NATURE MICROBIOLOGY 2, 17001 (2017) | DOI: 10.1038/nmicrobiol.2017.1 | www.nature.com/naturemicrobiology 1 PERSPECTIVE PUBLISHED: 22 FEBRUARY 2017 | VOLUME: 2 | ARTICLE NUMBER: 17001 A ntibiotics are a mainstay of modern clinical medicine. However, many bacterial pathogens have acquired multidrug resistance and can cause infections that are efectively untreatable, rep- resenting an increasing threat to public health 1,2 . Tis situation is especially troubling with respect to Gram-negative pathogens, such as Pseudomonas aeruginosa, Acinetobacter baumannii and members of the Enterobacteriaceae family 3–5 , with multiple examples classifed as ‘urgent’ or ‘serious’ threats in 2013 by the United States Centers for Disease Control and Prevention 2 . Improved understanding of the factors that render these pathogens difcult to target is a key step in addressing the rising unmet medical need in this area. Gram-negative bacteria are intrinsically resistant to many antibi- otics due to the permeability barrier that is provided by their unique cell envelope. Tis envelope consists of an outer membrane (OM) and inner membrane (IM), which are separated by a periplasmic space 6,7 . Te OM is a sophisticated asymmetric lipid bilayer in which phospholipids exclusively partition on the inner leafet, while the lipid A moiety of lipopolysaccharide (LPS) forms the outer leafet. Te LPS layer of the OM is an important component in providing a protective layer against harmful compounds in the extracellular envi- ronment 6–9 . Te IM is a traditional phospholipid bilayer 10 . Between the two membranes lies the periplasm, a viscous cellular compart- ment in which the peptidoglycan layer is situated. Although it is not thought to be a physical barrier for drugs or nutrients, this compart- ment deserves a mention as it is the site of action of the β-lactam anti- biotics and their potential deactivation by β-lactamases. In addition to these physical barriers for infux, multiple efux pumps are present in the cell envelope and can further reduce net permeability into the Gram-negative bacterial cell 11 . Logically, an antibacterial drug must reach its requisite site of action to be efective, and although there are examples of drugs on the market 12,13 and in development 14 that target the outer leafet of the OM, Mechanisms of envelope permeability and antibiotic infux and efux in Gram-negative bacteria Muriel Masi 1 , Matthieu Réfregiers 2 , Klaas M. Pos 3 and Jean-Marie Pagès 1 * Researchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are char- acterized by low permeability and often contain drug efux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or efux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identifed clear correlations between efux activ- ity and drug difusion and accumulation in both susceptible and resistant strains, and have also clarifed how certain chemical structures can afect drug entry and residence time within the cell. In this Perspective, we discuss the biological underpinnings of drug permeability and export using several prototypical infux and efux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid eforts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics. the majority of clinically used drugs needs to penetrate one or both of the membranes of the cell envelope 15 . Changes in the overall ability of drugs to pass through this envelope due to loss of porins and other transport systems or drug removal via upregulation of efux pumps can lead to clinical antibacterial resistance 7,8,11,16 . Tis Perspective summarizes current advances in understanding the molecular basis and impact of cell envelope permeability and antibiotic transloca- tion in Gram-negative bacteria, and ofers future prospects for an improved science base for antibacterial drug discovery. Outer membrane barrier and drug permeation Upon entry into Gram-negative bacteria via the OM, drugs are dis- tributed in diferent compartments (that is, the periplasm, the IM and the cytoplasm). Additional binding to components within each com- partment, including the intended drug target, adds to the complexity of this distribution. For most hydrophobic drugs with a mechanism of action inside the cytoplasm, the IM poses no serious uptake bar- rier 7,11 . In contrast, the OM protein channels, termed porins, that help small hydrophilic molecules to cross this compartment are the main factors regulating drug permeability as well as resistance. In Escherichia coli, the two general difusion porins OmpF and OmpC are among the most abundant OM proteins (>10 5  copies per cell) and are commonly used as prototypical structures for under- standing small molecule permeability. Tese porins consist of trimers of 16-stranded β-barrels, each of which allows the formation of a cen- tral channel 17,18 . Te amphipathic β-strands of porins are connected by short periplasmic turns and longer extracellular loops, which are generally exposed at the cell surface. However, the third loop (L3) folds inward at approximately half the height of the channel, and together with the opposite barrel wall, forms the so-called constric- tion zone (Fig. 1). Te presence of acidic residues in L3, which face a cluster of basic residues on the opposite side of the pore, creates a local 1 Transporteurs Membranaires, Chimiorésistance et Drug Design, Aix-Marseille Université, IRBA, UMR-MD1, Facultés de Médecine et de Pharmacie, 13385 Marseille cedex05, France. 2 DISCO beamline, Synchrotron Soleil, L’Orme des Merisiers Saint-Aubin - BP 48 91192, Gif-sur-Yvette cedex, France. 3 Membrane Transport Machineries, Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany. *e-mail: jean-marie.pages@univ-amu.fr ©2017MacmillanPublishersLimited,partofSpringerNature.Allrightsreserved.