Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling

Culp, Elizabeth; Waglechner, Nicholas; Wang, Wenliang; Fiebig‐Comyn, Aline; Hsu, Yen-Pang; Koteva, Kalinka; Sychantha, David; Coombes, Brian K.; Nieuwenhze, Michael S. Van; Brun, Yves V.; Wright, Gerard D.

doi:10.1038/s41586-020-1990-9

Cited by 224 publications

(218 citation statements)

References 53 publications

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“…Thus, ShyA-depleted ∆6 endo cells not only incorporate PG, but do so to higher levels than the WT, possibly reflecting the lack of EP-initiated turnover processes. Similar observations have been made in autolysin-inactivated B. subtilis, a Gram-positive bacterium (25,41,42). While we cannot rule out that residual ShyA remains in the cell following depletion (at levels too low to detect above background of the non-specific band we observed via Western Blot in the same size range as ShyA, Fig.…”

Section: Cell Wall Incorporation and Mass Increase Continue During Ensupporting

confidence: 81%

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Murphy

Murtha

Zhao

et al. 2020

Preprint

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AbstractThe bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and cellular structural integrity. PG is synthesized by two different types of PG synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by ‘autolytic’ enzymes (e.g., endopeptidases, EPs) to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, and a decline in viability, but continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, apparently without effectively inserting significant PG material. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, only the Rod system absolutely requires endopeptidase activity (but not necessarily direct interaction with EPs) for productive PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency.ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes (“autolysins”, e.g., endopeptidases, EPs) are required for activity of the different cell wall synthesis machines, the Rod system and the class A penicillin-binding proteins (aPBPs). Our results suggest that in Vibrio cholerae, endopeptidases are required only for cell expansion mediated by the Rod system, while the aPBPs maintain structural integrity during EP insufficiency. Overall, our results imply a complex relationship between cell wall synthesis and cleavage and suggest that aPBPs are more versatile than the Rod system in their ability to repair cell wall gaps formed by autolysins other than the major EPs, adding to our understanding of the co-ordination between autolysins and cell wall synthases.

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Section: Cell Wall Incorporation and Mass Increase Continue During Ensupporting

confidence: 81%

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Murphy

Murtha

Zhao

et al. 2020

Preprint

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“…The prevalence and mechanisms associated with antibiotic resistance are better characterized, but the mechanisms of MDR gene transfer in the environment are less understood. It is important to continue studying antibiotic resistance gene transfer, so that innovative solutions to the current antibiotic resistance crisis can be found (Bush et al, 2011;Culp et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Multidrug Resistant Acinetobacter Isolates Release Resistance Determinants Through Contact-Dependent Killing and Bacteriophage Lysis

et al. 2020

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Antimicrobial resistance is an ancient bacterial defense mechanism that has rapidly spread due to the frequent use of antibiotics for disease treatment and livestock growth promotion. We are becoming increasingly aware that pathogens, such as members of the genus Acinetobacter, are precipitously evolving drug resistances through multiple mechanisms, including the acquisition of antibiotic resistance genes. In this study, we isolated three multidrug resistant Acinetobacter species from birds on a free-range farm. Acinetobacter radioresistens, Acinetobacter lwoffii, and Acinetobacter johnsonii were isolated from hens, turkeys and ducks and were resistant to 14 clinically relevant antibiotics, including several listed by the World Health Organization as essential medicines. Co-culturing any of the three Acinetobacter species with Acinetobacter baumannii resulted in contact-dependent release of intact resistance determinants. We also isolated several lytic bacteriophages and selected two of these phages to be included in this study based on differences in plaquing characteristics, nucleic acid content and viral morphology. Both phages released host DNA, including antibiotic resistance genes during cell lysis and we demonstrated that these resistance determinants were transferable to a naïve strain of Escherichia coli. This study demonstrates that contact-dependent competition between bacterial species can readily contribute to DNA release into the environment, including antibiotic resistance determinants. We also highlight that the constant lysis and turnover of bacterial populations during the natural lifecycle of a lytic bacteriophage is an underappreciated mechanism for the liberation of DNA and subsequent genetic exchange.

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“…Corbomycin (Fig. 2a) is a Type V nonapeptide GPA discovered through phylogeny-guided genome mining 14 . While corbomycin shows clinical promise, it has a relatively low yield in the producing strain Streptomyces sp.…”

Section: Refactoring Of the Vector For Tar Cloning Of Large Np Bgcsmentioning

confidence: 99%

“…These challenges restrict the opportunities of securing GPAs for exploration as drug candidates 12,13 . While most GPAs target the D-Ala-D-Ala terminus of peptidoglycan, we recently established that a subgroup, termed Type V GPAs 12 , binds to the cell wall in a distinct manner 14 . Type V GPAs are characterized by a tryptophan (Trp) linked to the central 4-hydroxyphenylglycine (Hpg) and are not generally glycosylated.…”

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confidence: 99%

GPAHex-A synthetic biology platform for Type IV–V glycopeptide antibiotic production and discovery

Wang

Waglechner

et al. 2020

Nat Commun

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Glycopeptide antibiotics (GPAs) are essential for the treatment of severe infectious diseases caused by Gram-positive bacteria. The emergence and spread of GPA resistance have propelled the search for more effective GPAs. Given their structural complexity, genetic intractability, and low titer, expansion of GPA chemical diversity using synthetic or medicinal chemistry remains challenging. Here we describe a synthetic biology platform, GPAHex (GPA Heterologous expression), which exploits the genes required for the specialized GPA building blocks, regulation, antibiotic transport, and resistance for the heterologous production of GPAs. Application of the GPAHex platform results in: (1) a 19-fold increase of corbomycin titer compared to the parental strain, (2) the discovery of a teicoplanin-class GPA from an Amycolatopsis isolate, and (3) the overproduction and characterization of a cryptic nonapeptide GPA. GPAHex provides a platform for GPA production and mining of uncharacterized GPAs and provides a blueprint for chassis design for other natural product classes.

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Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling

Cited by 224 publications

References 53 publications

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

Multidrug Resistant Acinetobacter Isolates Release Resistance Determinants Through Contact-Dependent Killing and Bacteriophage Lysis

GPAHex-A synthetic biology platform for Type IV–V glycopeptide antibiotic production and discovery

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