Combining cell envelope stress reporter assays in a screening approach to identify BAM complex inhibitors

M. (Maurice) Steenhuis; F. Corona; Corinne ten Hagen-Jongman; M. Volmer; D. Lambin; P. Selhorst; H. Klaassen; M. Versele; P. Chaltin; Joen Luirink

doi:10.1021/acsinfecdis.0c00728

Combining cell envelope stress reporter assays in a screening approach to identify BAM complex inhibitors

M. (Maurice) Steenhuis
, F. Corona
, Corinne ten Hagen-Jongman
, M. Volmer
, D. Lambin
, P. Selhorst
, H. Klaassen
, M. Versele
, P. Chaltin
, Joen Luirink^*

^*Corresponding author for this work

AIMMS

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σE and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σE and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

Original language	English
Article number	34125508
Pages (from-to)	2250-2263
Number of pages	14
Journal	ACS Infectious Diseases
Volume	7
Issue number	8
Early online date	14 Jun 2021
DOIs	https://doi.org/10.1021/acsinfecdis.0c00728
Publication status	Published - 13 Aug 2021

Funding

We thank Thomas Silhavy (Princeton University, USA) for kindly providing E. coli bamA101. We thank Harris Bernstein (National Institutes of Health, USA) for providing the pJH114 plasmid. Arnaud Marchand is acknowledged for performing chemical analysis. We also thank Peter van Ulsen and Alexander Speer (VU University Amsterdam, The Netherlands) for critical reading of the manuscript and valuable input into the project. This study was supported by the NWO graduate program (022.005.031) awarded to MS. This project has also received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 721484 (International Training Network Train2Target) awarded to WV. Mass spectrometry was made possible by the support of the Hercules Foundation of the Flemish Government (Grant 20100225-7).

Funders	Funder number
Hercules Foundation of the Flemish Government	20100225-7
Marie Skłodowska-Curie	721484
Horizon 2020 Framework Programme
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	022.005.031

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title = "Combining cell envelope stress reporter assays in a screening approach to identify BAM complex inhibitors",

abstract = "The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σE and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σE and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.",

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AU - Steenhuis, M. (Maurice)

AU - Corona, F.

AU - ten Hagen-Jongman, Corinne

AU - Volmer, M.

AU - Lambin, D.

AU - Selhorst, P.

AU - Klaassen, H.

AU - Versele, M.

AU - Chaltin, P.

AU - Luirink, Joen

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N2 - The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σE and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σE and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

AB - The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σE and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σE and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

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JO - ACS Infectious Diseases

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M1 - 34125508

ER -

Combining cell envelope stress reporter assays in a screening approach to identify BAM complex inhibitors

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