Abstract
Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.
Highlights
Staphylococcus aureus is a leading cause of hospital and community-acquired infections by Gram-positive bacteria [1,2,3] and Staphylococcus epidermidis has emerged as the most common cause of biofilm infections on medical implant devices [4]
Staphylococcus aureus and Staphylococcus epidermidis cause life-threatening infections that are commonly acquired in hospitals as well as the community and remain difficult to treat with current antibiotics
This is due to the emergence of methicillin-resistant S. aureus and S. epidermidis (MRSA and MRSE), which exhibit broad resistance to β-lactams such as penicillin and other members of this important founding class of antibiotics
Summary
Staphylococcus aureus is a leading cause of hospital and community-acquired infections by Gram-positive bacteria [1,2,3] and Staphylococcus epidermidis has emerged as the most common cause of biofilm infections on medical implant devices [4]. The difficulty in treating these infections lies in their broad resistance to β-lactams, an otherwise powerful class of antibiotics that include methicillin, penicillin, cephalosporins and carbapenems such as imipenem [5]. Methicillin-resistant strains of S. aureus (MRSA) and S. epidermidis (MRSE), have acquired an exogenous PBP (Pbp2a) that exhibits low binding affinity to β-lactams, rendering such strains clinically resistant to most β-lactams [5, 7, 8]. Staphylococcal drug resistance is further exacerbated by the pathogen’s propensity to form a biofilm, in which many bacterial cells display a “persister”-like state of low metabolic activity and which renders antibiotics inactive, such as β-lactams that target active metabolic processes including growth and cell division [9, 10]. Biofilm formation mediates antibiotic drug resistance by providing a complex and extensive polysaccharide extracellular matrix that serves as an effective physical barrier to antibiotic penetration into the cell [11,12,13]
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