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Microorganisms
Volume 13
Issue 7
10.3390/microorganisms13071521
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Article

Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66

by
Samual C. Allgood
1,
Calvin A. Ewing
1,
Weiping Chu
2,
Steffen Porwollik
2,
Michael McClelland
2 and
Corrella S. Detweiler
1,*
1
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA
2
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(7), 1521; https://doi.org/10.3390/microorganisms13071521
Submission received: 28 May 2025 / Revised: 22 June 2025 / Accepted: 24 June 2025 / Published: 29 June 2025
(This article belongs to the Section Antimicrobial Agents and Resistance)
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Abstract

Anti-infectives include molecules that target microbes in the context of infection but lack antimicrobial activity under conventional growth conditions. We previously described D66, a small molecule that kills the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) within cultured macrophages and murine tissues, with low host toxicity. While D66 fails to inhibit bacterial growth in standard media, the compound is bacteriostatic and disrupts the cell membrane voltage gradient without lysis under growth conditions that permeabilize the outer membrane or reduce efflux pump activity. To gain insights into specific bacterial targets of D66, we pursued two genetic approaches. Selection for resistance to D66 revealed spontaneous point mutations that mapped within the gmhB gene, which encodes a protein involved in the biosynthesis of the lipopolysaccharide core molecule. E. coli and S. Typhimurium gmhB mutants exhibited increased resistance to antibiotics, indicating a more robust barrier to entry. Conversely, S. Typhimurium transposon insertions in genes involved in outer membrane permeability or efflux pump activity reduced fitness in the presence of D66. Together, these observations underscore the significance of the bacterial cell envelope in safeguarding Gram-negative bacteria from small molecules.
Keywords: AcrAB-TolC; anti-infective; efflux pump; gmhB/yaeD; inner membrane; lipopolysaccharide (LPS); outer membrane; Resistance Nodulation cell Division (RND); salmonella; ygeG AcrAB-TolC; anti-infective; efflux pump; gmhB/yaeD; inner membrane; lipopolysaccharide (LPS); outer membrane; Resistance Nodulation cell Division (RND); salmonella; ygeG

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MDPI and ACS Style

Allgood, S.C.; Ewing, C.A.; Chu, W.; Porwollik, S.; McClelland, M.; Detweiler, C.S. Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66. Microorganisms 2025, 13, 1521. https://doi.org/10.3390/microorganisms13071521

AMA Style

Allgood SC, Ewing CA, Chu W, Porwollik S, McClelland M, Detweiler CS. Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66. Microorganisms. 2025; 13(7):1521. https://doi.org/10.3390/microorganisms13071521

Chicago/Turabian Style

Allgood, Samual C., Calvin A. Ewing, Weiping Chu, Steffen Porwollik, Michael McClelland, and Corrella S. Detweiler. 2025. "Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66" Microorganisms 13, no. 7: 1521. https://doi.org/10.3390/microorganisms13071521

APA Style

Allgood, S. C., Ewing, C. A., Chu, W., Porwollik, S., McClelland, M., & Detweiler, C. S. (2025). Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66. Microorganisms, 13(7), 1521. https://doi.org/10.3390/microorganisms13071521

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