Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2016 |
| Idioma: | eng |
| Título da fonte: | Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) |
| Texto Completo: | http://hdl.handle.net/10451/26318 |
Resumo: | Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2016 |
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Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activityTeses de doutoramento - 2016Domínio/Área Científica::Ciências Médicas::Medicina BásicaTese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2016Bacteriophages, or phages, are viruses that strictly infect bacteria. Double-stranded DNA phages use the holin-endolysin system to lyse host cells, thus ensuring the release of the viral progeny and the realization of new infection cycles. The endolysin is an enzyme that degrades the peptidoglycan (PG), the main constituent of the bacteria cell wall (CW). The holin is a transmembrane protein that forms pores in the cytoplasmic membrane (CM), leading to dissipation of the proton motive force (pmf) and consequently to the cell death. The pmf is created by the electrochemical gradient across the CM and is responsible for driving many energy-requiring functions in the cell. According to the way how endolysins reach the CW, these can be classified into two types: i) canonical endolysins (c-endolysins), when access occurs through the holin pores, or ii) exported endolysins (e-endolysins), when transport is performed in a holin-independent pathway. It is considered that once synthesized c-endolysins immediately acquire their active conformation in the cytoplasm, thus having the capacity to effectively degrade the PG if the contact with the CW is allowed. Although exported to the CW by the host cell machinery, all e-endolysins described so far need to be activated by mechanisms that depend on the holin action. Due to their lytic activity, c-endolysins have gained great attention as potential antimicrobial agents for the elimination of pathogenic Gram-positive bacteria, especially in the actual context of increasing resistance to antibiotics. This approach relies on the observation that, at least under certain conditions, c-endolysins are able to efficiently lyse target bacteria when the enzymes are exogenously added in the form of recombinant proteins (enzybiotics). The main objective of the work here presented was to contribute with knowledge for increasing the potential of endolysins as antibacterial agents, namely by developing strategies to improve their production, solubility and lytic performance, and by deepening our understanding of factors and mechanisms that influence their enzymatic activity. This work began with the construction of chimerical endolysins with lytic activity against Staphylococcus aureus (Chapter 2). In addition to obtain enzymes displaying a broad lytic spectrum on clinical strains of this species, we also intended to overcome the problem of low solubility that is commonly observed when overproducing endolysins of S. aureus phages in Escherichia coli. We produced and purified two chimerical proteins (Lys168-87 and Lys170-87) by fusing the same cell wall binding domain (CWBD) of endolysin Lys87, produced by S. aureus phage F87s/06, to the catalytic domain (CD) of endolysins Lys168 or Lys170, produced by Enterococcus faecalis phages F168/08 and F170/08, respectively. This fusion between the CD of highly soluble endolysins and the CWBD of an endolysin with poor solubility, combined with optimized expression conditions, allowed the efficient production of chimerical enzymes in the soluble form. The lytic activity of the chimeras was initially assessed qualitatively by the "spot assay", using bacterial isolates from Portuguese community and hospital settings (n = 100). The endolysins showed high lytic potential, lysing more than 90% of the tested S. aureus isolates, including a high fraction that was methicillin-resistant (MRSA, n = 42). This lytic capacity was also observed in a collection of genetically characterized and typed S. aureus strains, which included representatives of the most relevant MRSA pandemic clones from different parts of the world (n = 30), and representative clones of the dominant methicillin-sensitive S. aureus (MSSA; n = 13). In semi-quantitative assays (lysis curves), Lys168-87 and Lys170-87 were effective in eliminating MRSA strain USA200 suspended in a physiological buffer, being observed a synergistic effect when the chimeras were simultaneously used. Interestingly, unlike the parental endolysins, the chimeras showed a wide lytic spectrum, being also active against Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, E. faecalis, Enterococcus faecium and Streptococcus pyogenes. Globally, Lys168-87 presented superior lytic performance than Lys170-87, being this result only inverted when the chimeras were tested on the enterococcal isolates. The results of our research and from many other laboratories support the lytic capacity of c-endolysins and chimeric derivatives when tested in vitro, particularly when the enzymes are added to cells previously suspended in physiological buffers. Nevertheless, we have found that lytic efficacy is often lost or greatly lessened when target bacteria are kept in nutritious media, i.e., in conditions that guarantee maintenance of the pmf and active cell growth. Thus, in a second part of this work we aimed at uncovering factors and mechanisms that underlie this phenomenon. Accordingly, we were led by the fact that in the phage infection context both c- and e-endolysins always act after cells have been killed by the holin. Based on this observation, we decided to study how maintenance or dissipation of the pmf could influence the activity of c-endolysins, either when they reach the CW from the cell inside or when applied externally. The c-endolysin of Bacillus subtilis phage SPP1 (LysSPP1) was chosen as study model, being in a first step transformed into an artificial e-endolysin. For this, the signal sequence (SP) of bacillopeptidase F of B. subtilis (Bpr protein) was fused to the N-terminal end of LysSPP1. The recombinant gene of the artificial e-endolysin SP-LysSPP1 was cloned in a B. subtilis replicative plasmid under the control of an inducible promoter. Surprisingly, the continued production and export of SP-LysSPP1 to the CW, through the secretion system (Sec system) of B. subtilis, produced no obvious effects on the viability and cell growth when compared with a strain that produced the native and not exported LysSPP1. The extracytoplasmic localization and lytic character of the mature form of SP-LysSPP1 was evidenced when cultures were treated with a pmf-dissipating ionophore, gramicidin D. As expected, the addition of the ionophore resulted in immediate growth interruption. However, only the culture producing SP-LysSPP1 showed quick cell lysis after addition of gramicidin D. Similarly, the prior sensitization of B. subtilis with gramicidin D or the maintenance of cells in a buffered medium without energy sources significantly enhanced the lytic activity of the endolysin when added exogenously. It was estimated that the amount of LysSPP1 necessary to lyse cells previously killed by the SPP1 holin action, or by a pmf-dissipating agent, was about 60 times lower than the amount needed to lyse exponentially growing cells. The results followed the same trend when the c-endolysin Lys11, of S. aureus phage 11, was tested in conditions promoting or decreasing the pmf/cell growth. Therefore, the results demonstrate that dissipation of the pmf, which in the context of phage infection occurs by the holin action, can have an activating or potentiating effect on the lytic action of c-endolysins, similarly to what was previously described for e-endolysins. Interestingly, this feature has also been observed with other PG hydrolases, such as autolysins, whose regulation is dependent on the maintenance of the pmf. These results may have implications on the selection and design of endolysins intended for enzybiotic therapy (see Chapter 3). Finally, in a third part of this work we aimed to identify and characterize the holin function of phage SPP1, a necessary condition to perform some of the studies presented in Chapter 3. It was previously proposed that the holin of SPP1 would be encoded by orf 26. Its deduced product (gp26) shares homology and hydrophobic characteristics with the XhlB protein, which was implicated in the holin function of the cryptic phage PBSX of B. subtilis. However, our analysis revealed the upstream orf 24.1 that encodes a holin-like protein analogous to XhlA, also involved in PBSX-mediated lysis. Therefore, we decided to study the role of gp24.1 and gp26 as possible SPP1 holins. Because of its potential toxicity, holins of Gram-positive systems are often studied in heterologous systems, typically E. coli. However, despite some advantages, it is sometimes difficult to extrapolate the results obtained to the native systems. Thus, to understand the contribution of gp24.1 and gp26 to the SPP1holin function, the corresponding orfs were cloned separately and as a transcriptional fusion in a B. subtilis replicative plasmid, under the control of an inducible promoter. The results showed that in the assay conditions the individual production of these proteins did not produce significant impact on B. subtilis cell growth, despite their insertion and accumulation in the CM. Growth cessation and cell death typical of the holin action were only observed after co-production of gp24.1 and gp26, suggesting that in SPP1 the holin function may involve the production of these two proteins. Surprisingly, a constitutive promoter was identified within orf 24.1, which we believe should correspond to the previously described early promoter PE5. The presence of this promoter raises questions regarding lysis regulation in phage SPP1 (see Chapter 4).São José, Carlos Jorge Sousa de, 1972-Repositório da Universidade de LisboaFernandes, Sofia2017-01-30T13:23:29Z201620162016-01-01T00:00:00Zdoctoral thesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10451/26318TID:101405685enginfo: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:RCAAP2025-03-17T13:35:27Zoai:repositorio.ulisboa.pt:10451/26318Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T02:48:40.154026Repositórios 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 |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| title |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| spellingShingle |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity Fernandes, Sofia Teses de doutoramento - 2016 Domínio/Área Científica::Ciências Médicas::Medicina Básica |
| title_short |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| title_full |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| title_fullStr |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| title_full_unstemmed |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| title_sort |
Endolysins as antibacterial agents : from engineering approaches to the uncovering of holin as a key factor influencing lytic activity |
| author |
Fernandes, Sofia |
| author_facet |
Fernandes, Sofia |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
São José, Carlos Jorge Sousa de, 1972- Repositório da Universidade de Lisboa |
| dc.contributor.author.fl_str_mv |
Fernandes, Sofia |
| dc.subject.por.fl_str_mv |
Teses de doutoramento - 2016 Domínio/Área Científica::Ciências Médicas::Medicina Básica |
| topic |
Teses de doutoramento - 2016 Domínio/Área Científica::Ciências Médicas::Medicina Básica |
| description |
Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2016 |
| publishDate |
2016 |
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2016 2016 2016-01-01T00:00:00Z 2017-01-30T13:23:29Z |
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doctoral thesis |
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info:eu-repo/semantics/publishedVersion |
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publishedVersion |
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http://hdl.handle.net/10451/26318 TID:101405685 |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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