Publicação
Targeting Acinetobacter species with bacteriophage derived proteins
| Resumo: | Acinetobacter species have become a major cause of nosocomial infections worldwide due to its inherent and adaptive resistance towards the majority of antibiotics, survival in hostile environments and virulence traits. The increased mortality rates associated with these hard-to-treat bacteria urgently demands the development of new therapeutic options. In the present work, bacteriophage endolysins and depolymerases were explored to tackle Acinetobacter infections. Five novel enzymes were identified, heterologously expressed in Escherichia coli and characterized to gain insights into their enzymatic and structural features, as well tested in vitro (Acinetobacter cells and human cell line A549) and in vivo (Galleria mellonella) to assess their therapeutic potential. Remarkably, endolysins exhibited intrinsic ability to kill Acinetobacter cells, with optimal activity at pH 5-7 and low ionic strength (20 mM), reducing approximately 3.5 log to > 5 log (below the detection limit) within 2 h. Interestingly, no antibacterial activity of endolysins could be observed in biomatrices such as culture medium. All depolymerases exhibited activity on extracellular polysaccharides in all ranges of pH values (pH 5 to 9) and ionic strengths (0 to 500 mM) and retained at least 50% of the activity at 70 ºC. Consistently, circular dichroism analysis showed moderate and high thermostability of endolysins (rich in α-helices) and depolymerases (rich in β-sheets), with melting temperatures around 56 ºC and > 68 ºC, respectively. Combinations of endolysin and depolymerase reduced 24 h-old bacterial biofilms in approximately 1 log. Further work conducted on depolymerases demonstrated that these enzymes degrade the bacterial capsular polysaccharides (observations confirmed by Atomic Force Microscope). The removal of the capsule via depolymerase activity reduced the virulence of Acinetobacter to human epithelial cells for 2 h. Complementary studies performed in vivo showed that depolymerase was able to rescue Galleria mellonella larvae from Acinetobacter baumannii infection in a time- and dose-dependent manner. Maximal difference survival between larvae injected with depolymerase-treated bacteria and non-treated bacteria was observed after 72 h with 5 μM of depolymerase (88% vs 10%; P < 0.01). In summary, the enzymes studied in this thesis revealed to be effective candidates to control problematic Acinetobacter infections, either as bacteriolytic or anti-virulence agents. |
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| Autores principais: | Ferreira, Alice Maria Fernandes |
| Assunto: | Acinetobacter endolysin depolymerase antibacterial activity anti-virulence activity Acinetobacter endolisina depolimerase atividade antibacteriana atividade anti-virulência |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | português |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Acinetobacter species have become a major cause of nosocomial infections worldwide due to its inherent and adaptive resistance towards the majority of antibiotics, survival in hostile environments and virulence traits. The increased mortality rates associated with these hard-to-treat bacteria urgently demands the development of new therapeutic options. In the present work, bacteriophage endolysins and depolymerases were explored to tackle Acinetobacter infections. Five novel enzymes were identified, heterologously expressed in Escherichia coli and characterized to gain insights into their enzymatic and structural features, as well tested in vitro (Acinetobacter cells and human cell line A549) and in vivo (Galleria mellonella) to assess their therapeutic potential. Remarkably, endolysins exhibited intrinsic ability to kill Acinetobacter cells, with optimal activity at pH 5-7 and low ionic strength (20 mM), reducing approximately 3.5 log to > 5 log (below the detection limit) within 2 h. Interestingly, no antibacterial activity of endolysins could be observed in biomatrices such as culture medium. All depolymerases exhibited activity on extracellular polysaccharides in all ranges of pH values (pH 5 to 9) and ionic strengths (0 to 500 mM) and retained at least 50% of the activity at 70 ºC. Consistently, circular dichroism analysis showed moderate and high thermostability of endolysins (rich in α-helices) and depolymerases (rich in β-sheets), with melting temperatures around 56 ºC and > 68 ºC, respectively. Combinations of endolysin and depolymerase reduced 24 h-old bacterial biofilms in approximately 1 log. Further work conducted on depolymerases demonstrated that these enzymes degrade the bacterial capsular polysaccharides (observations confirmed by Atomic Force Microscope). The removal of the capsule via depolymerase activity reduced the virulence of Acinetobacter to human epithelial cells for 2 h. Complementary studies performed in vivo showed that depolymerase was able to rescue Galleria mellonella larvae from Acinetobacter baumannii infection in a time- and dose-dependent manner. Maximal difference survival between larvae injected with depolymerase-treated bacteria and non-treated bacteria was observed after 72 h with 5 μM of depolymerase (88% vs 10%; P < 0.01). In summary, the enzymes studied in this thesis revealed to be effective candidates to control problematic Acinetobacter infections, either as bacteriolytic or anti-virulence agents. |
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