to target proteins to E. coli type III secretion pathway. A small 4xGS-linker is added for added avoidance of folding interference.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 85
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Map20-fusion protein expression plasmid construction

In order to be able to test the expression of N-terminal Map20-fusion protein, we constructed a plasmid consisting of a Map20-tagged reporter gene controlled by pBAD promoter, which can be activated by adding arabinose to the culture media. The Map20 T3SS signal is linked with the reporter protein by glycine-serine linker peptide to decrease folding interference. 6X-His tag has been added to the C-terminal to serve as an epitope tag for western blot. The plasmid map is shown in Figure 1.

Figure 1: Map20-mCherry expression plasmid.

To evaluate the function of CesF and CesT effector chaperone in the injectisome-dependent secretion of the fusion protein, the CesF-CesT chaperone cassette was inserted next to araC gene under a constitutive promoter pCAT as shown in Figure 2.

Figure 2: Map20-mCherry expression plasmid with CesF-CesT chaperone cassette.

Detection of signal peptide-dependent secretion without membrane

E. coli strains with different combinations of injectisome, signal, reporter, and chaperone was created for characterization. The reporter plasmids with and without chaperones were transformed into SIEC E. coli strain(2) which express injectisome under IPTG induction, and SIECΔP1 strain which does not. The E. coli strains with reporter plasmid were cultured in LB media overnight at 37 C. Reporter protein and injectisome expression was induced by adding 1% L-arabinose and 0.1mM IPTG respectively. The induced cultures were incubated at 16 C with 180 rpm shaking for 6 days. We collected 1 ml of sample from the induced culture every 24 hours and separated cells and supernatant by centrifugation. Cell pellets from each sample were resuspended in 500ul fresh LB media and measured for OD600 and mCherry fluorescent. After measurement, the resuspended cell pellets were lysed by sonication and separated into soluble and insoluble proteins by centrifugation. The supernatants, cellular soluble parts, and cellular insoluble parts from day 0, 3, and 6 were gel electrophoresed on 12% SDS-polyacrylamide gel (SDS-PAGE). The reporter protein was detected by western blot with anti-his tag antibody. All constructs tested contained 6xHIS-tagged mCherry or ꞵ-lactamase as a reporter protein.

Result

Map20-mCherry fusion protein expressing E. coli lack visible mCherry colour characteristic

Visual observation of E. coli cell pellet on day 6 shows a difference in color between E. coli that express mCherry and Map20-mCherry fusion protein. The mCherry-expressing E. coli pellet (tube no. 4 and 6) has pink color while Map20-mCherry fusion protein-expressing E. coli pellet (tube no. 1, 2, 3 and 5) has pale white-yellowish color as shown in Figure 3. This observation suggests that Map20 signal might decrease mCherry reporter protein expression or interferes with its folding.

Figure 3 Visual observation of different E. coli strains after 6 days of incubation in 16 ℃

Map20-mCherry fusion protein is expressed and present in both soluble and insoluble part of E. coli cell.

The SDS-PAGE(Figure 4) and Western blot(Figure 5) result shows that our mCherry reporter proteins with and without Map20 are present in both cellular soluble and insoluble part which confirm its expression in SIEC and SIECΔP1 E. coli cell. However, the expression level of mCherry with Map20 signal is significantly lower than normal mCherry. This data suggest that N-terminal Map20 signal might decrease translation rate or decrease stability of its fusion protein.

Figure 4: SDS-PAGE of cellular insoluble protein. M1: Stained ladder, M2: Unstained ladder Precision Plus Protein™ Unstained Protein Standards, (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

Figure 5: Western blot of cellular insoluble protein. mCherry bands are present in day 3 and day 6 of all groups that use mCherry as a reporter. Bands corresponding to mCherry protein are present in day 3 and day 6 of almost all groups that use mCherry as a reporter. Note that the beta-lactamase does not get a band. M1: Stained ladder, M2: Unstained ladder Precision Plus Protein™ Unstained Protein Standards, (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones. Map20+mCherry = 30-33 Kd.

Figure 6: SDS-PAGE of cellular soluble protein. In lanes 8/9 and 16/17, mCherry bands is readily visible as a prominent band, which indicate high expression level. The other lanes show no distinguishable bands for mCherry tagged with Map20. M1: Stained ladder, M2: Unstained ladder (UV visible)Precision Plus Protein™ Unstained Protein Standards, , D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones. Map20+mCherry = 30-33 Kd.

Figure 7.a: Western blot of cellular soluble protein. mCherry bands are present in day 3 and day 6 of all groups that use mCherry as a reporter. Note that the beta-lactamase does not get a band. M1: Stained ladder, M2: Unstained ladder (UV visible)Precision Plus Protein™ Unstained Protein Standards, , D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones. Map20+mCherry = 30-33 Kd.

Figure 7.b: Western blot of cellular soluble protein. mCherry bands are present in day 3 and day 6 of all groups that use mCherry as a reporter. Note: The shown sample was run on another gel than the rest of the samples from Figure 7.a Map20+mCherry = 30-33 Kd.

Figure 8: SDS-PAGE of supernatant with no distinguishable lanes other than identifiable markers M1 and M2.M1: Stained ladder, M2: Unstained ladder (UV visible)Precision Plus Protein™ Unstained Protein Standards, , D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones. The signal Map20 does not cause the Map20+mCherry to be secreted through the membrane and into the media - The signal does not cause the injectisome to "leak". Map20+mCherry = 30-33 Kd.

Figure 9: Western blot with anti-his tag antibody of supernatant.SDS-PAGE of cellular soluble protein. In lanes 8/9 and 16/17 an mCherry bands is visible as a prominent band, which indicate high expression level. The other lanes show no distinguishable bands for mCherry tagged with Map20. M1: Stained ladder, M2: Unstained ladder (UV visible)Precision Plus Protein™ Unstained Protein Standards, , D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones. The antibody binds to something unknown from possibly the media, which appears as a faint band. No other bands are visible. Map20+mCherry = 33 Kd.

Figure 10: Graph showing the OD600 of the bacterial cell collected on day 0, 3 and 6 after induction with 1% arabinose and 0.1 mM IPTG. A similar growth curve is seen for all strains.

Figure 11: Graph showing the fluorescence of the bacterial cell on day 0, 3 and 6 after induction with 1% arabinose and 0.1 mM IPTG. The two strains without signal peptide (I+ S- C+ mCherry and I- S- C- mCherry) shows remarkably stronger fluorescence compared to the other strains. For the other strains with mCherry as reporter protein ( I+ S+ C+ mCherry, I+ S+ C- mCherry, I- S+ C+ mCherry, I- S+ C- mCherry) a slight increasement in the fluorescence is seen. As expected, I+ S+ C+ beta-lactamase shows no fluorescence. This indicate that mCherry with Map20 signal sequence can, at least, partially fold and retain some fluorescent function.

Fluorescence confocal microscope

From the liposome experiment, where bacteria were incubated together with liposomes, we examined samples in a fluorescence confocal microscope. As seen in figure 12 and 13, we were able to visualize bacteria expressing mCherry and GFP indicating that the signal peptide not completely eliminate expression and folding of the reporter proteins inside the cell. However, no fluorescent is visible from the liposome, which suggests that secretion across liposomal membrane might not happen or the secreted protein cannot fold correctly and cannot express its fluorescent property.

Figure 12: Bacteria expressing Map20+mCherry visualized in a fluorescence confocal microscope. As seen in the lower left square, fluorescence is seen from the bacteria. The upper right square shows the bright field view with bacteria. Note, no fluorescence is seen in the lower left square (fluorescence from GFP). The results show that the Map20 signal+mCherry reporter protein is fluorescent and doesn't misfold enough to lose fluorescence because of the signal sequence.

Figure 13: Bacteria expressing Map20+GFP visualized in a fluorescence confocal microscope. As seen in the lower left square, fluorescence is seen from the bacteria. The upper right square shows the bright field view with bacteria. Note, no fluorescence is seen in the lower right square (fluorescence from mCherry). Again the results show that the Map20 signal+GFP reporter protein is fluorescent and doesn't misfold enough to lose fluorescence because of the signal sequence.

Conclusion

Map20-tagged mCherry is expressed in lower amount compared to mCherry without the tag. No Map20-tagged protein was detected in the culture supernatant of injectisome expressing SIEC strain, meaning the Map20 signal does not cause the tagged Map20+mCherry to be secreted into the media without membrane attachment. Figure 12 and 13 shows fluorescent reporter proteins tagged with the Map20 secretion signal, which indicates that the fusion-protein still retain its ability to folds correctly.
Map20 is a signal sequence which encodes for a signal peptide which is used as an N-terminal tag for a protein to be translocated by the Escherichia coli type-3-secretion system (T3SS). This 20 amino acid signal peptide derived from the map gene is sufficient to direct proteins to the type-3-secretion system (T3SS) pathway. Map20 is the signal sequence of Map protein, encoded in a chromosomal pathogenicity island called locus for enterocyte effacement (LEE). Map20 mediates secretion and translocation in a type III-dependent but chaperone-independent manner.[3]

References

1. Charpentier, X., Oswald, E. (2004) Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter. J Bacteriol, 186(16), pp 5486-5495.
2. Ruano-Gallego, D., Álvarez, B., & Fernández, L. Á. (2015). Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.5b00080
3.Charpentier X, Oswald E. Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter. J Bacteriol. 2004;186(16):5486-5495. doi:10.1128/JB.186.16.5486-5495.2004