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. Its short length seemed to be attractive to us because we wanted to minimize folding interference between the signal sequence and the intended protein fused to the signal sequence.

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.

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.

Detection of signal peptide-dependent secretion without membrane

To create an E. coli strain with a different combination of injectisome, signal, reporter, and chaperone. The reporter plasmids with and without chaperone cassette were transformed into SIEC E. coli strain which express injectisome, and SIECΔP1 strain which doesn’t have injectisome. 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 separate cell and supernatant by centrifugation. Cellular fraction were lysed by sonication and separated into soluble and insoluble parts by centrifugation. The supernatants, cellular soluble parts, and cellular insoluble parts from day 0, 3, and 6 were gel electrophoresis on 12% SDS-polyacrylamide gel (SDS-PAGE). The reporter protein was detected western blot with anti-his tag antibody. The constructs tested all contained the reporter protein, being mCherry or ꞵ-lactamase.

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 1.1 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 1.2) and Western blot(Figure 1.3) 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 1.2: SDS-PAGE of cellular insoluble protein. M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

Figure 1.3: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 (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

Figure 1.4: SDS-PAGE of cellular soluble protein. In lanes 8/9 and 16/17 an 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), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

Figure 1.5.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), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

Figure 1.5.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

Figure 1.6: SDS-PAGE of supernatant with no distinguishable lanes other than identifiable markers M1 and M2.M1: Stained ladder, M2: Unstained ladder (UV visible), 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".

Figure 1.7: 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), 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.

Fluorescence confocal microscope

From the liposome experiment, where bacteria were incubated together with liposomes, we examined samples in a fluorescence confocal microscope. As seen on figure 9 and 10, we were able to visualize bacteria expressing mCherry and GFP indicating that the signal peptide not completely eliminate expressing and folding of the reporter proteins.

Figure 2.1: 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 2.2: 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 tag. No protein secretion is detected in the supernate, meaning that the Map20 signal does not cause the tagged Map20+mCherry to be secreted into the media without membrane attachment. Figure 2.1 and 2.2 shows fluorescent reporter proteins tagged with the Map20 secretion signal, which indicates that the fusionprotein folds correctly.

2. Leakiness

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.

3. Protoplast experiment with plants

Figure 3.1 Onion protoplasts

After getting the production and preparation of the protoplasts and the bacteria right we incubated them together in a solution of 0.8M and 0.6M Mannitol ( for onion and tobacco respectively) in MgM-MES buffer with a PH of 5. samples from the solutions were taken after 2hours, 3 hours and the day after(approx 18hours). To evalute the samples we used a fluorescent microscope. The results seem to be negative or inconclusive.

Samples incubated with the strains that didn’t have the signal sequence showed bright fluorescence(of both GFP and Mcherry) but no apparent secretion into protoplasts, although some cell membranes appeared to have a greater fluorescence than their surroundings, it was too inconclusive for making any qualitative assumptions. Some of the protoplasts appeared to be full of bacteria in which case the protoplasts had probably burst, forming a “bag-like” structureand filled with bacteria.

Figure 3.2 and 3.3 Birght field vs fluorescence filter showing tobacco proplasts forming “bag-lige” structures filled with GFP.

Samples incubated with the strains containing the signal sequence showed a very weak fluorescence(of both GFP and Mcherry) and no clear fluorescence around or inside the protoplasts. We hoped to see some proteins inside the protoplasts and a greater fluorescence around the protoplasts.

We can therefore conclude that the preliminary results from the injection assay using onion and tobacco protoplasts, were inconclusive. It could be that the strains containing the signal sequence attached to the protoplasts and injected protein into them but the fluorescence emitted was to weak to observe it using a fluorescence microscope. it could also be that the signal sequence somehow disrupts the production of the proteins, perhaps during folding. Furthermore, it could simply be due to the fact that the injectisome does not recognize and bind to the membrane of the protoplasts.

4. Egg membrane experiment

In the first experiment 2 samples were taken, 20h after filling the chambers and after 47h. The bacterial load of lower chamber solution was tested by plating it on agarose gel plate with added chloramphenicol and arabinose. If the colonies appeared the following day and they were red in appearance (due to mCherry) that meant that the membrane was breached and lower chamber compromised.

As a control, a setup with LB media in both chambers was used.

Unfortunately all plates showed red colonies of different sizes the following days, meaning we got false positive results. Red colour came from produced mCherry protein (after induction with arabinose), since we were working with mCherry-expressing E coli in this experiment.

Figure 4.1: Presence of red violet colour meant that bacteria had indeed breached the membrane.

Troubleshooting

Few more tests were performed similar to the experiment described above. Increase of fluorescence was always connected with breaching of the membrane, however there were a few experiments where sterility of the collection chamber remained uncompromised, but also no increase in fluorescence was detected.

The strain used in that experiment was I+,S-,C+ meaning that it had the injectisome and chaperone but lacked the secretion signal. It was noted that the overnight culture turned red after incubation in 1% (w/w) arabinose already after 12 hours. However this strain was unable to secrete the protein through membrane - we speculated that this may be due to the lack of secretion signal.

To test this theory the only logical thing to do would have been to try the strain with secretion signal but the strain I+S+C+ didn't show enough fluorescence in overnight culture to even start the experiment. We believed that signal sequence was somehow disrupting the folding process of mCherry protein and therefore tried growing bacteria at lower temperature (28°C instead of 37°C as in the first experiments). Bacteria was allowed to grow for 5 days yet it still didn't produce enough of the fluorescent protein.

The same issue was encountered when trying to repeat the experiment with E coli producing sfGFP (superfolder green fluorescent protein). The strain without secretion signal produced enough fluorescent protein for experiments, yet the one with secretion signal produced protein below detectable levels for fluorescent plate reader. As the strains without secretin signal failed to secrete the fluorescent protein across the membrane and the strains with secretion signal failed to produce enough of it the experiments were eventually discontinued.

Conclusions

After initial difficulties with the "hardware" i.e. leaky membranes, the manufacturing process was eventually perfected to produce a reasonable number of leak free membrane systems of required quality. The protocols were also eventually optimized at least concerning the incubation time, volumes and similar, but the problem with signal interfering with the folding of the protein remained unsolved.

Nevertheless, had the issue with folding of the protein (with secretion signal) been solved the developed setup would be the best possible option to test whether the whole idea of secreting the proteins across membrane worked. Instead of egg yolk membrane, different substitutes could be used, either using membranes from nature or biomimetic membranes.

An interesting thing to investigate however would be how long can a membrane be exposed to bacteria before being damaged.

Team:UCopenhagen/Results

Overview of results for the different experiments

1. Liposome and biomimetic membrane