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, 6 (1), 106-16

Synthetic Lethal Compound Combinations Reveal a Fundamental Connection Between Wall Teichoic Acid and Peptidoglycan Biosyntheses in Staphylococcus Aureus

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Synthetic Lethal Compound Combinations Reveal a Fundamental Connection Between Wall Teichoic Acid and Peptidoglycan Biosyntheses in Staphylococcus Aureus

Jennifer Campbell et al. ACS Chem Biol.

Abstract

Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to β-lactam antibiotics. PBP2A cross-links nascent peptidoglycan when the native TPs are inhibited by β-lactams. Although mecA expression is essential for β-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes methicillin-resistant S. aureus (MRSA) strains to β-lactams even though the β-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and β-lactams is noteworthy not simply because strategies to overcome MRSA are desperately needed but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The "synthetic lethality" of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic-resistant bacterial infections.

Figures

Figure 1
Figure 1
Wall teichoic acid (WTA) and peptidoglycan synthesis begin with similar reactions. TarO catalyzes the first step in the WTA biosynthetic pathway and utilizes bactoprenol-phosphate as a substrate; MraY, a related enzyme, catalyzes an essential step in PG biosynthesis using the same carrier lipid (46). The chemical structure of the natural product tunicamycin is shown; D=UDP-activated substrate.
Figure 2
Figure 2
Tunicamycin selectively inhibits TarO in S. aureus. a) Representative gel showing levels of WTAs extracted from S. aureus strain Newman treated overnight with varying concentrations of tunicamycin. WTA expression is abolished at 0.1 μg mL−1; wt = Newman wildtype in the absence of tunicamycin. b) Growth curves of S. aureus strain Newman in the presence of various concentrations of tunicamycin. High concentrations of tunicamycin inhibit MraY, leading to growth inhibition. However, strains treated with lower concentrations of tunicamycin that abolish WTA expression grow similarly to the ΔtarO mutant.
Figure 3
Figure 3
Methicillin and tunicamycin comprise a synthetic lethal combination in methicillin-resistant S. aureus. a) Checkerboard drug-interaction analysis in MRSA clinical isolate MRSA+ shows that tunicamycin, at concentrations that abolish WTA synthesis without affecting growth, sensitizes MRSA to the beta-lactam oxacillin. b) Kill curve analysis of methicillin (400 μg mL−1) and tunicamycin (0.4 μg mL−1) in COL shows a bactericidal mechanism of killing; methicillin was added to the tunicamycin-treated culture after 3 h.
Figure 4
Figure 4
Electron micrographs of S. aureus strains show the effects of the lack of wall teichoic acid (WTA) expression on cell division. a) Wildtype MSSA RN6390 and b) its ΔtarO mutant. Cell division in WTA-null strains frequently occurs asymmetrically, and many cells contain duplicate septa. These septa can be parallel or occur at irregular angles, and many are incomplete. In addition, fields of cells contain numerous pseudomulticellular clusters with nonorthogonal division planes. c–f) A time course of electron micrographs of MSSA RN6390 treated with tunicamycin (0.4 μg mL−1) reveals that halting WTA expression is detrimental to cell division, leading to misplaced septa and cell separation defects; c) untreated control; d) 1 h; e) 2 h; f) 2 h (see Supplementary Figures 7 and 8 for time course images of MRSA strain COL treated with tunicamycin). Filled arrowheads point to the splitting system; empty arrowheads point to septa containing low density material; arrows point to secondary cell wall material that is peeling away from the cell surface; scale bars = 500 nm.
Figure 5
Figure 5
Models of S. aureus cell division. a) Wildtype S. aureus cell division model adapted from Beveridge and coworkers (32). b) The effect of tunicamycin treatment on septal formation; the dark staining WTA-rich layer does not extend into the newly formed septum; autolysins are dysregulated by the absence of WTAs and cell separation is hindered, resulting in pseudomulticellular clusters; scale bars = 100 nm.

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