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Characterization of the major autolysin in Staphylococcus aureus
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Para as células bacterianas crescerem e se dividirem, o peptidoglicano sofre clivagem por hidrolases específicas de modo a que novas subunidades possam ser incorporadas na parede celular. A hidrolase mais importante de Staphylococcus aureus, um agente patogénico humano oportunista, é a proteína bifuncional ATL composta por dois domínios, AM e GL, que são processados extracelularmente e se ligam à superfície da bactéria em locais precisos da superfície equatorial. Com base em observações que mostraram que para mutantes de atl, construídos em diferentes linhagens de S. aureus, a formação de biofilme não é em todos prejudicada, colocou-se a hipótese de que os papéis fisiológicos da ATL podem ser específicos da estirpe. Diferentes abordagens foram usadas para caracterizar a proteína ATL em diferentes estirpes de S. aureus: (i) o gene atl foi sequenciado com o intuito de encontrar diferenças de aminoácidos na proteína que poderiam alterar a atividade ou sofrer clivagem proteolítica diferente; (ii) analisou-se o tamanho das diferentes formas processadas do ATL, bem como o compartimento da célula em que o mesmo ocorre; (iii) a expressão de ATL foi analisada ao longo do tempo por Western Blot; (iv) determinou-se o impacto do DNA na atividade lítica do GL em células inativadas, na parede celular e no peptidoglicano, através de ensaios de lise com a proteína purificada GL.
Os resultados obtidos permitiram identificar padrões distintos de expressão da proteína de ATL e clivagem proteolítica que pode ser a base para as diferenças fenotípicas primárias.
For bacterial cells to enlarge and divide, peptidoglycan must be cleaved by specific hydrolases so that new subunits can be incorporated into the mature cell wall. The most important murein hydrolase of the opportunistic human pathogen Staphylococcus aureus is ATL, a 137.5 kDa bifunctional protein with two domains: AM and GL that are extracellularly processed and bind to the staphylococcal surface at precise locations of the equatorial surface rings. Based on observations that showed that atl mutants, constructed in different S. aureus genetic backgrounds, are not all impaired in biofilm formation, and that the GL-DNA interaction impacts biofilm formation in a strain specific way, we hypothesized that the physiological roles of ATL may be strain-specific. Different approaches were used to characterize ATL in different S. aureus strains: (i) the atl gene was sequenced to identify amino acid differences in the protein that could change its activity or undergo different proteolytic cleavage; (ii) the size of the different ATL processed forms and the cell compartment where they accumulate was assessed; (iii) the expression of ATL was analyzed over time by western blotting; (iv) the impact of DNA on GL lytic activity was determined for heat-inactivated cells, cell wall and peptidoglycan, through lytic assays with the GL purified protein. The results obtained allowed to identify distinct patterns of ATL protein expression and of proteolytic cleavage that may be the basis for the primary phenotypic differences.
For bacterial cells to enlarge and divide, peptidoglycan must be cleaved by specific hydrolases so that new subunits can be incorporated into the mature cell wall. The most important murein hydrolase of the opportunistic human pathogen Staphylococcus aureus is ATL, a 137.5 kDa bifunctional protein with two domains: AM and GL that are extracellularly processed and bind to the staphylococcal surface at precise locations of the equatorial surface rings. Based on observations that showed that atl mutants, constructed in different S. aureus genetic backgrounds, are not all impaired in biofilm formation, and that the GL-DNA interaction impacts biofilm formation in a strain specific way, we hypothesized that the physiological roles of ATL may be strain-specific. Different approaches were used to characterize ATL in different S. aureus strains: (i) the atl gene was sequenced to identify amino acid differences in the protein that could change its activity or undergo different proteolytic cleavage; (ii) the size of the different ATL processed forms and the cell compartment where they accumulate was assessed; (iii) the expression of ATL was analyzed over time by western blotting; (iv) the impact of DNA on GL lytic activity was determined for heat-inactivated cells, cell wall and peptidoglycan, through lytic assays with the GL purified protein. The results obtained allowed to identify distinct patterns of ATL protein expression and of proteolytic cleavage that may be the basis for the primary phenotypic differences.
Descrição
Palavras-chave
Microbiologia médica Staphylococcus aureus Atividade lítica ATL Autolisina Proteínas
Contexto Educativo
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Instituto de Higiene e Medicina Tropical