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Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
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The effect of aminoacid variability on the resistance phenotype in ARE subfamily of ABC proteins
Lenart, Jakub ; Balíková Novotná, Gabriela (advisor) ; Fišer, Radovan (referee)
ARE subfamily proteins belonging to ABC transporters confers a different degree of resistance to macrolides, linkosamides and streptogramins antibiotics. Among the most clinically ARE subfamily proteins in staphylococci is Vga(A) protein lead to the award resistance to streptogtramins A. In 2006, discovered the new variant called the Vga(A)LC, which in addition to streptogramins A resistance also confers linkosamides. Vga(A) and Vga(A)LC differ in only 7 amino acids, yet confer different resistance phenotypes. In previous experiments it was found that the central role in determining substrate specificity play a 4 amino acid differences that accumulate in the section of 15 amino acids within the linker connecting the two ABC domains (positions 212, 219, 220 and 226). The combination of amino acids LGAG Vga(A) increases resistance to streptogramins A while present in combination SVTS Vga(A)LC increased resistance to linkosamides. Although in this subfamily includes a large number of resistance proteins, the mechanism of resistance has not yet been established with certainty. The aim was to create a new Vga(A) variants that contain specific combinations of amino acids for Vga(A) and Vga(A)LC protein at positions 212, 219, 220 and 226 and compared their ability to grant resistance to linkosamides. We also...
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Subcellular localization of resistant proteins Vga(A)LC and Msr(A) using fluorescence microscopy
Nguyen Thi Ngoc, Bich ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
Vga(A)LC and Msr(A) are clinically significant resistant proteins in staphylococci that confer resistance to translational inhibitors. They belong to ARE ABC-F protein subfamily, which is part of ABC transporters. Unlike typical ABC transporters, ABC-F proteins do not have transmembrane domains that are responsible for the transport of substances through the membrane. Therefore, they do not have characteristic transport function but regulatory or resistance function. Their mechanism of action on the ribosome has been described only recently, where these proteins displace the antibiotic from the ribosome. However, some aspects of their function are still unclear. For example, what is the function of the Vga(A) location on a membrane that has been detected in the membrane fraction but not in the ribosomal. In this work, using fluorescence microscopy, I observed subcellular localization of the Vga(A)LC-mEos2, Vga(A)LC-GFP and Msr(A)-eqFP650 resistant fusion proteins in live cells of S. aureus under different culture conditions . It has been shown that Vga(A)LC-GFP and Msr(A)-eqFP650 occur in a foci near the membrane. Depending on ATPase activity or the presence of an antibiotic, the localization of Msr(A)-eqFP650 in the cell changes from focal to diffuse, presumably on ribosomes, suggesting a...
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The effect of aminoacid variability on the resistance phenotype in ARE subfamily of ABC proteins
Lenart, Jakub ; Balíková Novotná, Gabriela (advisor) ; Fišer, Radovan (referee)
ARE subfamily proteins belonging to ABC transporters confers a different degree of resistance to macrolides, linkosamides and streptogramins antibiotics. Among the most clinically ARE subfamily proteins in staphylococci is Vga(A) protein lead to the award resistance to streptogtramins A. In 2006, discovered the new variant called the Vga(A)LC, which in addition to streptogramins A resistance also confers linkosamides. Vga(A) and Vga(A)LC differ in only 7 amino acids, yet confer different resistance phenotypes. In previous experiments it was found that the central role in determining substrate specificity play a 4 amino acid differences that accumulate in the section of 15 amino acids within the linker connecting the two ABC domains (positions 212, 219, 220 and 226). The combination of amino acids LGAG Vga(A) increases resistance to streptogramins A while present in combination SVTS Vga(A)LC increased resistance to linkosamides. Although in this subfamily includes a large number of resistance proteins, the mechanism of resistance has not yet been established with certainty. The aim was to create a new Vga(A) variants that contain specific combinations of amino acids for Vga(A) and Vga(A)LC protein at positions 212, 219, 220 and 226 and compared their ability to grant resistance to linkosamides. We also...
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