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Latest revision as of 12:37, 29 September 2024

Conga antimicrobial peptide with extracellular localization sequence HSTII

Based on the research article by Ghimire et al. (2023) and a presentation by Prof. Dr. William Wimbley, iGEM Freiburg 2024 translated the peptide sequence of a synthethically produced antimicrobial peptide called d-CONGA-Q7 into a DNA sequence and optimized it for expression in E. coli. Since in the original source, the synthetic peptide was created with D-aminoacids, the name comes with a prefix "D-" referring to the molecular configuration of the aminoacids. In the experiments of team Freiburg 2024, the peptide was synthesized in E. coli using the usual L-amino acids.

Usage and Biology

D-CONGA-Q7 was created in a process of continued molecular evolution exhibiting a potent antimicrobial activity against planktonic or biofilm-forming Gram-negative bacteria such as P. aeruginosa and E. coli [1]. it is a short peptide with an overall cationic charge and as such can be classified as antimicrobial peptide (AMP).

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 the detergent-like manner [2]. 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 high specificity [3].

Ongoing research has led to the discovery of further categories of AMPs exhibiting inhibitory or disruptive effects on 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 [4, 5, 6, 7].

Due to their extraordinary 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 [8, 9, 10]. However, designing AMPs comes with several obstacles effectively preventing a wider use of AMPs in the medicine. As stated by Li et al.[2] 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, 3].

Experiments

To estimate the effect of synthetic D-Conga that we received from Prof. Dr. Wimbley, we performed a killing assay for E. coli BL21(DE3) and P. fluorescens DSM 50090. Additionally, we were provided with Conga in L-conformation. This experiment allowed us to see the effect of synthetic D-Conga in comparison to L-Conga on the bacterial growth of E. coli and P. fluorescens in liquid culture. We incubated 500 µL of bacterial culture with D- or L-Conga (40 µM) at 37 °C or 30°C respectively and 800 rpm shaking for 3 h. As a positive control kanamycin was used at 40 µg/mL for E. coli and 80 µg/mL for P. fluorescens. As a negative control 0,025% Acetic Acid, the solvent of D- and L-Conga, and bacteria without treatment were used. Every hour a 7x 10-fold serial dilution was plated on MH-Agar using the Drop-Count method.

The treatment with D-Conga resulted in a total growth inhibition of E.coli BL21(DE3) and P .fluorescens DSM 50090 comparable to the treatment with kanamycin. However, L-Conga at the same concentration had a much smaller effect on the growth of E.coli BL21(DE3) and did not inhibit the growth of P .fluorescens DSM 50090.

During our project we were able to compare the effect of the HSTII-Conga expression and our other constructs (HSTII-Sushi S1 and HSTII-Imitate) on bacterial growth over time. For our experiment, we used cultures possessing pET-HSTII-Sushi, pET-HSTII-Imitate, pET-HSTII-Conga and pET-HSTII-mCherry under IPTG-inducible promoter. Following the protocol established previously, overnight cultures with the respective constructs were pelleted and then grown in 100 mL MH-Amp to an OD600 of 0.6 at 37°C. All cultures were divided, one of the aliquots was induced with 0.5 mM IPTG. For the growth curve, all cultures were grown at 30°C, 200 rpm. OD600 was measured every hour for seven hours. Every hour and after 24 hours, 1 mL samples were taken for a Western Blot.

The expression of Conga had a comparable inhibitory effect to the expressed Sushi S1 AMP and the Sushi-imitating peptide within the first five hours. Uninduced HSTII-Conga in comparison showed similar growth to the controls with induced and uninduced HSTII-mCherry. E. coli expressing HSTII-Imitate were inhibited to the same magnitude as Conga and Sushi, exhibiting no growth within seven hours post induction. However, in comparison to uninduced cultures, Sushi, Conga and the Sushi-imitating peptide caused severe growth inhibition

Outlook

Due to time constraints, we were unable to finish the characterization of this AMP. Our future goals include the purification of Conga peptide fused to a His-tag and a temporal verification of its expression via Western Blot. Since the His-Tag might interfere with the activity of the peptide, we aim to test the activity of Conga-6xHis in a comparable killing assay performed with synthesized D- and L-Conga. In parallel, we consider testing the expression of the AMP in our model organism - P. fluorescens using pseudomonas specific plasmid with both a constitutive and inducible promoters. We hope that through our experiments, plasmid-encoded AMPs will become an effective strategy to combat antibiotics-resistant pathogens.

Sequence and Features

References

[1] 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 Infectious Diseases [Internet]. 2023 Mar 24 [cited 2024 Sep 23];9(4):952–65.

[2] Huan Y, Kong Q, Mou H, Yi H. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Frontiers in Microbiology [Internet]. 2020 Oct 16;11. ‌

[3] Li J, Koh JJ, Liu S, Lakshminarayanan R, Verma CS, Beuerman RW. Membrane Active Antimicrobial Peptides: Translating Mechanistic Insights to Design. Frontiers in Neuroscience. 2017 Feb 14;11. ‌

[4] Mardirossian M, Pérébaskine N, Benincasa M, Stefano Gambato, Hofmann S, Huter P, et al. The Dolphin Proline-Rich Antimicrobial Peptide Tur1A Inhibits Protein Synthesis by Targeting the Bacterial Ribosome. 2018 May 17;25(5):530-539.e7. ‌

[5] He SW, Zhang J, Li NQ, 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 Immunology [Internet]. 2017 Jan 1;60:466–73. ‌

[6] Lutkenhaus J. Regulation of cell division in E. coli. Trends in Genetics. 1990;6:22–5. ‌

[7] 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. Applied Microbiology and Biotechnology. 2016 Jan 8;100(7):3245–53. ‌

[8] Goormaghtigh E, 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. European Journal of Biochemistry. 1991 Jan;195(2):421–9. ‌

[9] Fjell CD, Hiss JA, Hancock REW, Schneider G. Designing antimicrobial peptides: form follows function. Nature Reviews Drug Discovery. 2011 Dec 16;11(1):37–51. ‌

[10] Jenisha G, 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 Infectious Diseases [Internet]. 2023 Mar 24;(4):952–65. ‌

[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. 2017 Nov 28;50(1):39–68. ‌