Part:BBa_K5311006

CAP 10-3 Endolysin

Cutibacterium acnes has great potential to be used as a chassis for various applications. In our project, we demonstrate this by developing a system to optimize the release of Cry proteins. Given its potential, it was essential for our project—as well as for future uses of C. acnes as a chassis in other iGEM projects—to establish a reliable biocontainment strategy. In our case, we achieved this through the incorporation of an endolysin, which allows for controlled cell lysis and ensures safe use by minimizing the risks associated with prolonged contact of GMOs with the skin.

We will combine its implementation with a regulation mechanism by using an RNA thermometer (BBa_K115002) that controls endolysin expression. While our current RNA thermometer activates at around 37°C, slightly higher than normal skin temperature (~30°C), this could be fine-tuned in future iterations to match physiological conditions more closely. Additionally, the endolysin facilitates the release of therapeutic Cry proteins, which is a key component of our project, breaking down the bacterial cell walls and enhancing the effectiveness of the treatment against skin infestations. This dual function ensures safety while improving protein release.

Biology

In the case of the endolysin derived from the CAP 10-3 bacteriophage, its action specifically targets the degradation of the cell wall of Cutibacterium acnes, a Gram-positive bacterium with a cell wall rich in peptidoglycan, which is a crucial structural component. This endolysin is classified as an N-acetylmuramoyl-L-alanine amidase, which cleaves the bond between the N-acetylmuramic acid and the L-alanine residue in the peptidoglycan backbone.

By breaking down the peptidoglycan, the endolysin disrupts the rigid structure of the cell wall, leading to a loss of osmotic integrity. As a result, the bacterial cell can no longer withstand the internal pressure, causing rapid cell lysis. This mechanism is particularly effective against Gram-positive bacteria like C. acnes because they have a thick peptidoglycan layer that is more accessible to endolysins, unlike Gram-negative bacteria, which possess an additional outer membrane that restricts endolysin access.[1]

Advantages of Endolysins Against C. acnes:

High Specificity: The CAP 10-3 Endolysin is known for its high specificity towards C. acnes, often recognizing unique peptidoglycan motifs present in the bacterial cell wall. This ensures that they selectively lyse the pathogenic bacteria without affecting other beneficial members of the skin microbiota [2].
Low Risk of Resistance: Unlike traditional antibiotics, which typically target essential processes such as protein synthesis or DNA replication, endolysins act on the structural components of the bacterial cell wall, such as the peptidoglycan layer. The peptidoglycan is a highly conserved and essential component that is less prone to mutation, reducing the likelihood of bacteria developing resistance.
Safety and reduced side effects: Endolysins have a favorable safety profile compared to traditional antibiotics, as they are proteins that are rapidly degraded in the human body without inducing toxic side effects. Furthermore, since they specifically target bacterial cell wall components, they do not interact with human cells, making them safe for topical or systemic applications [2].

Design and Usage

Design

We extracted the source sequence from the paper that is referenced in [1] and started with the design for our part. We had performed a codon optimization for the original sequence in order to optimize it for C. acnes, this was achieved with the ATG.me database and a python script developed by our instructors.

Once we designed the initial sequence we added the common prefixes and suffixes adapted for Gibson Assembly, which we have used for constructing our sequences of interest. We also added a His-tag sequence at the end of our part to be able to detect it with a Western Blot.

On the images below you can find the final plasmid designs, these parts were achieved and used to characterize the behavior of the endolysin:

Figure 1 Plasmid design for the final construct including endolysin.

Usage

Since we wanted to use this part on C. acnes to characterize its behavior and effect on its growth we had to develop an action plan to achieve a proper transformation. This was done because C. acnes contains defense mechanism systems called RM (Restriction-Modification) that contains endonucleases that recognize non-methylated DNA to cut it. While the restriction endonuclease recognizes specific DNA sequences and cleaves any foreign DNA lacking the appropriate methylation pattern, the modification methyltransferase, methylates the bacterium’s own DNA at these same recognition sites, ensuring that the bacterial genome is not targeted by its own restriction enzymes.

To surpass this mechanisms we firstly transformed our plasmid of interest inside E. coli EC24 strain [3] since has specific engineered methyllation mechanisms that are targeted to allow transformation on our chassis of interest. Once we had our colonies transformed on C. acnes we wanted to demonstrate that this transformation was achieved we confirmed our results with an agarose gel for electrophoresis and 16S sequencing to confirm that we haven't had any contamination.

Figure 2 PCR colony of C. acnes transformation, analyzing both our region of integration, and the 16S region.

Characterization

As we have described previously one of the main objectives on using endolysin was to implement a regulation mechanism with an RNA Thermometer that allows transcription once the ideal temperature is reached (37°C). Previous to this transformation we succesfully characterized the behavior of this part on C. acnes strain KPA171202. In order to do that designed our construct with a His-tag thanks to this we were able to perform a Western Blot that allowed us to quantify the expression in both of our chassis of interest C. acnes and E. coli and we also conducted an experiment to quantify the effect of endolysin on the growth of C. acnes.

Western Blot

The Western Blot performed by our team was conducted to characterize the endolysin expression with the combination of different chassis and different promoters. Since we also wanted to evaluate how was the protein being expressed we conducted the western blot with both pellet and supernatant. Finally on the Western Blot the wells were organized with the following order:

Sample 1: Not rellevant for this part documentation
Sample 2: E. coli NZY 5α with Endolysin+MG10 (pellet)
Sample 3: C. acnes with Endolysin+MG10 (pellet)
Sample 4: C. acnes with Endolysin+MG26 (pellet)
Sample 5: Not relevant for this part documentation
Sample 6: C. acnes with Endolysin+MG10 (supernatant)
Sample 7: C. acnes with Endolysin+MG26 (supernatant)
Sample 8: Not relevant for this part documentation

Where MG10 (Part: BBa_J23119) and MG26 (which is a C. acnes 16S rRNA promoter) are constitutive promoters that allow expression on C acnes (while MG10 also allows it in E. coli) and the expected mollecular weight for the endolysin is For E. coli, proteins are primarily intracellular, requiring cell lysis to isolate the proteins from the pellet. For C. acnes, we anticipated protein expression both within the cell and in the supernatant. Thus, we evaluated both pellet and supernatant samples to capture the full protein profile.

For the loading of the SDS-PAGE we used the SeeBlue Pre-Stained Standard Western blot ladder which has its reference attached below with the obtained results:

Results: After revealing the Western blot, we noted the following:

Sample 1: Showed no significant protein bands, indicating no detectable protein expression.
Sample 2: E. coli Endolysin+MG10 (pellet) showed two clear bands, one around 76 kDa and another around 31 kDa. This suggests that Cry3Aa protein (Part:BBa_K5311002) contaminated this well, leading us to conclude that both Cry3Aa and Endolysin+MG10 are expressed.
Sample 3: C. acnes Endolysin+MG10 (pellet) showed a faint band at 31 kDa, suggesting some expression of Endolysin+MG10.
Sample 4: C. acnes Endolysin+MG26 (pellet) displayed a strong band at 31 kDa, confirming successful protein expression of endolysin MG26.
Samples 5–8: No detectable protein bands were observed. Specifically, for the C. acnes supernatants (wells 6–8), the lack of protein expression may be due to insufficient time for the protein to be secreted into the supernatant since we only left the liquid cultures growing for 48 hours.

In conclusion, protein expression was confirmed for E. coli Endolysin+MG10 and C. acnes Endolysin+MG26. We would have liked to perform another Western Blot with newly transformed C.acnes cells, however, we did not have enough time to do this.

Characterization of the effect of CAP 10-3 Endolysin on C. acnes growth

Once we had determined and characterized protein expression on C. acnes we wanted to evaluate the effectiveness of the CAP 10-3 endolysin in reducing its viability. In order to do that we conducted an experiment comparing survival rates of C. acnes across different conditions. We distributed the plates in three different grous: one expressing an endolysin-coding plasmid, one with a plasmid providing only antibiotic resistance (WT), and another one with a plasmid with constitutive GFP expression.

We did take into account different experimental conditions such as the growth temperature (Constant at 37°C)at 110 rpm, anaerobiosis incubation, finally the experiment was conducted during 5 days and the measurement of the results was performed every 1-2 days.

Finally, the obtained results were presented with the plots below

La imagen de la derecha es más pequeña que la de la izquierda, quiero que tengan el mismo tamaño porfa:

Endolysin 10 — Figure 4: MG10 Promoter + Endolysin

Endolysin 26 — Figure 5: MG26 Promoter + Endolysin

The endolysin-expressing strain exhibited a lag phase that was noticeably longer than that of both the WT and GFP strains. This extended lag phase suggests that the presence of endolysin may require additional time for the cells to acclimate before starting to grow. Once the lag phase concluded, the growth rate of the endolysin strain appeared slightly slower than that of the WT and GFP strains. The OD600 measurements indicated that, although the endolysin strain eventually increased in cell density, it did so at a reduced rate, further supporting the fact that endolysin affects cellular viability.

Interestingly, this decline in bacterial growth does not appear to be related to metabolic burden. This is evidenced by the fact that cells expressing GFP, which would also be expected to experience some metabolic load, displayed normal growth patterns compared to the endolysin-expressing strain. Our results indicate that the reduced growth rate is specific to the presence of endolysin and not due to general stress from protein expression.

These findings are important as they support the idea that endolysin may assist in the delivery of the Cry proteins. Future research should focus on investigating the specific role of endolysin in facilitating protein release, and evaluate whether this mechanism enhances the therapeutic effectiveness against the targeted skin infestations.

In a future experiment, the fluorescence quantification of GFP and mCherry in Cutibacterium acnes will be investigated under the control of the RNA thermometer. Fluorescence will be measured at three distinct temperatures: room temperature, 30°C, and 37°C. By comparing fluorescence levels at each temperature, the study aims to determine how the RNA thermometer modulates gene expression in C. acnes and explore the potential for temperature-sensitive control of protein production in this organism.

References

[1] Kim JI, Hasnain MA, Moon GS. Expression of a recombinant endolysin from bacteriophage CAP 10-3 with lytic activity against Cutibacterium acnes. Sci Rep. 2023 Sep 30;13(1):16430. doi: 10.1038/s41598-023-43559-z. PMID: 37777575; PMCID: PMC10542754.

[2] Rahman MU, Wang W, Sun Q, Shah JA, Li C, Sun Y, Li Y, Zhang B, Chen W, Wang S. Endolysin, a Promising Solution against Antimicrobial Resistance. Antibiotics (Basel). 2021 Oct 20;10(11):1277. doi: 10.3390/antibiotics10111277. PMID: 34827215; PMCID: PMC8614784.

[3] Knödlseder, N., Fábrega, MJ., Santos-Moreno, J. et al. Delivery of a sebum modulator by an engineered skin microbe in mice. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-02072-4