ABSTRACT
The N-acetylmuramic acid α-1-phosphate (MurNAc-α1-P) uridylyltransferase MurU catalyzes the synthesis of uridine diphosphate (UDP)-MurNAc, a crucial precursor of the bacterial peptidoglycan cell wall. MurU is part of a recently identified cell wall recycling pathway in Gram-negative bacteria that bypasses the general de novo biosynthesis of UDP-MurNAc and contributes to high intrinsic resistance to the antibiotic fosfomycin, which targets UDP-MurNAc de novo biosynthesis. To provide insights into substrate binding and specificity, we solved crystal structures of MurU of Pseudomonas putida in native and ligand-bound states at high resolution. With the help of these structures, critical enzyme-substrate interactions were identified that enable tight binding of MurNAc-α1-P to the active site of MurU. The MurU structures define a "minimal domain" required for general nucleotidyltransferase activity. They furthermore provide a structural basis for the chemical design of inhibitors of MurU that could serve as novel drugs in combination therapy against multidrug-resistant Gram-negative pathogens.
Subject(s)
Nucleotidyltransferases/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/pathogenicity , Magnesium/chemistry , Models, Molecular , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Protein Conformation , Protein Structure, Tertiary , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Substrate Specificity , Uridine Diphosphate N-Acetylmuramic Acid/biosynthesisABSTRACT
BACKGROUND: Genetically determined variation in the expression of innate defense molecules may explain differences in the propensity to be colonized with Staphylococcus aureus. METHODS: We determined S. aureus nasal carriage in 603 volunteers; analyzed polymorphisms in the DEFB1 promoter at positions -52 G>A (rs1799946), -44 C>G (rs1800972), and -20 G>A (rs11362); and measured the content of human ß-defensin 1 (hBD-1) and hBD-3 messenger RNA (mRNA) in 192 samples of healthy and experimentally wounded human skin. RESULTS: Compared with GGG at the positions -52/-44/-20, the ACG haplotype was more common among persistent S. aureus nasal carriers (odds ratio, 1.93; 95% confidence interval [CI], 1.2-3.1; P = .006) and was associated with reduced expression of hBD-1 (GGG>ACG>GCA; P < .001) and hBD-3 (GGG>GCA>ACG; P = .04) in skin when measured 72 hours after wounding. Furthermore, a 50% decrease in hBD-1 and hBD-3 mRNA expression in wounded skin increased the odds of persistent carriage by 1.45 (95% CI, .93-2.26; P = .1) and 1.48 (95% CI, 1.01-2.17; P = .04), respectively. Adjustment for known risk factors of persistent S. aureus carriage did not substantially change the associations of both DEFB1 haplotypes and ß-defensin expression with S. aureus colonization. CONCLUSIONS: DEFB1 polymorphisms may promote persistent S. aureus colonization by altering ß-defensin expression in keratinocytes of human skin.
