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From proteins to Bacteria: a multidisciplinary approach for the rational development of new anti-biofilm and antibacterial agents
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O aumento de estirpes resistentes a antibióticos, associado à capacidade das bactérias de forma-rem biofilmes, que frequentemente resultam em infeções crónicas e recorrentes, são dois pro-blemas urgentes que necessitam de ser abordados nos próximos anos. Existe uma necessidade premente de encontrar novos antibióticos e as primeiras terapêuticas direcionados para os biofi-lmes bacterianos. Os glicopolímeros da parede celular (GPPCs) são constituintes essenciais da parede celular em bactérias Gram-positivas e desempenham um papel fundamental na sobrevi-vência e/ou patogenicidade bacteriana. Ao privar as bactérias dos GPPCs, o seu crescimento e divisão podem ser dificultados, aumentando a sua suscetibilidade ao tratamento com antibióticos e facilitando o combate às infeções. A via de biossíntese de todos os GPPCs é muito complexa, mas o passo final, que consiste na transferência do precursor lipídico para o peptidoglicano, ocorre fora da célula e é mediado pela família de proteínas LytR-CpsA-Psr (LCP), tornando esta família um alvo para intervenção terapêutica.
Os principais objetivos desta tese foram a caracterização das duas proteínas LCP (LytR e Psr) presentes no patógeno bovino Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) e a procu-ra de potenciais inibidores desta família de proteínas que possam dificultar o desenvolvimento e a formação de biofilmes de SDSD.
Para responder a estas questões, foi utilizada uma abordagem multidisciplinar, integrando crista-lografia de raios-X e SAXS para a caracterização estrutural, métodos in silico como virtual scree-ning e simulações de dinâmica molecular para identificar e analisar potenciais inibidores, técni-cas biofísicas para avaliar a atividade das proteínas e ensaios biológicos para avaliar a inibição e erradicação de biofilmes. Esta abordagem multidisciplinar permitiu obter a estrutura cristalográ-fica da proteína LytR, validar experimentalmente o modelo AlphaFold da proteína Psr e identifi-car 10 moléculas promissoras que podem interagir com a proteína LytR. Ensaios de inibição e erradicação de biofilmes onde foram utilizadas estas moléculas mostraram resultados muito inte-ressantes, destacando o potencial destas moléculas em abordagens terapêuticas contra biofilmes. Além disso, esta tese demonstra pela primeira vez o potencial da utilização de ADP como subs-trato para monitorizar a atividade da família de proteínas LCP, utilizando o ensaio de malachite greee, uma abordagem colorimétrica, ou espectroscopia de RMN de ¹H. No geral, esta tese apre-senta os primeiros passos no desenvolvimento dos primeiros inibidores para a família de proteí-nas LCP e o potencial destas moléculas contra biofilmes.
The rise of antibiotic-resistant strains associated with the ability of bacteria to form biofilms, which lead many times to chronic and recurrent infections, are two very pressing issues that need to be addressed in the years to come. There is an urgent need to find new antibiotics and the first therapeutics to target bacterial biofilms. Cell wall glycopolymers (CWGPs) are essential constituents of the cell wall in Gram-positive bacteria and play a key role in bacterial survival and/or phatogenesis. By depleting bacteria of CWGPs, their growth and division can be ham-pered, increasing their susceptibility to antibiotic treatment and facilitating the fight against infec-tions. The biosynthesis pathway for all CWGPs is very complex, but the final step, which con-sists of the transference from the lipidic precursor into the peptidoglycan, takes place outside the cell and is mediated by the LytR-CpsA-Psr (LCP) family of proteins, making this family a target for therapeutic intervention. This thesis's main objectives were the characterization of the two LCP proteins (LytR and Psr) present in the bovine pathogen Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) and the search for putative inhibitors for this family of proteins that could hamper the development and biofilm formation of SDSD. To address these questions a multidisciplinary approach was employed, integrating X-ray crys-tallography and Small Angle X-ray Scattering (SAXS) for structural characterization, in silico methods such as virtual screening and molecular dynamics simulations to identify and analyze potential inhibitors, biophysical techniques to assess protein activity, and biological assays to evaluate biofilm inhibition and eradication. This multidisciplinary approach allowed to obtain the crystal structure for the LytR protein, to experimental validate the AlphaFold model of the Psr protein and to find 10 promising molecules that can putatively interact with the LytR protein. Biofilm inhibition and eradication assays using the same molecules showed very interesting re-sults, highlighting the potential of these molecules in therapeutic approaches against biofilms. Also, this thesis shows for the first time the potential of using ADP as a substrate to follow pro-tein activity for this family of proteins, using the malachite green assay, a colorimetric approach, or 1H NMR, a spectroscopic approach. Overall, this thesis shows the first steps in the develop-ment of the first inhibitors for the LCP family of proteins and the potential of these molecules against biofilms.
The rise of antibiotic-resistant strains associated with the ability of bacteria to form biofilms, which lead many times to chronic and recurrent infections, are two very pressing issues that need to be addressed in the years to come. There is an urgent need to find new antibiotics and the first therapeutics to target bacterial biofilms. Cell wall glycopolymers (CWGPs) are essential constituents of the cell wall in Gram-positive bacteria and play a key role in bacterial survival and/or phatogenesis. By depleting bacteria of CWGPs, their growth and division can be ham-pered, increasing their susceptibility to antibiotic treatment and facilitating the fight against infec-tions. The biosynthesis pathway for all CWGPs is very complex, but the final step, which con-sists of the transference from the lipidic precursor into the peptidoglycan, takes place outside the cell and is mediated by the LytR-CpsA-Psr (LCP) family of proteins, making this family a target for therapeutic intervention. This thesis's main objectives were the characterization of the two LCP proteins (LytR and Psr) present in the bovine pathogen Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) and the search for putative inhibitors for this family of proteins that could hamper the development and biofilm formation of SDSD. To address these questions a multidisciplinary approach was employed, integrating X-ray crys-tallography and Small Angle X-ray Scattering (SAXS) for structural characterization, in silico methods such as virtual screening and molecular dynamics simulations to identify and analyze potential inhibitors, biophysical techniques to assess protein activity, and biological assays to evaluate biofilm inhibition and eradication. This multidisciplinary approach allowed to obtain the crystal structure for the LytR protein, to experimental validate the AlphaFold model of the Psr protein and to find 10 promising molecules that can putatively interact with the LytR protein. Biofilm inhibition and eradication assays using the same molecules showed very interesting re-sults, highlighting the potential of these molecules in therapeutic approaches against biofilms. Also, this thesis shows for the first time the potential of using ADP as a substrate to follow pro-tein activity for this family of proteins, using the malachite green assay, a colorimetric approach, or 1H NMR, a spectroscopic approach. Overall, this thesis shows the first steps in the develop-ment of the first inhibitors for the LCP family of proteins and the potential of these molecules against biofilms.
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Palavras-chave
Antibiotic resistance Biofilms LytR-CpsA-Psr family of proteins Streptococcus dysgalactiae subsp. dysgalactiae X-ray Crystallography Virtual Screening