Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB
LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting <i>Pseudomonas aeruginosa</i>. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immu...
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MDPI AG
2021-06-01
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| Online Access: | https://www.mdpi.com/2076-0817/10/6/765 |
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| author | Sirijan Santajit Thida Kong-ngoen Manas Chongsa-Nguan Usa Boonyuen Pornpan Pumirat Nitat Sookrung Wanpen Chaicumpa Nitaya Indrawattana |
| author_facet | Sirijan Santajit Thida Kong-ngoen Manas Chongsa-Nguan Usa Boonyuen Pornpan Pumirat Nitat Sookrung Wanpen Chaicumpa Nitaya Indrawattana |
| author_sort | Sirijan Santajit |
| collection | DOAJ |
| description | LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting <i>Pseudomonas aeruginosa</i>. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the <i>P. aeruginosa.</i> This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs (<i>huscfvs</i>) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the <i>huscfv</i>-plasmid transformed <i>Escherichia coli</i>. HuscFvs of two transformed <i>E. coli</i> clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities <i>in vitro</i>. Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by <i>P. aeruginosa</i>. |
| first_indexed | 2024-03-10T10:19:13Z |
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| language | English |
| last_indexed | 2024-03-10T10:19:13Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
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| series | Pathogens |
| spelling | doaj.art-2010560fcf8a4bbca2e2afa2f5d897a52023-11-22T00:32:14ZengMDPI AGPathogens2076-08172021-06-0110676510.3390/pathogens10060765Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasBSirijan Santajit0Thida Kong-ngoen1Manas Chongsa-Nguan2Usa Boonyuen3Pornpan Pumirat4Nitat Sookrung5Wanpen Chaicumpa6Nitaya Indrawattana7Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, ThailandDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, ThailandFaculty of Public Health and Environment, Pathumthani University, Pathum Thani 12000, ThailandDepartment of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, ThailandDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, ThailandCenter of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandCenter of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, ThailandLasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting <i>Pseudomonas aeruginosa</i>. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the <i>P. aeruginosa.</i> This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs (<i>huscfvs</i>) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the <i>huscfv</i>-plasmid transformed <i>Escherichia coli</i>. HuscFvs of two transformed <i>E. coli</i> clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities <i>in vitro</i>. Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by <i>P. aeruginosa</i>.https://www.mdpi.com/2076-0817/10/6/765elastaseelastolytic activityhuman single-chain antibodyHuscFv<i>Pseudomonas aeruginosa</i>LasB |
| spellingShingle | Sirijan Santajit Thida Kong-ngoen Manas Chongsa-Nguan Usa Boonyuen Pornpan Pumirat Nitat Sookrung Wanpen Chaicumpa Nitaya Indrawattana Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB Pathogens elastase elastolytic activity human single-chain antibody HuscFv <i>Pseudomonas aeruginosa</i> LasB |
| title | Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB |
| title_full | Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB |
| title_fullStr | Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB |
| title_full_unstemmed | Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB |
| title_short | Human Single-Chain Antibodies That Neutralize Elastolytic Activity of <i>Pseudomonas aeruginosa</i> LasB |
| title_sort | human single chain antibodies that neutralize elastolytic activity of i pseudomonas aeruginosa i lasb |
| topic | elastase elastolytic activity human single-chain antibody HuscFv <i>Pseudomonas aeruginosa</i> LasB |
| url | https://www.mdpi.com/2076-0817/10/6/765 |
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