Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29

Monika Szadkowska; Aleksandra Maria Kocot; Daria Sowik; Dariusz Wyrzykowski; Elzbieta Jankowska; Lukasz Pawel Kozlowski; Joanna Makowska; Magdalena Plotka

doi:10.3389/fmicb.2023.1303794

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29

Front Microbiol. 2024 Jan 19:14:1303794. doi: 10.3389/fmicb.2023.1303794. eCollection 2023.

Authors

Monika Szadkowska¹, Aleksandra Maria Kocot¹, Daria Sowik², Dariusz Wyrzykowski³, Elzbieta Jankowska², Lukasz Pawel Kozlowski⁴, Joanna Makowska³, Magdalena Plotka¹

Affiliations

¹ Laboratory of Extremophiles Biology, Department of Microbiology, University of Gdańsk, Gdańsk, Poland.
² Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland.
³ Department of General and Inorganic Chemistry, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland.
⁴ Institute of Informatics, Faculty of Mathematics, Informatics and Mechanics, University of Warsaw, Warsaw, Poland.

Abstract

Introduction: In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.

Methods: Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3'-Dipropylthiacarbocyanine iodide (DiSC₃(5)).

Results and discussion: The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo_105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

Keywords: antimicrobial peptide; bacteriophage; extremophile; lysin; thermostable endolysin.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. Funding was provided by the National Science Centre (Poland) OPUS20 grant: UMO-2020/39/B/NZ6/00589 to MP.