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1. Conserved lipid-facing basic residues promote the insertion of the porin OmpC into the E. coli outer membrane

   https://doi.org/10.1128/mbio.03319-24  

   Peterson, Janine H; Yang, Lixinhao; Gumbart, James C; Bernstein, Harris D (March 2025, mBio)

   Trent, M Stephen; Konovalova, Anna (Ed.)

   ABSTRACT Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet (“β barrel”) that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called thebarrelassemblymachine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of theEscherichia coliOmpC protein into the OMin vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called thebarrelassemblymachine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues inEscherichia coliOmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane. 

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2. Development of the computational antibiotic screening platform (CLASP) to aid in the discovery of new antibiotics

   https://doi.org/10.1039/D0SM02035D  

   Dai, Yinghui; Ma, Huilin; Wu, Meishan; Welsch, Tory Alane; Vora, Soor Rajiv; Ren, Dacheng; Nangia, Shikha (January 2021, Soft Matter)

   null (Ed.)

   Bacterial colonization of biotic and abiotic surfaces and antibiotic resistance are grand challenges with paramount societal impacts. However, in the face of increasing bacterial resistance to all known antibiotics, efforts to discover new classes of antibiotics have languished, creating an urgent need to accelerate the antibiotic discovery pipeline. A major deterrent in the discovering of new antibiotics is the limited permeability of molecules across the bacterial envelope. Notably, the Gram-negative bacteria have nutrient specific protein channels (or porins) that restrict the permeability of non-essential molecules, including antibiotics. Here, we have developed the Computational Antibiotic Screening Platform (CLASP) for screening of potential drug molecules through the porins. The CLASP takes advantage of coarse grain (CG) resolution, advanced sampling techniques, and a parallel computing environment to maximize its performance. The CLASP yields comprehensive thermodynamic and kinetic output data of a potential drug molecule within a few hours of wall-clock time. Its output includes the potential of mean force profile, energy barrier, the rate constant, and contact analysis of the molecule with the pore-lining residues, and the orientational analysis of the molecule in the porin channel. In our first CLASP application, we report the transport properties of six carbapenem antibiotics—biapenem, doripenem, ertapenem, imipenem, meropenem, and panipenem—through OccD3, a major channel for carbapenem uptake in Pseudomonas aeruginosa . The CLASP is designed to screen small molecule libraries with a fast turnaround time to yield structure–property relationships to discover antibiotics with high permeability. The CLASP will be freely distributed to enable accelerated antibiotic drug discovery. 

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3. Synthesis of Lysine Mimicking Membrane Active Antimicrobial Polymers

   Ankita Arora; Wan Zheng; Hongjun Liang; Abhijit Mishra (November 2018, Advances in Polymer Sciences and Technology)

   Bhuvanesh Gupta; Anup K.Ghosh; Atsushi Suzuki; Sunita Rattan (Ed.)

   Antibiotic resistance in bacteria is a major health concern. Antimicrobial Peptides (AMPs) are efficient in killing most microbes and yet the development of resistance to AMPs is rare. Although AMPs show promising antimicrobial activities, commercializing them as antibiotics is difficult as in vitro extraction and purification of AMPs is complicated and expensive. AMP mimicking antimicrobial polymers can overcome such problems while maintaining the necessary features of AMPs. Here, we have developed meth-acrylamide based polymers to mimic AMPs which possess high antimicrobial activities with low cytotoxicity. Bactericidal and scanning electron microscopy studies show that the synthesized polymers are effective against Gram-positive and Gram-negative bacteria. We find that these polymers are lethal to bacteria and at the same time, they are also non-cytotoxic to mammalian cells, thereby increasing the potential of these polymers to be used as antibiotics. 

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4. Structure‐Activity Relationships in Supramolecular Hosts Targeting Bacterial Phosphatidylethanolamine (PE) Lipids

   https://doi.org/10.1002/chem.202402698  

   Ojah, Emmanuel_O; Gneid, Hassan; Herschede, Sarah_R; Busschaert, Nathalie (October 2024, Chemistry – A European Journal)

   Abstract The World Health Organization has described the antimicrobial resistance crisis as one of the top ten global public health threats. New antimicrobial agents that can fight infections caused by antimicrobial resistant pathogens are therefore needed. A potential strategy is the development of small molecules that can selectively interact with bacterial membranes (or membranes of other microbial pathogens), and thereby rapidly kill the bacteria. Here, we report the structure‐activity relationship within a group of 22 compounds that were designed to bind the bacterial lipid phosphatidylethanolamine (PE). Liposome‐based studies reveal that the lipophilicity of the compounds has the strongest effect on both the affinity and selectivity for PE. The best results were obtained for compounds with logP≈3.75, which showed a 5x–7x selectivity for bacterial PE lipids over human PC (phosphatidylcholine) lipids. Furthermore, these compounds also showed potent antibacterial activity against the Gram‐positive bacteriumB. cereus, with minimum inhibitory concentrations (MICs) below 10 μM, a concentration where they showed minimal hemolytic activity against human red blood cells. These results not only show the possibility of PE‐binding small molecules to function as antibiotics, but also provide guidelines for the development of compounds targeting other types of biologically relevant membrane lipids. 

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5. Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study

   https://doi.org/10.1038/s41598-024-52536-z  

   Smith, Brooke L; Fernando, Sandun; King, Maria D (December 2024, Scientific Reports)

   Abstract While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism ofEscherichia coli (E. coli)cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand–protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand–protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showingE. colicells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms. 

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