Chemical Genetic Identification of Peptidoglycan Inhibitors Potentiating Carbapenem Activity against Methicillin-Resistant Staphylococcus aureus

Huber, John D.; Donald, Robert G. K.; Lee, Sang Ho; Jarantow, Lisa Wang; Salvatore, Michael J.; Meng, Xin; Painter, Ronald E.; Onishi, Russell; Occi, James; Dorso, Karen; Young, Katherine; Park, Young Hwan; Skwish, Stephen; Szymonifka, Michael J.; Waddell, Tim S.; Miesel, Lynn; Phillips, John W.; Roemer, Terry

doi:10.1016/j.chembiol.2009.05.012

Cited by 98 publications

(93 citation statements)

References 44 publications

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“…Orthologs in some Gram-positive bacteria are also required for PG biosynthesis (29,30). A key finding strengthening the proposal that MurJ is the lipid II flippase in E. coli is the fact that MurJ is a member of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily (14,16,31).…”

Section: ) (7)mentioning

confidence: 60%

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

et al. 2013

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Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.T he cell envelope of most bacteria contains a cell wall exoskeleton composed of peptidoglycan (PG) that surrounds the cytoplasmic membrane (1, 2). The rigid PG structure protects the bacterium from osmotic rupture, serves as a scaffold onto which other envelope components are attached, and defines cell shape. Underscoring the essentiality of the PG cell wall is the fact that many antibiotics target PG biosynthesis (3).Bacteria build their PG sacculus by polymerizing an N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) disaccharide-pentapeptide into long glycan chains that are cross-linked by peptide bonds between stem peptides (2). This GlcNAcMurNAc disaccharide-pentapeptide is synthesized at the cytoplasmic side of the membrane as a polyisoprenyl lipid-linked precursor known as lipid II (Fig. 1A) (4). Because lipid II polymerization occurs at the extracytoplasmic side of the membrane, an obligatory step in PG biosynthesis is the translocation of the lipidlinked disaccharide-pentapeptide across the cytoplasmic membrane.The use of polyisoprenyl lipid-linked precursors in the biogenesis of envelope glycopolymers is widespread in bacteria. Examples include the biogenesis of PG, certain capsules and exopolysaccharides, and O antigens (5, 6). In these systems, bacteria build each precursor ...

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Section: ) (7)mentioning

confidence: 60%

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

et al. 2013

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“…However, some key differences in the composition of the stem peptide exist between lipid II from Gram-positive and Gram-negative bacteria (5,34). In addition, there is a low level of conservation between MurJ and YtgP, the putative MurJ ortholog in Gram-positive bacteria (22,35). Given that we have shown that 8 residues within the MurJ cavity region are critical for MurJ function in E. coli, we tested whether these requirements are conserved in YtgP by taking advantage of the fact that ytgP from S. pyogenes complements the depletion of MurJ in E. coli (22).…”

Section: Resultsmentioning

confidence: 99%

Charge Requirements of Lipid II Flippase Activity in Escherichia coli

et al. 2014

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Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport. Most bacteria produce a rigid peptidoglycan (PG) layer that is essential for defining cell shape and providing protection against osmotic lysis (1, 2). This mesh-like PG macromolecule consists of glycan strands of repeating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units that are bridged by the cross-linking of peptide stems extending from the MurNAc moiety. The PG layer is localized outside the cytoplasmic membrane, and as the cell grows, newly synthesized PG material must be incorporated into the preexisting polymer. Since the disaccharide-pentapeptide building blocks are made in the cytoplasm, the cytoplasmic membrane separates PG precursor synthesis from its utilization. Therefore, transport of the disaccharide-pentapeptide across the membrane is an essential step in PG biogenesis.In Escherichia coli, PG is present in the periplasm, the aqueous compartment between the inner and outer membranes (3). Like in all PG-producing bacteria, the disaccharide-pentapeptide PG precursor is synthesized onto the lipid carrier undecaprenol at the inner leaflet of the inner membrane (IM) (reviewed in references 4-7). This lipid-linked precursor, known as lipid II (undecaprenyl-pyrophosphoryl-MurNAc-[pentapeptide]-GlcNAc), must be flipped across the IM and delivered to the outer leaflet of the IM. Once lipid II is exposed to the periplasm, t...

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“…Resistance in MRSA involves the acquired gene, mecA, which encodes PBP2a, a transpeptidase that functions to cross-link peptidoglycan strands when native transpeptidases are inactivated by β-lactams (4)(5)(6). Many other genes native to S. aureus must be present for mecAmediated resistance to operate (7)(8)(9)(10). Understanding how different factors participate in β-lactam resistance is of paramount importance for developing new approaches to combat MRSA.…”

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confidence: 99%

Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids

Brown

Xia

Luhachack

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

250

295

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Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d -alanylation, α- O -GlcNAcylation, and β- O -GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches β- O -GlcNAc (β- O - N -acetyl- d -glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to β-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to β-lactams, Δ tarS strains have no growth or cell division defects. Because neither α- O -GlcNAc nor β- O -Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, β- O -GlcNAc residues. These data suggest β- O -GlcNAcylated WTAs scaffold factors required for MRSA resistance. The β- O -GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to β-lactams.

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Chemical Genetic Identification of Peptidoglycan Inhibitors Potentiating Carbapenem Activity against Methicillin-Resistant Staphylococcus aureus

Cited by 98 publications

References 44 publications

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

Charge Requirements of Lipid II Flippase Activity in Escherichia coli

Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids

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