Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy

• Author(s): Proença, Daniela Sofia Moreira, 1983-
• Date: 2015
• Persistent ID: http://hdl.handle.net/10451/16174
• Origin: Repositório da Universidade de Lisboa
• Subject(s): Teses de doutoramento - 2015
• Document Type: Doctoral thesis
• Language: English
• Advisor(s): São José, Carlos Jorge Sousa de, 1972-; Garcia, Miguel Angelo da Costa, 1968-
• Contributor(s): Repositório da Universidade de Lisboa

Description

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2015

Increasing 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.

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