Structure and reconstitution of a hydrolase complex that releases peptidoglycan from the membrane after polymerization

Schaefer, Kaitlin; Owens, Tristan W.; Page, Julia E; Santiago, Marina; Kahne, Daniel; Walker, Suzanne

doi:10.1101/2020.05.21.109470

Cited by 2 publications

(3 citation statements)

References 54 publications

(68 reference statements)

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“…16 Transporter proteins flip the extended WTA polymer to the extracellular surface, 17 where it is linked to PG. 6,18 D-Alanylation of ribitol units is thought to occur extracellularly and is mediated by the DltA, DltB, DltC, and DltD proteins. 19 Lipoteichoic acid (LTA) is also produced by Gram-positive bacteria but is integrally associated with the cell membrane.…”

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confidence: 99%

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance

Romaniuk

Cegelski

2018

Biochemistry

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Gram-positive bacteria surround themselves with a multilayered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of Staphylococcus aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present C cross polarization magic angle spinning solid-state nuclear magnetic resonance (NMR) spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole-cellC NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl α-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing N spectral signatures include glycyl amide nitrogens in PG and the esterified d-alanyl amine nitrogens in WTA.C NMR analysis was performed with samples at natural abundance and included 10 whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole-cell activity.

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confidence: 99%

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance

Romaniuk

Cegelski

2018

Biochemistry

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“…24−26 Additionally, WTA recruits PBP4 to the cell division septum, which allows PBP4 to participate in β-lactam resistance. 2,3,31,54 Because WTA synthesis is essential for a robust cell wall architecture, 55 previous studies that disrupt WTA biosynthesis with mutants were not accurately representing the MRSA cell wall. Furthermore, disrupting WTA biosynthesis causes biomolecular precursors and Lipid II fragments to accumulate, which downregulates vital biochemical pathways.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…Furthermore, disrupting WTA biosynthesis causes biomolecular precursors and Lipid II fragments to accumulate, which downregulates vital biochemical pathways. 55 To avoid these confounding variables, we used wild-type MRSA cells (which have a completely intact envelope composition and architecture) to investigate the role of WTA in cell division and β-lactam resistance. We found that hydrophilic, amineterminated, 600 Da branched polyethylenimine (BPEI) binds to and disables WTA within wild-type MRSA cells.…”

Section: ■ Conclusionmentioning

confidence: 99%

BPEI-Induced Delocalization of PBP4 Potentiates β-Lactams against MRSA

et al. 2019

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With its high morbidity rate and increasing resistance to treatment, methicillin-resistant Staphylococcus aureus (MRSA) is a grave concern in the medical field. In methicillin-susceptible strains, β-lactam antibiotics disable the penicillin binding proteins (PBPs) that cross-link the bacterial cell wall. However, methicillin-resistant strains have PBP2a and PBP4, which continue enzymatic activity in the presence of β-lactam antibiotics. The activity of PBP2a and PBP4 is linked to the presence of wall teichoic acid (WTA); thus, WTA has emerged as a target for antibiotic drug discovery. In this work, we disable WTA in situ using its anionic phosphodiester backbone to attract cationic branched polyethylenimine (BPEI). Data show that BPEI removes βlactam resistance in common MRSA strains and clinical isolates. Fluorescence microscopy was used to investigate this mechanism of action. The results indicate that BPEI prevents the localization of PBP4 to the cell division septum, thereby changing the cellular morphology and inhibiting cell division. Although PBP4 is not required for septum formation, proper cell division and morphology require WTA; BPEI prevents this essential function. The combination of BPEI and β-lactams is bactericidal and synergistic. Because BPEI allows us to study the role of WTA in the cell wall without genetic mutation or altered translocation of biomolecules and/or their precursors, this approach can help revise existing paradigms regarding the role of WTA in prokaryotic biochemistry at every growth stage.

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Structure and reconstitution of a hydrolase complex that releases peptidoglycan from the membrane after polymerization

Cited by 2 publications

References 54 publications

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance

BPEI-Induced Delocalization of PBP4 Potentiates β-Lactams against MRSA

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