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Mechanistic assessment of DNA ligase as an antibacterial target in Staphylococcus aureus.

ABSTRACT: We report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD(+)-dependent DNA ligase (LigA) as an antibacterial target in Staphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [K(i)] = 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC(50)] = 28 nM) or its isolated adenylation domain (IC(50) = 36 nM). Antistaphylococcal activity was confirmed against methicillin-susceptible and -resistant S. aureus (MSSA and MRSA) strains (MIC = 1.0 ?g/ml). Analysis of spontaneous resistance potential revealed a high frequency of emergence (4 × 10(-7)) of high-level resistant mutants (MIC > 64) with associated ligA lesions. There were no observable effects on growth rate in these mutants. Of 22 sequenced clones, 3 encoded point substitutions within the catalytic adenylation domain and 19 in the downstream oligonucleotide-binding (OB) fold and helix-hairpin-helix (HhH) domains. In vitro characterization of the enzymatic properties of four selected mutants revealed distinct signatures underlying their resistance to inhibition. The infrequent adenylation domain mutations altered the kinetics of adenylation and probably elicited resistance directly. In contrast, the highly represented OB fold domain mutations demonstrated a generalized resistance mechanism in which covalent LigA activation proceeds normally and yet the parameters of downstream ligation steps are altered. A resulting decrease in substrate K(m) and a consequent increase in substrate occupancy render LigA resistant to competitive inhibition. We conclude that the observed tolerance of staphylococcal cells to such hypomorphic mutations probably invalidates LigA as a viable target for antistaphylococcal chemotherapy.

SUBMITTER: Podos SD

PROVIDER: S-EPMC3421614 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Mechanistic assessment of DNA ligase as an antibacterial target in Staphylococcus aureus.

Podos Steven D SD Thanassi Jane A JA Pucci Michael J MJ

Antimicrobial agents and chemotherapy 20120514 8

We report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD(+)-dependent DNA ligase (LigA) as an antibacterial target in Staphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [K(i)] = 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC(50)] = 28 nM) or its isolated adenylation domain (IC(50) = 36 nM). Antistaphyloco ...[more]

PMID: 22585221

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Project description:A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Dataset Information

Mechanistic assessment of DNA ligase as an antibacterial target in Staphylococcus aureus.

Publications

Mechanistic assessment of DNA ligase as an antibacterial target in Staphylococcus aureus.

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