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Critical sites determining the resistance phenotype of ABC proteins from the ARE subfamily and the molecular mechanism of their function
Lenart, Jakub
Vga(A) and Msr(A) are resistance proteins belonging to the ARE subfamily of ABC -F proteins. They confer resistance to inhibitors of the peptidyltransferase center. It has been proposed that the mechanism of resistance is based on interaction with a transmembrane partner that forms the functional transporter. Their ribosomal function has been described by cryoelectron microscopy of ribosome complexes with ABCF mutants unable to hydrolyze ATP. However, the exact mechanism of resistance is not yet known. We have produced the mutant proteins combining the four amino acid residues in Vga(A) and Vga(A)LC at the linker tip, and we were the first to describe the effects of substrate specificity of the single mutants. Amino acid positions 212 and 220 are important for resistance to lincosamides and pleuromutilins, respectively, while position 219 is responsible for resistance to streptogramin A. Each amino acid property plays a critical role in conferring antibiotic specificity, as confirmed by the fact that amino acid substitution at position K218T in the Vga(A) protein causes the shift in resistance from streptogramins to lincosamides and pleuromutilins. The mechanism of resistance conferred by Vga(A) is ribosomal protection. This is supported by the fact that the rate of [3H]-lincomycin accumulation in...
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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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Critical sites determining the resistance phenotype of ABC proteins from the ARE subfamily and the molecular mechanism of their function
Lenart, Jakub ; Balíková Novotná, Gabriela (advisor) ; Melter, Oto (referee) ; Branny, Pavel (referee)
Vga(A) and Msr(A) are resistance proteins belonging to the ARE subfamily of ABC -F proteins. They confer resistance to inhibitors of the peptidyltransferase center. It has been proposed that the mechanism of resistance is based on interaction with a transmembrane partner that forms the functional transporter. Their ribosomal function has been described by cryoelectron microscopy of ribosome complexes with ABCF mutants unable to hydrolyze ATP. However, the exact mechanism of resistance is not yet known. We have produced the mutant proteins combining the four amino acid residues in Vga(A) and Vga(A)LC at the linker tip, and we were the first to describe the effects of substrate specificity of the single mutants. Amino acid positions 212 and 220 are important for resistance to lincosamides and pleuromutilins, respectively, while position 219 is responsible for resistance to streptogramin A. Each amino acid property plays a critical role in conferring antibiotic specificity, as confirmed by the fact that amino acid substitution at position K218T in the Vga(A) protein causes the shift in resistance from streptogramins to lincosamides and pleuromutilins. The mechanism of resistance conferred by Vga(A) is ribosomal protection. This is supported by the fact that the rate of [3H]-lincomycin accumulation in...
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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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Influence of expression of lmr(C) on the biosynthesis of lincomycin in Streptomyces lincolnensis: Resistance or production?
Veselá, Ludmila ; Balíková Novotná, Gabriela (advisor) ; Beranová, Jana (referee)
The genus Streptomyces produces more than a half of the known bioactive substances, ranking it among the most important bacterial taxons. Streptomyces lincolnensis ATCC 25466 encodes a biosynthetic gene cluster for lincomycin biosynthesis in its genome. Apart from the biosynthetic and regulatory genes, the cluster also contains three resistance genes, lmr(A), lmr(B) a lmr(C), which could protect of the host from the toxicity of a synthesized antibiotic. The Lmr(C) protein belongs to ARE proteins which generaly confer resistance to clinically important classes of antibiotics: macrolides, streptogramins, lincosamides and pleuromutilins. In addition to antibiotic producers, ARE proteins are also present in pathogenic microorganisms. However, the resistance mechanism conferred by these protins which belong to ABC transporters, even though they lack the transmembrane domain, have not been characterized yet. This makes the ARE proteins an interesting subject of the research. Using deletion mutants in resistance genes lmr(A), lmr(B) a lmr(C) we studied their effect on the lincomycin production and resistance to lincosamides, lincomycin and clindamycin with special focus on the function of the lmr(C). We have found that deletion of lmr(C) does not significantly influence lincomycin production and...
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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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