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Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension

Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied...

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Autores principales: Plotka, Magdalena, Szadkowska, Monika, Håkansson, Maria, Kovačič, Rebeka, Al-Karadaghi, Salam, Walse, Björn, Werbowy, Olesia, Kaczorowska, Anna-Karina, Kaczorowski, Tadeusz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404271/
https://www.ncbi.nlm.nih.gov/pubmed/32664473
http://dx.doi.org/10.3390/ijms21144894
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author Plotka, Magdalena
Szadkowska, Monika
Håkansson, Maria
Kovačič, Rebeka
Al-Karadaghi, Salam
Walse, Björn
Werbowy, Olesia
Kaczorowska, Anna-Karina
Kaczorowski, Tadeusz
author_facet Plotka, Magdalena
Szadkowska, Monika
Håkansson, Maria
Kovačič, Rebeka
Al-Karadaghi, Salam
Walse, Björn
Werbowy, Olesia
Kaczorowska, Anna-Karina
Kaczorowski, Tadeusz
author_sort Plotka, Magdalena
collection PubMed
description Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied earlier in our laboratory. Both these enzymes originate from Thermus scotoductus bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from Clostridium intestinale URNW was found to have the highest amino acid sequence similarity to the bacteriophage proteins and was chosen for further analysis. The recombinant enzyme showed strong activity against its host bacteria C. intestinale, as well as against C. sporogenes, Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus, on average causing a 5.12 ± 0.14 log reduction of viable S. aureus ATCC 25923 cells in a bactericidal assay. Crystallographic studies of the protein showed that the catalytic site of LysC contained a zinc atom coordinated by amino acid residues His(50), His(147), and Cys(155), a feature characteristic for type 2 amidases. Surprisingly, neither of these residues, nor any other of the four conserved residues in the vicinity of the active site, His(51), Thr(52), Tyr(76), and Thr(153), were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged N-terminal region of the enzyme. Potential antibacterial activity of this part of the sequence, predicted by the Antimicrobial Sequence Scanning System, AMPA, was confirmed in our experimental studies; the truncated version of LysC (LysCΔ2–23) completely lacked antibacterial activity. Moreover, a synthetic peptide, which we termed Intestinalin, with a sequence identical to the first thirty amino acids of LysC, displayed substantial anti-staphylococcal activity with IC(50) of 6 μg/mL (1.5 μM). This peptide was shown to have α-helical conformation in solution in the presence of detergents which is a common feature of amphipathic α-helical antimicrobial peptides.
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spelling pubmed-74042712020-08-18 Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension Plotka, Magdalena Szadkowska, Monika Håkansson, Maria Kovačič, Rebeka Al-Karadaghi, Salam Walse, Björn Werbowy, Olesia Kaczorowska, Anna-Karina Kaczorowski, Tadeusz Int J Mol Sci Article Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied earlier in our laboratory. Both these enzymes originate from Thermus scotoductus bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from Clostridium intestinale URNW was found to have the highest amino acid sequence similarity to the bacteriophage proteins and was chosen for further analysis. The recombinant enzyme showed strong activity against its host bacteria C. intestinale, as well as against C. sporogenes, Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus, on average causing a 5.12 ± 0.14 log reduction of viable S. aureus ATCC 25923 cells in a bactericidal assay. Crystallographic studies of the protein showed that the catalytic site of LysC contained a zinc atom coordinated by amino acid residues His(50), His(147), and Cys(155), a feature characteristic for type 2 amidases. Surprisingly, neither of these residues, nor any other of the four conserved residues in the vicinity of the active site, His(51), Thr(52), Tyr(76), and Thr(153), were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged N-terminal region of the enzyme. Potential antibacterial activity of this part of the sequence, predicted by the Antimicrobial Sequence Scanning System, AMPA, was confirmed in our experimental studies; the truncated version of LysC (LysCΔ2–23) completely lacked antibacterial activity. Moreover, a synthetic peptide, which we termed Intestinalin, with a sequence identical to the first thirty amino acids of LysC, displayed substantial anti-staphylococcal activity with IC(50) of 6 μg/mL (1.5 μM). This peptide was shown to have α-helical conformation in solution in the presence of detergents which is a common feature of amphipathic α-helical antimicrobial peptides. MDPI 2020-07-11 /pmc/articles/PMC7404271/ /pubmed/32664473 http://dx.doi.org/10.3390/ijms21144894 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Plotka, Magdalena
Szadkowska, Monika
Håkansson, Maria
Kovačič, Rebeka
Al-Karadaghi, Salam
Walse, Björn
Werbowy, Olesia
Kaczorowska, Anna-Karina
Kaczorowski, Tadeusz
Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension
title Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension
title_full Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension
title_fullStr Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension
title_full_unstemmed Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension
title_short Molecular Characterization of a Novel Lytic Enzyme LysC from Clostridium intestinale URNW and Its Antibacterial Activity Mediated by Positively Charged N-Terminal Extension
title_sort molecular characterization of a novel lytic enzyme lysc from clostridium intestinale urnw and its antibacterial activity mediated by positively charged n-terminal extension
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404271/
https://www.ncbi.nlm.nih.gov/pubmed/32664473
http://dx.doi.org/10.3390/ijms21144894
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