Beta-Lactam Antibiotics Induce a Lethal Malfunctioning of the Bacterial Cell Wall Synthesis Machinery

Cho, Hongbaek; Uehara, Tsuyoshi; Bernhardt, Thomas G.

doi:10.1016/j.cell.2014.11.017

Cited by 546 publications

(629 citation statements)

References 55 publications

(77 reference statements)

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“…5A, TG‐TP‐) with meropenem, the survival rate is almost the same as the single TG‐mutant. This may correlate with other cell biology data that show the expression of a TP allele is more toxic than a TG‐allele or a TG‐TP‐allele (and that the latter phenocopy each other) if the cells also undergo inhibition of crosslinking 11, 15. One model in E. coli explains that this may be due to cells experiencing a buildup of uncrosslinked glycan strands 15.…”

Section: Resultssupporting

confidence: 71%

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

et al. 2016

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Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic‐resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin‐binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor‐free form and in complex with penicillin V are reported. We used site‐directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β‐lactams. Structural comparison of the PonA1 apo‐form and the antibiotic‐bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4′‐α3 surrounding the penicillin‐binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β‐lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall‐targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections.DatabaseStructural data are available in the PDB database under the accession numbers 5CRF and 5CXW.

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Section: Resultssupporting

confidence: 71%

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

et al. 2016

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“…We, therefore, replaced the bend-induced termination hypothesis with a transpeptidation-facilitated translocation hypothesis specifying that the probability of translocation was low, once every 2 · 10 6 steps, if the last-formed peptide stem was not crosslinked, but increased to once every 3 · 10 4 steps after the stem was crosslinked. The effect, however, might be different on uncrosslinked strands, for example, when transglycosylases synthesize glycan strands in vitro (67) or β-lactam treatment blocks transpeptidation (68). Because uncrosslinked strands can move freely, transpeptidation might not be needed to facilitate translocation.…”

Section: Resultsmentioning

confidence: 99%

“…Our simulations suggest a different explanation for how PG is released: glycosidases cleave loose uncrosslinked PG tails. Interestingly, while this manuscript was under revision, Cho et al (68) published evidence identifying Slt as a hydrolase that removes uncrosslinked PG strands, thus providing strong experimental support for the tail hydrolysis hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape

Nguyen

Gumbart

Beeby

et al. 2015

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

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Bacteria are surrounded by a peptidoglycan (PG) cell wall that must be remodeled to allow cell growth. While many structural details and properties of PG and the individual enzymes involved are known, how the process is coordinated to maintain cell integrity and rod shape is not understood. We have developed a coarse-grained method to simulate how individual transglycosylases, transpeptidases, and endopeptidases could introduce new material into an existing unilayer PG network. We find that a simple model with no enzyme coordination fails to maintain cell wall integrity and rod shape. We then iteratively analyze failure modes and explore different mechanistic hypotheses about how each problem might be overcome by the macromolecules involved. In contrast to a current theory, which posits that long MreB filaments are needed to coordinate PG insertion sites, we find that local coordination of enzyme activities in individual complexes can be sufficient to maintain cell integrity and rod shape. We also present possible molecular explanations for the existence of monofunctional transpeptidases and glycosidases (glycoside hydrolases), trimeric peptide crosslinks, cell twisting during growth, and synthesis of new strands in pairs.coarse-grained modeling | cell wall synthesis | morphogenesis T he cytoplasmic membrane of Gram-negative rod-shaped bacteria is surrounded by a peptidoglycan (PG) sacculus that protects the cell from internal turgor pressure, and its architecture determines the cell's shape (1, 2). The sacculus is composed of long glycan strands crosslinked by peptides into a mesh-like network. The glycan repeating unit is a disaccharide of an N-acetylglucosamine and an N-acetylmuramic acid attached to a stem pentapeptide L-Ala-D-iGlu-m-A 2 pm-D-Ala-D-Ala. Crosslinks are formed between peptides on adjacent strands-most at the fourth (D-Ala) residues of the donors and the third (m-A 2 pm) residues of the acceptors. While it is now understood that, in Gram-negative cells, the glycan strands run parallel to the cell surface, how the strands are arranged in this plane is still debated. While recent atomic force microscopy images of purified sacculi were interpreted to indicate that glycan strands had random orientations (3), electron cryotomography of sacculi (4) and other evidence from both Gramnegative (1, 5) and -positive (6, 7) sacculi have, instead, pointed to a universal "circumferential" model, in which the stiff glycan strands are circumferentially around the rod and the flexible peptide crosslinks parallel to the rod's long axis.Cell growth requires that the sacculus be elongated without losing its integrity or characteristic shape. This remodeling process requires the presence of not only transglycosylases and transpeptidases, also known as penicillin-binding proteins (PBPs), to polymerize and crosslink new glycan strands into the existing network (2, 8) but also, endopeptidases to cleave covalent bonds to open space for the new material (9). How these small enzymes work together to maintain the order and...

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“…It is formally possible that WigK directly senses antibiotics or depletion of PG synthesis precursors, which has recently been described as a lethal consequence of exposure to beta-lactam antibiotics (31). However, direct sensing of antibiotics is highly unlikely because structurally diverse agents (beta-lactams, D-cycloserine, fosfomycin) induce WigR and WigR governs cell width even in the absence of antibiotics.…”

Section: Discussionmentioning

confidence: 99%

A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance

Dörr

Álvarez

Delgado

et al. 2015

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

101

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The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall–acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall–acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

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Beta-Lactam Antibiotics Induce a Lethal Malfunctioning of the Bacterial Cell Wall Synthesis Machinery

Cited by 546 publications

References 55 publications

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape

A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance

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