Antibiotic resistance has emerged as a serious threat to global public health in recent years. Lack of novel antimicrobials, especially new classes of compounds, further aggravates the situation. For Gram-negative bacteria, their double layered cell envelope and an array of efflux pumps act as formidable barriers for antimicrobials to penetrate. While cytoplasmic targets are hard to reach, proteins in the periplasm are clearly more accessible, as the drug only needs to breach the outer membrane. In this review, we summarized recent efforts on the validation and testing of periplasmic proteins as potential antimicrobial targets and the development of related inhibitors that either inhibit the growth of a bacterial pathogen or reduce its virulence during interaction with host cells. We conclude that the periplasm contains a promising pool of novel antimicrobial targets that should be scrutinized more closely for the development of effective treatment against multidrug-resistant Gram-negative bacteria.

The cell envelope structure of Gram-negative bacteria is unique, composed of two lipid bilayer membranes and an aqueous periplasmic space sandwiched in between. The outer membrane constitutes an extra barrier to limit the exchange of molecules between the cells and the exterior environment. Donnan potential is a membrane potential across the outer membrane, resulted from the selective permeability of the membrane, which plays a pivotal role in the permeability of many antibiotics. In this review, we discussed factors that affect the intensity of the Donnan potential, including the osmotic strength and pH of the external media, the osmoregulated periplasmic glucans trapped in the periplasmic space, and the displacement of cell surface charges. The focus of our discussion is the impact of Donnan potential on the cellular permeability of selected antibiotics including fluoroquinolones, tetracyclines, β-lactams, and trimethoprim.

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

Efflux pumps, combined with low permeability of the outer membrane of gram negative bacteria play a crucial role in bacteria multi‐drug resistance. There are five classes of efflux pumps but the major and most studied of the efflux system in E. coli is the resistance nodulation cell division (RND) pump. This pump is a complex of three proteins: the inner membrane transporter AcrB, the periplasmic adaptor AcrA, and the outer membrane protein channel TolC. Of the three proteins, the structure and dynamic behavior of AcrA remain to be fully elucidated. In this study the functional roles of the unstructured regions at the N‐ and C‐termini of the protein were examined. These structurally unresolved regions include residues 26–37 at the N‐terminus, and residues 376 to 397 at the C‐terminus. We conducted truncation mutation to systematically remove residues from the C‐terminus of AcrA, and found that residues after Q376 are indeed not functionally important. The truncated AcrA, in which E377 is replaced by a stop codon, is fully active. On the N‐terminus, we created a mutant in which four amino acid residues (Q31QGG34) were deleted. The mutant was nonfunctional. To understand the mechanism of function disruption, we modified the membrane anchoring of AcrA, and determined the role of these first 13 residues at the N‐terminus of the mature AcrA sequence.Support or Funding InformationNSF grant CHE‐1709381This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

the structural and dynamic changes affecting the protein as it associates with different types of lipid bilayers. We previously reported that the activation of lipid peroxidase activity occurs with the native fold largely preserved. Spectral signatures of the membrane-bound protein are clearly distinct from those of denatured cytochrome c. Remarkably, localized spectral changes indicate selective dynamics in the protein-lipid complex that respond to the membrane fluidity. Different dynamic modulations occur dependent on the lipid composition, in terms of acyl chain and headgroup type. Thus, lipids act not only as preferred substrates but also as crucial dynamic regulators of this lethal interplay of proteins and lipids at the mitochondrial inner membrane.