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Edited by Christopher T. Walsh, Stanford University, Stanford, CA, and approved April 19, 2018 (received for review March 16, 2018)
Significance
The outer membrane of gram-negative bacteria contains LPS on the cell surface. The presence of LPS creates an effective permeability barrier that protects gram-negative bacteria from small hydrophobic molecules. Because the entire LPS biogenesis pathway, including biosynthesis and transport, is highly conserved, proteins involved are attractive targets for antibiotic discovery. Historically, it has been challenging to target LPS biogenesis since many of the components are membrane proteins with hard-to-assay activities. Utilizing the nonessentiality of this pathway in a gram-negative pathogen, we developed a cell-based screen specific to LPS biogenesis. We identified a small-molecule inhibitor targeting an essential component of the pathway, MsbA, and validated it as an antibacterial target using a combination of genetics, biochemistry, and cellular assays.
Abstract
New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter. We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.
Footnotes
- ↵1To whom correspondence may be addressed. Email: schultz{at}scripps.edu, txm50{at}psu.edu, or kahne{at}chemistry.harvard.edu.
Author contributions: G.Z., V.B., T.C.M., and D.K. designed experiments; G.Z. and T.C.M. constructed mutant strains in A. baumannii; V.B. constructed and characterized all reported A. baylyi strains; G.Z. optimized the A. baumannii assay for high throughput with help of V.B.; G.Z. performed the high-throughput screen and hit confirmation; N.S.R., A.K.C., C.W.M., T.S.Y., and P.G.S. analyzed the screening hits; G.Z. characterized drug–drug interactions, selected for lpx resistance mutations, and analyzed whole-genome sequences; V.B. developed the strategy to isolate on-target resistant mutants; G.Z. and V.B. selected for msbA resistance mutations and analyzed whole genome sequences; G.Z., V.B., and K.S.P. measured MICs; G.Z. constructed complementation strains with help from V.B.; V.B. generated E. coli strains reported in this work; D.T. developed the protocol for expressing and purifying the MsbA protein; K.S.P. synthesized analogs and performed biochemical reconstitution; E.M. optimized conditions with G.Z. for microscopy and performed fluorescent imaging; G.Z. prepared and submitted samples for TEM; T.C.M. and D.K. designed and supervised the project; G.Z., V.B., K.S.P., and D.K. wrote the manuscript with input for specific sections from E.M., P.G.S, T.S.Y., C.W.M., and T.C.M.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Commentary on page 6530.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1804670115/-/DCSupplemental.
Published under the PNAS license.
Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors
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