Part:BBa_K5057007

Sushi S1 antimicrobial peptide with extracellular signal peptide HSTII

This composite part is composed of the signal peptide, Heat-Stable Enterotoxin II (HSTII, BBa_K5057002 ) and the antimicrobial peptide Sushi S1 ( BBa_K5057004 )

We, the iGEM Team Freiburg 2024, used this composite part in our project CAPTURE (link to wiki). to investigate the effect of intracellular expression of HSTII-Sushi S1 on the viability of bacterial cultures.

Usage and Biology</i>

The aim of our project

Exploiting the revolutionary potential of AMPs, our project CAPTURE aims to deliver a plasmid encoding Sushi S1 - a bactericidal peptide inside of lipid-based nanocarriers or outer membrane vesicles equipped with targeting mechanisms designed to bind and fuse with Pseudomonas aeruginosa - a multidrug-resistant opportunistic pathogen responsible for most cases of hospital-acquired pneumonia. As a result of the highly specific targeting mechanisms and supported by the selected Pseudomonas-specific promoter the AMP will be expressed solely in the target bacteria. Once expressed, the AMPs will kill the bacteria, treating the infection directly at the infection site. Through the continuous expression of the peptide directly in the bacteria we aim to circumvent high production costs and the proteolytic activity of synthetic AMPs.

Antimicrobial peptides

Antimicrobial peptides (AMPs) are a diverse class of small, naturally occurring peptides playing a crucial role in the innate immune response of various organisms. These peptides consist of 10 to 60 amino acids and are generally characterized by their net positive charge and the ability to disrupt microbial membranes, thereby exhibiting potent activity against a wide range of pathogens, including bacteria, fungi, viruses and parasites. Most AMPs target bacterial membranes by creating pores or disrupting the whole membranes in a detergent-like manner [1]. These modes of action rely on both the cationic properties of the AMP itself, and negatively charged bacterial membranes. The differences in lipid composition between the host and pathogen membranes enable the AMPs to achieve comparably low cytotoxicity [2].

Ongoing research has led to the discovery of AMPs with diverse mechanisms of action. While many primarily target membranes, others exhibit inhibitory or disruptive effects on intracellular processes such as protein and DNA synthesis, cell division and biofilm formation. These peptides rely on various mechanisms involving enzyme inactivation, signaling disruption or induction of degradation processes [3, 4, 5, 6].

Due to their extraoridinary characteristics, AMPs constitute a promising research field for the development of new therapeutics to combat antibiotic resistance. Many attempts have been made to create synthetic AMPs de novo, mimicking the design of already existing peptides [7, 8, 9, 10]. However, designing AMPs comes with several obstacles effectively preventing a wider use of AMPs in the medicine. As stated by Liu et al. AMPs often have a hemolytic effect on eukaryotic cells, they lack stability due to limited pH tolerance and proteolyse susceptibility and experience reduced activity in the presence of iron and different serums. A further limitation is also high costs of AMP production typically by chemical synthesis [11,2].

Sushi S1

Sushi S1 is an antimicrobial cationic peptide composed of 34 amino acids, derived from the lipopolysaccharide (LPS)-binding region of Factor C found in horseshoe crabs. It targets bacterial membranes through four successive steps in the bactericidal process: Binding, primarily mediated by charged residues in the peptide; Peptide association; Membrane disruption, during which lipopolysaccharides remain intact; and Lysis, resulting from the leakage of cytosolic contents through large membrane defects.

Sushi S1 neutralizes LPS biotoxicity and mitigates the severe effects of septic shock - a condition that arises from bacterial infections and consecutive antibiotics treatment and is characterized by critical health complications, particularly in vulnerable populations. Furthermore, it has been shown that Sushi S1 has low cytotoxic activity against mammalian erythrocytes and remains active in a physiologically relatively broad pH range (pH 6-8) and osmolarity (50 to 300 mM)[1].

These properties make Sushi S1 an ideal candidate for our project, as it combines potent antimicrobial activity with low cytotoxicity and stability under physiological conditions.

Design

The peptide sequence Sushi 1 [1] was reverse translated and optimized for expression in E. coli with the IDT codon optimization tool [1]. We cloned our insert into the pET22b(+) [Biobrick: BBa_K5022002 ] plasmid under an IPTG-inducible promoter with the HSTII signal peptide the N-term of Sushi S1.

Characterization and Optimization

1. HSTII Characterization

To characterize our best composite part we first started to test the functionality of HSTII as an extracellular signal sequence. To facilitate the verification process, we decided to use mCherry as a reporter protein. Expression of mCherry was determined by measuring the fluorescence intensity in the bacterial pellet and supernatant of pET-HSTII-Cherry (Biobrick: BBa_K5057009 ) and control pET-mCherry (periplasmic expression)

Fig. 1: Expression of mCherry and HSTII-mCherry in E. coli BL21(DE3) pRARE2 LysS cells Expression was induced with 0.1 mM IPTG at 18°C in Mueller-Hinton-medium with Ampicillin and Chloramphenicol. Fluorescence of pellet and supernatant (SN) was measured with plate reader at an excitation of 570 nm and emission 610 nm. The experiment was performed in technical duplicates that were individually measured three times. Pellets were washed with Phosphate-buffered saline (PBS) before measurement. The OD600 was normalized for all cultures before measurement.

This comparative study between HSTII-mCherry and mCherry provided strong evidence for the functionality of the HSTII signal peptide. The higher fluorescence signal in the supernatant of HSTII-mCherry cultures compared to mCherry cultures suggests successful export of the fusion protein mediated by the HSTII signal peptide. The comparable fluorescence levels in the pellets indicate that the presence of the HSTII signal does not significantly affect overall mCherry expression or folding. These results validate our approach of using HSTII as a signal peptide for extracellular localization of our target proteins.

2. Signal Peptide Evaluation

Sushi is known to disrupt bacterial membranes when applied externally. The first critical question we sought to answer was the optimal cellular location for AMP expression: Would the AMP be effective when expressed inside the bacterial cell, or would it need to be secreted to the extracellular space? We compared the growth of bacteria expressing Sushi 1 with different signal peptides (Fig. 2):

1. Sushi without a signal peptide for intracellular expression

2. Sushi fused to the pelB signal peptide for periplasmic localization, and

3. Sushi linked to the Heat Stable Toxin II (HSTII) signal peptide for extracellular secretion.

BBa_K5057009 ) and control pET-mCherry (periplasmic expression)

Fig. 2: Experimental Set-Up for expression of Sushi 1 with different signal peptides: pET-pelB-Sushi (periplasmic), pET-HSTII-Sushi (extracellular), pET-Sushi (intracellular), pET (negative control).

Our experiments revealed significant differences in bacterial growth inhibition among the various Sushi constructs and controls (Fig. 3). In all cases, induced cultures exhibited higher growth inhibition compared to uninduced cultures.

<b>Growth inhibition in liquid cultures: When comparing the effect of Sushi at different cellular localizations, we observed the strongest growth inhibition with HSTII-Sushi (secreted), followed by pelB-Sushi (periplasm) and Sushi (intracellular). Notably, cultures expressing HSTII-Sishi did not surpass an OD₆₀₀ of 0.9 post-induction.

Fig 3: Comparison of Signal Peptides fused to Sushi Growth curve of E. coli BL21(DE3) containing plasmids: pET-HSTII-Sushi (extracellular signal peptide), pET-pelB-Sushi (periplasmic signal peptide), pET-Sushi (without signal peptide). Cultures were introduced with 0.5 mM IPTG at 30°C and 200 rpm. Error bars represent biological duplicates.

Viability assessment via drop count assay:

The growth inhibition could not only be observed in liquid cultures but also for the drop count assay performed two hours post-induction (Fig. 4). The assay revealed distinct differences in colony-forming ability among the constructs: For pET-HSTII-Sushi colonies were observed up to the dilution of 10 ^-3, indicating moderate growth inhibition. Colonies for pET-pelB-Sushi, pET-Sushi and pET (control) were visible up to 10^-4 dilution, suggesting lower growth inhibition compared to HSTII-Sushi.

Fig. 4: Drop count assay of E. coli BL21(DE3) containing indicated plasmids. Cultures were serially diluted (from 10^-1 to 10^-5) and 5 µL of each dilution was spotted on LB-Amp agar plates, two hours post-induction with 0.5 mM IPTG. Plates were incubated on LB-Amp at 37°C overnight.

The extracellular signal peptide HSTII, when fused to Sushi, achieved the lowest survival rate of E. coli BL21(DE3) in liquid culture. This finding aligns with previous studies indicating that Sushi exhibits enhanced antimicrobial activity when applied externally to bacterial cells.

3. Expression Optimization

We aimed to determine the most effective temperature and IPTG concentration for HSTII-Sushi expression and bacterial growth inhibition.

HSTII-Sushi expression at 18°C:

During our experiments, we realized that the IPTG-inducible T7 promoter in our constructs has a background expression in case of mCherry (pink overnight cultures prior to induction). Therefore, we tested our constructs in a different strain of E. coli BL21 (DE3) possessing pRARE2 LysS plasmids that reduces the background activity through the expression of lysozyme. Based on our experiments of HSTII-mCherry expression we decided to use these conditions (18°C, 0.1 mM IPTG) as a starting point to evaluate the expression of HSTII-Sushi in E. coli BL21 (DE3) pRARE LysS cells.

Fig. 5: Growth curve of HSTII-Sushi compared to HSTII-mCherry in *E. coli pRARE2 LysS cells. Expression was induced with 0.1 mM IPTG Mueller-Hinton-medium containing Ampicillin and Chloramphenicol at 18°C, 200 rpm. Error bars represent biological duplicates.

All cultures (induced and uninduced) showed similar growth patterns over 8 hours (Fig. 5). The conditions that worked best for HSTII-mCherry (18°C, 0.1 mM IPTG) were not effective for HSTII-Sushi expression or activity in E. coli BL21(DE3) pRARE2 LysS cells. With Western-Blot analysis we did not detect any Sushi peptide (data not shown). We concluded that under these conditions, Sushi is either not expressed or in too low concentrations. This led us to explore alternative expression conditions.

HSTII-Sushi expression at 30°C:

Following our initial experiments at 18°C, which failed to demonstrate the expected growth inhibition of E. coli BL21(DE3) expressing HSTII-Sushi, we hypothesized that the optimal expression conditions might differ between HSTII-mCherry and HSTII-Sushi. This led us to investigate the effect of higher temperatures on HSTII-Sushi expression and activity. We used the same experimental set up as before, but changed the induction condition to 0.5 mM IPTG at 30°C using E. coli BL21(DE3) cells in MH medium.

Induced pET-HSTII-Sushi cultures showed a significant growth inhibition, while uninduced HSTII-Sushi and HSTII-mCherry cultures achieved an OD₆₀₀ of 6.18. Induced mCherry cultures showed slower but continuous growth, in contrast to induced HSTII-Sushi cultures, which stagnated at OD₆₀₀ of around 0.7 at 8 hours post-induction.

Fig. 6; Growth curve of HSTII-Sushi compared to HSTII-mCherry in *E. coli BL21(DE3). Expression was induced with 0.5 mM IPTG in MH-Amp at 30°C, 200 rpm. Error bars represent biological duplicates.

This experiment demonstrates that HSTII-Sushi expression in E. coli BL21(DE3) at 30°C effectively inhibits bacterial growth.

Optimization of IPTG concentration for HSTII-Sushi expression:

From our previous experiments we have learned that optimal expression conditions are protein-specific and cannot be universally applied across different proteins. By systematically testing a range of IPTG concentrations while maintaining a constant temperature of 30°C, we seek to optimize the expression conditions for HSTII-Sushi

All induced HSTII-Sushi cultures showed growth inhibition within the first hour post-induction (Fig. 8). In the first six hours the cultures showed no growth above an OD₆₀₀ of 0.9. After six hours, the cultures began to recover. After 24 hours, the induced HSTII-Sushi cultures reached similar OD₆₀₀ values as the controls.

Figure 7. Experimental setup to test different IPTG concentration for expression of HSTII-Sushi in E. coli BL21(DE3) cells at 30°C. pET-HSTII-Sushi cultures were induced with 0.1, 0.5, 0.7 and 1.0 mM IPTG. pET-HSTII-mCherry (control) was induced with 0.5 mM IPTG.

To assess the impact of IPTG concentration on HSTII-Sushi expression and bacterial growth inhibition, we focused our analysis on the first eight hours post-induction. During this period, all induced cultures showed significant growth inhibition compared to the control cultures (Fig. 9). Notably, we observed no substantial differences in growth patterns among the various IPTG concentrations tested (0.1 mM, 0.5 mM, 0.7 mM, and 1.0 mM). This suggests that within the range of concentrations examined, the induction of HSTII-Sushi expression and its subsequent antimicrobial effect are not highly sensitive to IPTG concentration.

Fig. 8: Test various IPTG concentrations. Growth curve of *E. coli* BL21(DE3) transformed with pET-HSTII-Sushi and pET-HSTII-mCherry (control), respectively. IPTG concentrations: A: 0.1 mM B: 0.5 mM, C: 0.7 mM, D: 1.0 mM. pET-HSTII-mCherry induced with 0.5 mM IPTG. Error bars represent biological duplicates.

Our experiments demonstrate that HSTII-Sushi expression in E. coli BL21(DE3) at 30°C effectively inhibits bacterial growth. The antimicrobial effect is most pronounced in the first 6 hours post-induction, indicating a potent but time-limited activity of the expressed HSTII-Sushi. The recovery of bacterial growth after 24 hours could be due to plasmid loss following ampicillin degradation. The similar results observed across different IPTG concentrations (0.1-1.0 mM) indicate that HSTII-Sushi expression and activity are not highly sensitive to inducer concentration within this range. This robustness allows flexibility in experimental design and potential applications. Our series of experiments demonstrate that the expression and activity of HSTII-Sushi in E. coli is highly dependent on temperature and strain characteristics.

4. Protein Expression Confirmation

Western blot analysis was performed to detect the expression of HSTII-Sushi-6xHis (Biobrick: BBa_K50570012 ) containing a C-terminal 6xHis-tag. Expression was induced with 0.5 mM IPTG at 30°C, 200 rpm.

Fig. 9: Induction with different IPTG concentrations of pET-HSTII-Sushi in E. coli BL21(DE3)Induction conditions: 30°C, 200rpm, 0.1/0.5/0.7/1.0 mM IPTG. Error bars from biological duplicates.

Western blot analysis revealed distinct bands at approximately 5 kDa confirming the expression of HSTII-Sushi-6xHis (Biobrick: BBa_K50570012 ) in the pelleted bacterial cultures (Fig. 10). Notably, HSTII-Sushi-6xHis expression was predominantly observed in the initial hours post-induction, becoming undetectable in later stages. This pattern suggests potential protein degradation over time or the formation of complexes that alter protein mobility. The transient expression profile of HSTII-Sushi-6xHis aligns with the previously observed growth recovery at 24 hours post-induction (Fig. 8), suggesting a potential correlation between protein expression dynamics and bacterial growth patterns.

5. Comparative Expression Analysis

After successfully characterizing Sushi, the next step was to compare the antimicrobial efficacy of expressed Sushi with our other AMP CONGA (Biobrick: BBa_K5057008 ). Additionally, we assess the activity of Imitate (Biobrick: BBa_K5057006 ), a designed peptide intended to be non-functional.

Fig. 10: Western Blot analysis of HSTII-Sushi-6xHis expression in E. coli BL21 (DE3). Cells were induced with 0.5 mM IPTG at 30°C and samples were collected at indicated times points post-induction. HSTII-mCherry-His (36 kDa) expressed under identical conditions served as a positive control. Proteins were separated on a 15% SDS-PAGE gel and detected using anti-His mouse primary antibodies.

Fig. 11 Growth curve of *E. coli* BL21(DE3) expressing A: pET-HSTII-Sushi, B: pET-HSTII-CONGA, C: pET-HSTII-Imitate, A,B,C: pET-HSTII-mCherry (negative control), Induction with 0.5 mM IPTG in MH-Amp at 30°C, 200 rpm. Error bars represent biological duplicates.

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Fig. 12 Western Blot analysis of HSTII-Sushi-6xHis (4.7 kDa) and HSTII-Imitate-6xHis (4.1 kDa) expression in E. coli BL21 (DE3) pellets. Cells were induced with 0.5 mM IPTG at 30°C and samples were collected at indicated times points post-induction. Proteins were separated on a 15% SDS-PAGE gel and detected using anti-His mouse primary antibodies.

Western blot analysis confirmed the expression of HSTII-Sushi-6xHis and pET-HSTII-Imitate in bacterial pellets within the first four hours post-induction (Fig. 12). However, both Sushi and Imitat, were not detectable in the corresponding supernatants, possibly due to insufficient protein concentration in these samples.

Bactericidal assay:

We additionally tested if the supernatant of cultures expressing Sushi S1, Imitate peptide, Sushi-6xHis and CONGA, respectively, could have an bactericidal effect on plated bacteria.

None of the supernatants affected the growth of E. coli and P. fluorescens on the plates. Considering the results of the Western blot analysis this observation could be due to low concentration of the peptides in the media.

Fig. 13 Killing Assay of AMPs in Supernatant (SN)A: E. coli BL21(DE3), B: P. fluorescens DSM 50900 1: kanamycin (positive control) and 2: MH-medium (negative control), 3: SN of HSTII-Sushi, 4: SN of HSTII-CONGA, 5: SN of HSTII-Imitate, 6: SN of HSTII-Sushi-6xHis.

We repeated the killing assay with purified Sushi-6xHis and Imitate-6xHis. However, there was no growth inhibition for plated E. coli and P. fluorescens measurable.

Conclusion

Our novel composite part was designed to combat antimicrobial resistance by introducing a plasmid expressing a potent antimicrobial peptide (AMP) directly into target pathogens. This approach aimed to create a low-cost, effective, and adaptable system for various pathogenic microorganisms.

While we were unable to reproduce the results published by Yau et al. (2001) and Lepthin et al.(2009) in the research group of Prof. Dr. Jeak Ling Ding, our project yielded promising outcomes. Among our multiple growth curves and killing assays, we observe a similar inhibitory effect with the expression of a different AMP - CONGA, introduced to us by Prof. Wimley. This AMP demonstrated efficacy as an synthesized peptide in D- and L-configuration against E. coli and in D-configuration against P. fluorescens.

Due to time constraints, we were unable to continue our work with further AMPs, but we took some initial steps in the direction that we believe can bring a new solution to the emerging global threat of antimicrobial resistance.

This new composite part serves as a foundation for future iGEM teams eager to continue and improve our research. The antimicrobial peptide database of the medical center of the university of Nebraska contains 3940 AMPs (stand: 23.09.2024), including 3146 naturally occurring peptides from different kingdoms of life and thus provide the perfect opportunity to start your research!

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

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[2] Li J, Koh J-J, Liu S, Lakshminarayanan R, Verma CS, Beuerman RW. Membrane active antimicrobial peptides: Translating mechanistic insights to design. Front Neurosci [Internet]. 2017;11:73. Available from: http://dx.doi.org/10.3389/fnins.2017.00073 ‌

[3] Mardirossian M, Pérébaskine N, Benincasa M, Gambato S, Hofmann S, Huter P, et al. The dolphin proline-rich antimicrobial peptide Tur1A inhibits protein synthesis by targeting the bacterial ribosome. Cell Chem Biol [Internet]. 2018;25(5):530-539.e7. Available from: http://dx.doi.org/10.1016/j.chembiol.2018.02.004 ‌

[4] He S-W, Zhang J, Li N-Q, Zhou S, Yue B, Zhang M. A TFPI-1 peptide that induces degradation of bacterial nucleic acids, and inhibits bacterial and viral infection in half-smooth tongue sole, Cynoglossus semilaevis. Fish Shellfish Immunol [Internet]. 2016;60:466–73. Available from: http://dx.doi.org/10.1016/j.fsi.2016.11.029 ‌

[5] Lutkenhaus J. Regulation of cell division in E. coli. Trends Genet [Internet]. 1990;6(1):22–5. Available from: http://dx.doi.org/10.1016/0168-9525(90)90045-8 ‌

[6] Li L, Sun J, Xia S, Tian X, Cheserek MJ, Le G. Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest. Appl Microbiol Biotechnol [Internet]. 2016;100(7):3245–53. Available from: http://dx.doi.org/10.1007/s00253-015-7265-y ‌

[7] Goormaghtigh E, De Meutter J, Szoka F, Cabiaux V, Parente RA, Ruysschaert JM. Secondary structure and orientation of the amphipathic peptide GALA in lipid structures. An infrared-spectroscopic approach. Eur J Biochem [Internet]. 1991;195(2):421–9. Available from: http://dx.doi.org/10.1111/j.1432-1033.1991.tb15721.x ‌

[8] Fjell CD, Hiss JA, Hancock REW, Schneider G. Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov [Internet]. 2011;11(1):37–51. Available from: http://dx.doi.org/10.1038/nrd3591 ‌

[9] Kim H, Jang JH, Kim SC, Cho JH. Development of a novel hybrid antimicrobial peptide for targeted killing of Pseudomonas aeruginosa. Eur J Med Chem [Internet]. 2019;185:111814. Available from: http://dx.doi.org/10.1016/j.ejmech.2019.111814 ‌

[10] Ghimire J, Hart RJ, Soldano A, Chen CH, Guha S, Hoffmann JP, et al. Optimization of host cell-compatible, antimicrobial peptides effective against biofilms and clinical isolates of drug-resistant bacteria. ACS Infect Dis [Internet]. 2023;9(4):952–65. Available from: http://dx.doi.org/10.1021/acsinfecdis.2c00640 ‌

[11] Jaradat DMM. Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation. Amino Acids [Internet]. 2017;50(1):39–68. Available from: http://dx.doi.org/10.1007/s00726-017-2516-0 ‌