Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-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...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-07-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/21/14/4894 |
| _version_ | 1797562733087948800 |
|---|---|
| author | Magdalena Plotka Monika Szadkowska Maria Håkansson Rebeka Kovačič Salam Al-Karadaghi Björn Walse Olesia Werbowy Anna-Karina Kaczorowska Tadeusz Kaczorowski |
| author_facet | Magdalena Plotka Monika Szadkowska Maria Håkansson Rebeka Kovačič Salam Al-Karadaghi Björn Walse Olesia Werbowy Anna-Karina Kaczorowska Tadeusz Kaczorowski |
| author_sort | Magdalena Plotka |
| collection | DOAJ |
| 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 <i>Thermus scotoductus</i> bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from <i>Clostridium intestinale</i> 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 <i>C. intestinale</i>, as well as against <i>C. sporogenes</i>, <i>Bacillus cereus</i>, <i>Micrococcus luteus,</i> and <i>Staphylococcus aureus</i>, on average causing a 5.12 ± 0.14 log reduction of viable <i>S. aureus</i> 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<sup>50</sup>, His<sup>147</sup>, and Cys<sup>155</sup>, 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<sup>51</sup>, Thr<sup>52</sup>, Tyr<sup>76</sup>, and Thr<sup>153</sup>, were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged <i>N</i>-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<sub>50</sub> 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. |
| first_indexed | 2024-03-10T18:33:08Z |
| format | Article |
| id | doaj.art-fe357e4b7c3644e09650276ce66e2860 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T18:33:08Z |
| publishDate | 2020-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-fe357e4b7c3644e09650276ce66e28602023-11-20T06:29:26ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-07-012114489410.3390/ijms21144894Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal ExtensionMagdalena Plotka0Monika Szadkowska1Maria Håkansson2Rebeka Kovačič3Salam Al-Karadaghi4Björn Walse5Olesia Werbowy6Anna-Karina Kaczorowska7Tadeusz Kaczorowski8Laboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, 80-822 Gdansk, PolandLaboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, 80-822 Gdansk, PolandSARomics Biostructures, SE-223 81 Lund, SwedenSARomics Biostructures, SE-223 81 Lund, SwedenSARomics Biostructures, SE-223 81 Lund, SwedenSARomics Biostructures, SE-223 81 Lund, SwedenLaboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, 80-822 Gdansk, PolandCollection of Plasmids and Microorganisms, Faculty of Biology, University of Gdansk, 80-308 Gdansk, PolandLaboratory of Extremophiles Biology, Department of Microbiology, Faculty of Biology, University of Gdansk, 80-822 Gdansk, PolandPeptidoglycan 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 <i>Thermus scotoductus</i> bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from <i>Clostridium intestinale</i> 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 <i>C. intestinale</i>, as well as against <i>C. sporogenes</i>, <i>Bacillus cereus</i>, <i>Micrococcus luteus,</i> and <i>Staphylococcus aureus</i>, on average causing a 5.12 ± 0.14 log reduction of viable <i>S. aureus</i> 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<sup>50</sup>, His<sup>147</sup>, and Cys<sup>155</sup>, 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<sup>51</sup>, Thr<sup>52</sup>, Tyr<sup>76</sup>, and Thr<sup>153</sup>, were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged <i>N</i>-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<sub>50</sub> 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.https://www.mdpi.com/1422-0067/21/14/4894bacteriophageendolysinautolysinantimicrobial peptidepeptidoglycan<i>Staphylococcus aureus</i> |
| spellingShingle | Magdalena Plotka Monika Szadkowska Maria Håkansson Rebeka Kovačič Salam Al-Karadaghi Björn Walse Olesia Werbowy Anna-Karina Kaczorowska Tadeusz Kaczorowski Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal Extension International Journal of Molecular Sciences bacteriophage endolysin autolysin antimicrobial peptide peptidoglycan <i>Staphylococcus aureus</i> |
| title | Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal Extension |
| title_full | Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal Extension |
| title_fullStr | Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal Extension |
| title_full_unstemmed | Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal Extension |
| title_short | Molecular Characterization of a Novel Lytic Enzyme LysC from <i>Clostridium intestinale</i> URNW and Its Antibacterial Activity Mediated by Positively Charged <i>N</i>-Terminal Extension |
| title_sort | molecular characterization of a novel lytic enzyme lysc from i clostridium intestinale i urnw and its antibacterial activity mediated by positively charged i n i terminal extension |
| topic | bacteriophage endolysin autolysin antimicrobial peptide peptidoglycan <i>Staphylococcus aureus</i> |
| url | https://www.mdpi.com/1422-0067/21/14/4894 |
| work_keys_str_mv | AT magdalenaplotka molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT monikaszadkowska molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT mariahakansson molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT rebekakovacic molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT salamalkaradaghi molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT bjornwalse molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT olesiawerbowy molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT annakarinakaczorowska molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension AT tadeuszkaczorowski molecularcharacterizationofanovellyticenzymelyscfromiclostridiumintestinaleiurnwanditsantibacterialactivitymediatedbypositivelychargediniterminalextension |