Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy

Bibliographic Details
Main Author: Proença, Daniela Sofia Moreira, 1983-
Publication Date: 2014
Language: eng
Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
Download full: http://hdl.handle.net/10451/16174
Summary: Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2015
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spelling Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacyTeses de doutoramento - 2015Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2015Increasing antibiotic resistance among bacterial pathogens has been promoting the studyof bacteriophage (phage) lytic enzymes (bacterial cell wall hydrolases) asalternatives/complements to antibiotics. Phages can employ two types of these enzymesduring their life cycle: i) virion-associated lysins (VALs), which promote a local cleavageof cell wall bonds to facilitate phage genome entry into the host cell; and ii) endolysinsthat destroy the wall at the end of infection, leading to cell burst and release of virionprogeny. We studied the lytic activity of two enterococcal endolysins, Lys168 andLys170, towards clinical isolates of different Gram-positive bacterial pathogens. In theconditions tested, both enzymes showed broad antimicrobial activity against E. faecalis,including vancomycin-resistant strains, and to less extent against E. faecium.We show that lys170 expression results in the production of the expected full lengthpolypeptide (Lys170FL, 32.6 kDa) and of a C-terminal fragment of the enzyme(CWB170, 12 kDa), with both proteins co-eluting in the purification steps. Furtheranalysis revealed that CWB170 corresponded to the Lys170 cell wall binding domain,which is independently produced from an in-frame, secondary translational start site.Biochemical and biophysical analysis indicated that the fully active Lys170 is a complexmost likely corresponding to one subunit of Lys170FL associated to three of CWB170.Study of Lys170 has thus uncovered a new strategy of increasing the number of CWBdomains in this type of enzymes.A frequently reported problem when working with phage lytic enzymes is theirpropensity to become insoluble. Further, the activity of endolysins is rarely studied inconditions that promote robust growth of target bacteria. With the goal of supplantingthese limitations we engineered a chimerical lysin, EC300, aimed at lysing E. faecalisgrowing in rich culture media. EC300 resulted from the fusion of a M23 endopeptidasedomain of a VAL to the CWB170 domain of Lys170. The bacteriolysin-like proteinexhibited a clear enhanced lytic activity when compared to the parental endolysin,particularly when assayed in a rich culture medium, thus having the potential to be usedas an anti-E. faecalis therapy.Fundação para a Ciência e a Tecnologia (FCT)São José, Carlos Jorge Sousa de, 1972-Garcia, Miguel Angelo da Costa, 1968-Repositório da Universidade de LisboaProença, Daniela Sofia Moreira, 1983-2015-03-03T11:57:07Z201520142015-01-01T00:00:00Zdoctoral thesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10451/16174TID:101375689enginfo:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2025-03-17T13:16:56Zoai:repositorio.ulisboa.pt:10451/16174Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T02:39:44.297629Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse
dc.title.none.fl_str_mv Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
title Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
spellingShingle Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
Proença, Daniela Sofia Moreira, 1983-
Teses de doutoramento - 2015
title_short Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
title_full Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
title_fullStr Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
title_full_unstemmed Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
title_sort Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy
author Proença, Daniela Sofia Moreira, 1983-
author_facet Proença, Daniela Sofia Moreira, 1983-
author_role author
dc.contributor.none.fl_str_mv São José, Carlos Jorge Sousa de, 1972-
Garcia, Miguel Angelo da Costa, 1968-
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Proença, Daniela Sofia Moreira, 1983-
dc.subject.por.fl_str_mv Teses de doutoramento - 2015
topic Teses de doutoramento - 2015
description Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2015
publishDate 2014
dc.date.none.fl_str_mv 2014
2015-03-03T11:57:07Z
2015
2015-01-01T00:00:00Z
dc.type.driver.fl_str_mv doctoral thesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/16174
TID:101375689
url http://hdl.handle.net/10451/16174
identifier_str_mv TID:101375689
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron:RCAAP
instname_str FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron_str RCAAP
institution RCAAP
reponame_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
collection Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository.name.fl_str_mv Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
repository.mail.fl_str_mv info@rcaap.pt
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