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A new target inside an old molecule: Glutamate amidation of peptidoglycan
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Staphylococcus aureus é uma bactéria responsável por várias infeções humanas devido à sua capacidade de expressar vários fatores de virulência. Este organismo torna-se ainda mais perigoso devido à sua capacidade de adquirir resistência a todos os antibióticos utilizados na prática clínica. O complexo MurT-GatD é responsável pela amidação do segundo aminoácido do pentapéptido, onde D-iso-glutamato é convertido em D-iso-glutamina. A inibição desta modificação afeta vários processos fisiológicos que são importantes para a sua sobrevivência, como o crescimento celular, resistência a antibióticos β-lactâmicos e à lisozima, tornando este complexo um potencial alvo para o desenvolvimento de uma molécula com atividade antimicrobiana. No entanto, o mecanismo através do qual as proteínas MurT-GatD interagem e como realizam a reação catalítica, bem como o impacto da amidação do peptidoglicano na interação com o hospedeiro são questões que ainda faltam responder. Esta tese teve como objetivo descrever como se processa a interação física entre MurT e GatD e compreender o potencial de virulência da amidação do peptidoglicano. Através de várias técnicas genéticas e bioquímicas concluímos que o domínio C-terminal do MurT (DUF1727) é o domínio de ligação entre o MurT e GatD. Além disso, identificámos quatro aminoácidos do DUF1727 importantes para a formação do complexo (W352, Y354, L381 e R375), dois importantes para a estabilidade do MurT (D346 e D349) e três importantes para a atividade de MurT-GatD (D355, Y418 e L313). Esta informação contribuirá para o futuro desenvolvimento de moléculas que impeçam a interação física entre MurT e GatD e consequentemente possibilite a eliminação de S. aureus. Em relação ao impacto da amidação do peptidoglicano, em contexto de virulência, procedeu-se á infeção de macrófagos com vários mutantes murT-gatD construídos em diferentes estirpes de S. aureus. Após inibição dos genes murT-gatD observaram-se diferentes fenótipos consoante a origem genética das estirpes. Demonstrou-se que estes genes são importantes para a evasão de macrófagos nas estirpes COL e MW2 e para a sobrevivência dentro dos macrófagos para a estirpe COL, contudo não para a estirpe HDE288. Para correlacionar estes resultados com o impacto específico da amidação do peptidoglicano nas várias origens genéticas de S. aureus estudadas, o impacto da agregação celular, formação do biofilme e resistência à lisozima foram analisados. Para além disso, o efeito de condições específicas relacionadas com o hospedeiro como diferentes valores de pH, níveis de oxigénio e azoto na amidação do peptidoglicano foram também analisadas. Em relação à expressão do murT-gatD, a atividade do promotor do operão foi analisado. Observou-se que a ausência do substrato de GatD, pH externo ácido e a presença de cobre resultam em diferentes alterações no padrão de expressão deste operão. Determinar como a amidação do peptidoglicano afeta a interação de S. aureus com as células do hospedeiro e como pode contribuir para a virulência desta bactéria será fundamental para validar o complexo MurT-GatD como um alvo para o desenvolvimento de um novo antibiótico.
Staphylococcus aureus causes multiple human infections due to its capacity to express a wide range of virulence factors. S. aureus became an even more dangerous pathogen due to its capacity to acquire resistance to all the antibiotics clinically available. The MurT-GatD complex is responsible for a secondary modification of the peptidoglycan, the amidation of the second amino acid of the stem peptide, D-iso-glutamate, into D-iso-glutamine. Inhibition of peptidoglycan amidation was described to impair physiological processes important for bacterial survival, such as cell growth, β-lactam and lysozyme resistance, making this complex an attractive target for the design of new molecules with antibacterial activity. However, how MurT and GatD proteins physically interact and perform catalysis and how this surface modification interacts with the host are unanswered questions. The aim of this Thesis was to describe the physical link between MurT and GatD and on the other hand, to understand the virulence potential of peptidoglycan amidation. Through several genetic and biochemical techniques, we concluded that the MurT C-terminal domain (DUF1727) is the physical linker between MurT and GatD. Furthermore, eleven conserved amino acids were identified and substituted to alanine, through site-directed mutagenesis; four were found to be involved in complex formation (W352, Y354, L381 and R375), two important for MurT stability (D346 and D349) and three important for MurT-GatD activity (D355, Y418 and L313). By providing this information we paved the way for the future development of molecules that hinder the physical interaction between MurT and GatD and consequently kill S. aureus. Regarding the impact of peptidoglycan amidation in virulence, macrophages were infected with several murT-gatD mutants in different S. aureus genetic backgrounds. Upon impairment of murT-gatD genes we observed different behaviors according to the S. aureus background. We showed that these genes are important to avoid macrophage engulfment in strains COL and MW2 and for survival inside macrophages in COL, but not in strain HDE288. To correlate these results with the specific impact of peptidoglycan amidation in the S. aureus backgrounds studied, we determined its involvement in cell aggregation, biofilm formation and lysozyme resistance. Furthermore, the effect of specific host-related conditions, such as different pH, oxygen levels and nitrogen sources, on peptidoglycan amidation, was analysed. Regarding murT-gatD expression, the activity of the promoter of the operon was analyzed. We observed that the absence of GatD substrate, an acidic external pH and the presence of copper, result in different alterations to the regular expression pattern. Elucidation of how peptidoglycan amidation impacts S. aureus interaction with host cells and how it contributes to the virulence potential of S. aureus is a vital step to validate MurT-GatD complex as a promising target for the design of a new antibiotic.
Staphylococcus aureus causes multiple human infections due to its capacity to express a wide range of virulence factors. S. aureus became an even more dangerous pathogen due to its capacity to acquire resistance to all the antibiotics clinically available. The MurT-GatD complex is responsible for a secondary modification of the peptidoglycan, the amidation of the second amino acid of the stem peptide, D-iso-glutamate, into D-iso-glutamine. Inhibition of peptidoglycan amidation was described to impair physiological processes important for bacterial survival, such as cell growth, β-lactam and lysozyme resistance, making this complex an attractive target for the design of new molecules with antibacterial activity. However, how MurT and GatD proteins physically interact and perform catalysis and how this surface modification interacts with the host are unanswered questions. The aim of this Thesis was to describe the physical link between MurT and GatD and on the other hand, to understand the virulence potential of peptidoglycan amidation. Through several genetic and biochemical techniques, we concluded that the MurT C-terminal domain (DUF1727) is the physical linker between MurT and GatD. Furthermore, eleven conserved amino acids were identified and substituted to alanine, through site-directed mutagenesis; four were found to be involved in complex formation (W352, Y354, L381 and R375), two important for MurT stability (D346 and D349) and three important for MurT-GatD activity (D355, Y418 and L313). By providing this information we paved the way for the future development of molecules that hinder the physical interaction between MurT and GatD and consequently kill S. aureus. Regarding the impact of peptidoglycan amidation in virulence, macrophages were infected with several murT-gatD mutants in different S. aureus genetic backgrounds. Upon impairment of murT-gatD genes we observed different behaviors according to the S. aureus background. We showed that these genes are important to avoid macrophage engulfment in strains COL and MW2 and for survival inside macrophages in COL, but not in strain HDE288. To correlate these results with the specific impact of peptidoglycan amidation in the S. aureus backgrounds studied, we determined its involvement in cell aggregation, biofilm formation and lysozyme resistance. Furthermore, the effect of specific host-related conditions, such as different pH, oxygen levels and nitrogen sources, on peptidoglycan amidation, was analysed. Regarding murT-gatD expression, the activity of the promoter of the operon was analyzed. We observed that the absence of GatD substrate, an acidic external pH and the presence of copper, result in different alterations to the regular expression pattern. Elucidation of how peptidoglycan amidation impacts S. aureus interaction with host cells and how it contributes to the virulence potential of S. aureus is a vital step to validate MurT-GatD complex as a promising target for the design of a new antibiotic.
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Staphylococcus aureus MurT-GatD Host cell-bacteria infection