Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin

Abstract

Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are β-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. IMPORTANCE Due to the growing antibiotic resistance crisis, there is a critical need to develop new antibiotics, particularly compounds capable of targeting high-priority antibiotic-resistant Gram-negative pathogens. In order to develop new compounds capable of overcoming resistance a greater understanding of how Gram-negative bacteria are able to prevent the uptake and accumulation of many antibiotics is required. This study used a novel genome wide approach to investigate the significance of a primary amine group as a chemical feature that promotes the uptake and accumulation of compounds in the Gram-negative model organism Escherichia coli. The results support previous biochemical observations that the primary amine promotes passage through the outer membrane porin OmpF, but also highlight active efflux as a major resistance factor.

Keywords: Gram-negative bacteria; efflux pumps; outer membrane.

FIG 1

Challenging BW25113 transposon library with subinhibitory concentrations of ampicillin and benzylpenicillin. Volcano plots of the fold change in transposon insertions in genes between antibiotic challenges and the untreated control are shown for 6.25 μg/mL PEN (A), 1 μg/mL AMP (B), and 1 μg/mL PEN challenges (C). Red datapoints are insertion changes with a ≥2-fold decrease and q.value <0.05, blue datapoints are insertion changes with a ≥2-fold increase and q.value <0.05, and gray datapoints are insertion changes with q.value ≥0.05. Inserts show the chemical structures for PEN (A) and AMP (B), with the primary amine group circled in red. (D) Venn diagram of overlap between genes with significant changes in insertion numbers in different challenge conditions.

FIG 2

Insertion frequency changes following ampicillin and benzylpenicillin treatment. Transposon insertion site numbers within each gene are presented as log₂(FC), compared to the control group for ampicillin (AMP) and benzylpenicillin (PEN). Only genes with a q.value <0.05 and a fold change ≥abs(1) are shown. Genes are functionally grouped according to their COG categories: [C] Energy production and conversion, [D] Cell cycle control, cell division, chromosome partitioning, [G] Carbohydrate transport and metabolism, [H] Coenzyme transport and metabolism, [I] Lipid transport and metabolism, [J] Translation, ribosomal structure and biogenesis, [K] Transcription, [L] Replication, recombination and repair, [M] Cell wall/membrane/envelope biogenesis, [O] Posttranslational modification, protein turnover, and chaperones, [P] Inorganic ion transport and metabolism, [S] Function unknown, [T] Signal transduction mechanisms, [U] Intracellular trafficking, secretion, and vesicular transport, [V] Defense mechanisms.