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Enzibióticos contra Pseudomonas aeruginosa resistentes a antibióticos
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Resumo(s)
Enzimas sintetizadas pelos fagos, como as endolisinas responsáveis por degradarem a camada de peptidoglicano (PG) das bactérias, estão a ser estudadas como uma terapia alternativa devido ao crescente número de bactéria multirresistente a antibióticos. Neste trabalho, de um conjunto de 16 genomas de P. aeruginosa pertencentes à Faculdade de Farmácia da Universidade de Lisboa (FFUL), foram identificados 24 profagos diferentes, aos quais se somaram outros 7 de um trabalho realizado por Mageeney e equipa (2020) e mais 72 identificados em estirpes de P. aeruginosa presentes na base de dados PathoSystems Resource Integration Center (PATRIC). Nestes genomas fágicos foram identificadas um total de 25 endolisinas putativas, selecionando-se 5 (7; 13; 15; 119 e 542) mediante a sua percentagem de identidade (identidade inferior 20%) e da família identificada. Foi então induzida a sua expressão, não se conseguindo apenas induzir a expressão da proteína 542. A atividade bacteriolítica das restantes 4 proteínas foi avaliada contra a bactéria Gram-positiva Micrococcus luteus, com todas a exibirem resultados positivos. Posteriormente, foi realizado um zimograma, onde se usou como substrato células autoclavadas de P. aeruginosa com todas as endolisinas putativas a hidrolisar o PG da bactéria alvo. Avaliada a ação sinérgica das endolisinas mais promissoras, a 7 e 119, com permeabilizadores membranares contra P. aeruginosa, ambas levaram à morte bacteriana em sinergia com o ácido etilenodiamino tetra-acético (EDTA), o que não se observou para o ácido cítrico. Quando encapsuladas em lipossomas observou-se a morte da bactéria P. aeruginosa ao fim de 5 dias para a composição lipídica DOPE: DMPC: CHEMS (rácio 4:4:2). Relativamente aos profagos, apresentaram uma elevada variabilidade entre si, com a maioria a pertencer à família Siphoviridae. Foram ainda identificados outros genes de interesse e determinados os locais de inserção, observando-se que os genes de tRNA são os locais de inserção preferências dos profagos.
The global increase in multidrug-resistant (MDR) bacteria has led to phage therapy being refocused upon. Applying bioinformatics tools in 16 newly sequenced genomes of P. aeruginosa were identified 24 prophages to which add up more 7 of another project accomplished by Mageeney et al. (2020). Then 6 phylogenetic distant prophages (identity < 50%) were selected to identify homologues prophages in P. aeruginosa strains deposited in the database PathoSystems Resource Integration Center (PATRIC) with defined criteria. In total were identified 61 prophages homologues to the 6 of reference prophages and 11 different from the prophages already in study in 50 strains of P. aeruginosa. We have identified 25 putative endolysins and based on sequence and structure distinctiveness we selected 5 (7; 13; 15; 119 and 542) to study their activity. We try to induce their expression in Escherichia coli BL21(DE3) but just 4 of them were expressed. These 4 putative endolysins showed in vitro activity against the Gram positive bacteria Micrococcus luteus. Zymogram analysis using autoclaved cells of P. aeruginosa showed that these 4 putative endolysins exhibit activity against P. aeruginosa peptidoglycan (PG). Then the activity of 2 of the most promising putative endolysins was analysed together with outer membrane permeabilizers: ethylenediaminetetra-acetic acid (EDTA) and citric acid. Only EDTA allowed permeabilizing the P. aeruginosa outer membrane, leading to the observation of cell lysis after endolysin application. Then these 2 putative endolysins were encapsulated in liposomes, with the composition DOPE: DMPC: CHEMS (ratio 4:4:2) and was observed the cell death after 5 days. The studied prophages genomes reveal to have small intergenic regions and exhibit high variability. Besides endolysins, other genes with therapeutic and virulence interests were identified. The insertion sites were also analysed, and it was possible to conclude that tRNA genes are the most common.
The global increase in multidrug-resistant (MDR) bacteria has led to phage therapy being refocused upon. Applying bioinformatics tools in 16 newly sequenced genomes of P. aeruginosa were identified 24 prophages to which add up more 7 of another project accomplished by Mageeney et al. (2020). Then 6 phylogenetic distant prophages (identity < 50%) were selected to identify homologues prophages in P. aeruginosa strains deposited in the database PathoSystems Resource Integration Center (PATRIC) with defined criteria. In total were identified 61 prophages homologues to the 6 of reference prophages and 11 different from the prophages already in study in 50 strains of P. aeruginosa. We have identified 25 putative endolysins and based on sequence and structure distinctiveness we selected 5 (7; 13; 15; 119 and 542) to study their activity. We try to induce their expression in Escherichia coli BL21(DE3) but just 4 of them were expressed. These 4 putative endolysins showed in vitro activity against the Gram positive bacteria Micrococcus luteus. Zymogram analysis using autoclaved cells of P. aeruginosa showed that these 4 putative endolysins exhibit activity against P. aeruginosa peptidoglycan (PG). Then the activity of 2 of the most promising putative endolysins was analysed together with outer membrane permeabilizers: ethylenediaminetetra-acetic acid (EDTA) and citric acid. Only EDTA allowed permeabilizing the P. aeruginosa outer membrane, leading to the observation of cell lysis after endolysin application. Then these 2 putative endolysins were encapsulated in liposomes, with the composition DOPE: DMPC: CHEMS (ratio 4:4:2) and was observed the cell death after 5 days. The studied prophages genomes reveal to have small intergenic regions and exhibit high variability. Besides endolysins, other genes with therapeutic and virulence interests were identified. The insertion sites were also analysed, and it was possible to conclude that tRNA genes are the most common.
Descrição
Tese de Mestrado, Biologia Humana e Ambiente, 2021, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Pseudomonas aeruginosa Profagos Endolisinas Resistência antibióticos Lipossomas Teses de mestrado - 2021