Publicação
Enhancing phage endolysins enzybiotic potential through their combined action with antimicrobial peptides
| dc.contributor.author | Gouveia, Ana Isabel Ricacho | |
| dc.contributor.institution | Faculty of Pharmacy | |
| dc.contributor.supervisor | José, Carlos Jorge Sousa de São | |
| dc.date.accessioned | 2026-02-19T17:35:05Z | |
| dc.date.available | 2026-02-19T17:35:05Z | |
| dc.date.issued | 2026-01-14 | |
| dc.description | Tese de doutoramento em Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2026 | |
| dc.description.abstract | Com o aumento do aparecimento de estirpes bacterianas resistentes a múltiplos antibióticos, há o perigo de haver um regresso a uma era pré-antibióticos se não forem tomadas medidas para mitigar o problema. A descoberta dos antibióticos foi um ponto de viragem na medicina, permitindo reduzir as taxas de mortalidade associada a infeções bacterianas, aumentando também a qualidade e a esperança de vida. No entanto, o aparecimento e desenvolvimento de bactérias resistentes a antibióticos põe em causa a eficácia dos antibióticos. A resistência a antibióticos é considerada pela Organização Mundial de Saúde (OMS) uma das maiores ameaças à saúde humana a uma escala mundial. Devido à redução da eficácia dos antibióticos no tratamento de infeções bacterianas, é necessário que se desenvolvam terapias alternativas à antibioterapia convencional. Neste sentido, nas últimas duas décadas, o potencial das enzimas líticas derivadas dos bacteriófagos como antibacterianos tem sido intensamente estudado e desenvolvido. As enzimas líticas derivadas dos bacteriófagos incluem as endolisinas (na sua forma nativa ou derivados sintéticos) e, ainda que menos exploradas, as lisinas associadas ao virião (VALs, de virion associated lysins), sendo os principais exemplos de uma vasta classe de antibacterianos à base de proteínas, conhecida como enzibióticos. As endolisinas são enzimas bacteriolíticas produzidas por bacteriófagos (ou, simplesmente, fagos), os vírus que infetam, especificamente, bactérias. As endolisinas são responsáveis pela degradação da parede celular das bactérias hospedeiras, após a replicação viral, permitindo, deste modo, a libertação da descendência viral. No contexto da infeção fágica, as endolisinas são produzidas no citoplasma e translocadas para o compartimento da parede celular através da membrana citoplasmática, atuando assim de dentro para fora da célula hospedeira. Contudo, as endolisinas também podem ter ação bacteriolítica quando aplicadas exogenamente, atuando de fora para dentro, desde que tenham acesso à parede celular. Devido a esta atividade exolítica, as endolisinas são consideradas enzibióticos, tendo grande potencial antibacteriano contra bactérias patogénicas. Os primeiros estudos que demonstraram o potencial antibacteriano das endolisinas focaramse principalmente em bactérias patogénicas Gram-positivas. Considera-se que as bactérias Grampositivas são alvos mais suscetíveis à aplicação exógena das endolisinas, uma vez que não possuem uma membrana externa, que reveste e protege a parede celular (característica das bactérias Gram-negativas). No entanto, têm de ser feitas algumas considerações importantes. A caracterização da atividade lítica das endolisinas é tipicamente realizada em soluções-tampão que não fornecem nutrientes às bactérias, mantendo-as num estado viável, mas não suportando o seu crescimento ativo (estado metabólico e fisiológico desfavorável à divisão celular). Na última década, vários estudos demonstraram que várias espécies de bactérias Gram-positivas, quando mantidas em meios nutritivos capazes de manter as bactérias num estado de crescimento ativo e com uma membrana polarizada, apresentam um certo grau de tolerância à ação lítica das endolisinas, quando estas atuam de fora para dentro. Ainda assim, os determinantes celulares e enzimáticos que regulam a ação lítica das endolisinas e a tolerância bacteriana a estas ainda são pouco conhecidos. No entanto, os dados disponíveis indicam que o potencial da membrana citoplasmática (ou força proto-motriz, PMF, de proton-motive force), e alguns polímeros secundários da parede celular são os principais factores que regulam a ação das autolisinas bacterianas (enzimas que são semelhantes quer estruturalmente quer funcionalmente às endolisinas) e que também estão envolvidos no fenómeno de tolerância bacteriana às endolisinas. | pt |
| dc.description.abstract | With an antimicrobial resistance crisis on the rise, alternatives to traditional antibiotics are needed. Over the past two decades, intensive research has been focused on the development of bacteriophage lytic enzymes as antibacterials. These include endolysins (in their native form or engineered derivatives) and the still underexplored virion associated lysins (VALs). Together, they represent the leading examples of a broader class of protein-based antibacterials known as enzybiotics. Endolysins are bacteriolytic enzymes produced by bacteriophages (or simply phages), the viruses that infect bacteria. Endolysins are responsible for host cell wall (CW) degradation after viral replication, a key event underlying the lysis of the infected cell for virion progeny release. In the phage infection context endolysins act from within, reaching the CW after being translocated across the cytoplasmic membrane (CM). Importantly, they can also exert they bacteriolytic action from outside the cell (from without), if access to the CW is granted. This is at the basis of the exploration of endolysins as enzybiotics. Initial research in this field has principally focused on endolysins targeting Gram-positive pathogens, as they were considered better targets for exogenously added endolysins due to the fact they lack an outer membrane (OM) shielding the CW. However, endolysin antimicrobial characterization assays are typically performed in nutrient-depleted, buffered solutions, in which cells are maintained in a “growth-arrested” state. More recently, several studies have revealed that different Gram-positive bacteria maintained in nutrient-rich media, supporting an energized CM and bacterial growth, can display a certain degree of tolerance to endolysin-mediated lysis from without. Yet, the cellular and enzymatic factors regulating endolysins’ lytic action and tolerance remain poorly understood. Evidence to date has pinpointed the bacterial membrane potential (or proton motive force, PMF) and certain CW secondary polymers of Gram-positive bacteria as key factors regulating bacterial autolysins and contributing to tolerance against endolysins. In the natural context of phage infection, endolysins activity is tightly coordinated with that of holins, which are small phage-encoded proteins that lead to CM permeabilization. Holins form “holes” in the CM and in most studied systems they provide the pathway for endolysin passage from the cytoplasm to the CW. Of note, these holes have also the transversal role of causing abrupt and extensive PMF collapse, with consequent cell death. Interestingly, PMF collapse appears to be an important determinant of bacterial susceptibility to endolysins. Indeed, studies have shown that the holin or ionophores mimicking its PMF-dissipation action can significantly enhance the lytic action of endolysins, regardless of whether they access the CW from within or without. Building on these observations, we proposed that antimicrobial peptides (AMPs) - another class of alternative antimicrobials that frequently act by disrupting the CM and abolishing the PMF – could be combined with endolysins, in order to enhance their antibacterial potential. Using as model the high-priority pathogen Staphylococcus aureus and anti-staphylococcal endolysins, we demonstrated that in nutrient rich media S. aureus cells become much more susceptible to the exolytic action of endolysins in presence of selected AMPs. The combined action yielded a stronger bacteriolytic and bactericidal effect than either agent alone, across a range of S. aureus strains, which included methicillin-resistant S. aureus (MRSA) from different clonal complexes. The results indicated that AMP-mediated PMF dissipation simultaneously stimulated endolysin binding to the cell surface and subsequent CW degradation. Using the same model, we next sought to understand in more detail the determinants and mechanisms governing bacterial tolerance to a specific endolysin, Lys11, and how they impacted the enzyme’s functional domains. The PMF across the CM has two components: the electrical potential (Δψ) and the proton gradient (ΔpH). In this study we have used selective membrane ionophores to abolish either or both components of the PMF and assess how they impacted Lys11 lytic action. We have shown that the pH and electrical gradients of the PMF affect distinctively the catalytic and binding domains of Lys11. The ΔpH component was preponderant in restraining the endolysin lytic action and its dissipation enhanced CW cleavage primarily via the enzyme’s peptidase domain. Interestingly, Δψ elimination boosted Lys11 binding to cells through the enzyme’s amidase domain. Thus, the two PMF components appear to regulate distinct functional domains of Lys11. In addition, the drug tunicamycin and a panel of S. aureus mutants allowed us to study the role of the major S. aureus CW polymers, and their modifications, in tolerance. The negatively charged wall teichoic acids were confirmed as the key CW polymers contributing to tolerance by severely impairing Lys11 association to the CW through its canonical cell binding domain and by restraining the enzyme’s CW cleavage activity, thereby restraining endolysin action by a dual mechanism. In conclusion, this work has contributed to deepening our understanding of how bacterial physiology and CW composition affect the susceptibility of Gram-positive bacteria to the exolytic action of endolysins, providing new insights into the mechanisms of bacterial tolerance. The results open new venues for strategies aiming at increasing the enzybiotic potential of endolysins. | en |
| dc.format | application/pdf | |
| dc.identifier.tid | 101741650 | |
| dc.identifier.uri | http://hdl.handle.net/10400.5/117218 | |
| dc.language.iso | eng | |
| dc.subject | Antibiotic resistance | |
| dc.subject | endolysins | |
| dc.subject | antimicrobial peptides | |
| dc.subject | cell wall | |
| dc.subject | membrane potential | |
| dc.subject | Resistência a antibióticos | |
| dc.subject | endolisinas | |
| dc.subject | péptidos antimicrobianos | |
| dc.subject | parede celular | |
| dc.subject | potencial de membrana | |
| dc.title | Enhancing phage endolysins enzybiotic potential through their combined action with antimicrobial peptides | en |
| dc.type | doctoral thesis | |
| dspace.entity.type | Publication | |
| rcaap.rights | openAccess |
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