Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains
Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for ac...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-03-01
|
| Series: | Antibiotics |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6382/12/4/667 |
| _version_ | 1797606717334224896 |
|---|---|
| author | Jin-Mi Park Dae-Sung Ko Hee-Soo Kim Nam-Hyung Kim Eun-Kyoung Kim Young-Hye Roh Danil Kim Jae-Hong Kim Kang-Seuk Choi Hyuk-Joon Kwon |
| author_facet | Jin-Mi Park Dae-Sung Ko Hee-Soo Kim Nam-Hyung Kim Eun-Kyoung Kim Young-Hye Roh Danil Kim Jae-Hong Kim Kang-Seuk Choi Hyuk-Joon Kwon |
| author_sort | Jin-Mi Park |
| collection | DOAJ |
| description | Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via <i>E. coli</i> expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in <i>E. coli</i> showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the <i>S. aureus</i> strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis. |
| first_indexed | 2024-03-11T05:19:02Z |
| format | Article |
| id | doaj.art-824c7a63afa5478dabf454ae13c674fe |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-11T05:19:02Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-824c7a63afa5478dabf454ae13c674fe2023-11-17T18:02:15ZengMDPI AGAntibiotics2079-63822023-03-0112466710.3390/antibiotics12040667Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> StrainsJin-Mi Park0Dae-Sung Ko1Hee-Soo Kim2Nam-Hyung Kim3Eun-Kyoung Kim4Young-Hye Roh5Danil Kim6Jae-Hong Kim7Kang-Seuk Choi8Hyuk-Joon Kwon9Laboratory of Poultry Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of KoreaLaboratory of Poultry Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of KoreaLaboratory of Poultry Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of KoreaLaboratory of Poultry Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of KoreaDepartment of Farm Animal Medicine, College of Veterinary Medicine, Seoul National University, Pyeongchang-gun 25354, Republic of KoreaDepartment of Farm Animal Medicine, College of Veterinary Medicine, Seoul National University, Pyeongchang-gun 25354, Republic of KoreaDepartment of Farm Animal Medicine, College of Veterinary Medicine, Seoul National University, Pyeongchang-gun 25354, Republic of KoreaLaboratory of Poultry Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of KoreaResearch Institute for Veterinary Science, College of Veterinary Medicine, BK21 for Veterinary Science, Seoul 08826, Republic of KoreaLaboratory of Poultry Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of KoreaChimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via <i>E. coli</i> expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in <i>E. coli</i> showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the <i>S. aureus</i> strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.https://www.mdpi.com/2079-6382/12/4/667<i>Staphylococcus aureus</i>chimeric lysinscreening test methodantibacterial activityautolysincell-free expression system |
| spellingShingle | Jin-Mi Park Dae-Sung Ko Hee-Soo Kim Nam-Hyung Kim Eun-Kyoung Kim Young-Hye Roh Danil Kim Jae-Hong Kim Kang-Seuk Choi Hyuk-Joon Kwon Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains Antibiotics <i>Staphylococcus aureus</i> chimeric lysin screening test method antibacterial activity autolysin cell-free expression system |
| title | Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains |
| title_full | Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains |
| title_fullStr | Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains |
| title_full_unstemmed | Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains |
| title_short | Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against <i>Staphylococcus aureus</i> Strains |
| title_sort | rapid screening and comparison of chimeric lysins for antibacterial activity against i staphylococcus aureus i strains |
| topic | <i>Staphylococcus aureus</i> chimeric lysin screening test method antibacterial activity autolysin cell-free expression system |
| url | https://www.mdpi.com/2079-6382/12/4/667 |
| work_keys_str_mv | AT jinmipark rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT daesungko rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT heesookim rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT namhyungkim rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT eunkyoungkim rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT younghyeroh rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT danilkim rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT jaehongkim rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT kangseukchoi rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains AT hyukjoonkwon rapidscreeningandcomparisonofchimericlysinsforantibacterialactivityagainstistaphylococcusaureusistrains |