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Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open–closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF β-barrel. Mutation of key residues important for the loop’s conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 μs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost. Carbapenem-resistance in Klebsiella pneumoniae sequence type ST258 can be enhanced by modification of the porins OmpK35 and OmpK36. Here, Wong et al. solve the crystal structure of a clinical ST258 OmpK36 variant, elucidating the mechanism of resistance and consequences on pathogenicity in vivo.

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy. The efficacy of stimuli-responsive enzyme delivery systems is usually limited to in vitro applications. Here the authors form artificial organelles by inserting stimuli-responsive protein gates in membranes of polymersomes loaded with enzymes and obtain a triggered functionality both in vitro and in vivo.

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

The 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 the b arrel a ssembly m achine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues in Escherichia coli OmpC, 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.

Key Points Porin modification is a major bacterial resistance strategy that restricts the influx of β-lactam and fluoroquinolone antibiotics. Clinical multidrug resistant enterobacterial isolates that exhibit modified membrane permeability are highly prevalent. Interactions between antibiotic molecules and porin channels govern translocation efficiency. New physico-chemical techniques are being developed to assess drug–channel interactions and to quantify translocation through porins. Computer modelling of the pathway of substrates through porins provides information on the orientation and interaction of substrates in the channel. Quantification of antibiotic translocation provides new insights into how to optimize drug molecules so that they have sufficient permeation rates to circumvent multidrug resistance mechanisms. The outer membrane of Gram-negative bacteria contains many protein channels, called porins. These channels mediate the influx of various compounds, including antibiotics. Adaptations that reduce influx contribute to the emergence and dissemination of antibiotic resistance. This Review outlines recent advances in our understanding of the physico-chemical parameters that govern antibiotic translocation through porin channels. Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.

As human population density and antibiotic exposure increase, specialised bacterial subtypes have begun to emerge. Arising among species that are common commensals and infrequent pathogens, antibiotic-resistant 'high-risk clones' have evolved to better survive in the modern human. Here, we show that the major matrix porin (OmpK35) of Klebsiella pneumoniae is not required in the mammalian host for colonisation, pathogenesis, nor for antibiotic resistance, and that it is commonly absent in pathogenic isolates. This is found in association with, but apparently independent of, a highly specific change in the co-regulated partner porin, the osmoporin (OmpK36), which provides enhanced antibiotic resistance without significant loss of fitness in the mammalian host. These features are common in well-described 'high-risk clones' of K. pneumoniae, as well as in unrelated members of this species and similar adaptations are found in other members of the Enterobacteriaceae that share this lifestyle. Available sequence data indicate evolutionary convergence, with implications for the spread of lethal antibiotic-resistant pathogens in humans.

The increasing resistance of Gram-negative bacteria to last resort antibiotics, such as carbapenems, is particularly of concern as it is a significant cause of global health threat. In this context, there is an urgent need for better understanding underlying mechanisms leading to antimicrobial resistance in order to limit its diffusion and develop new therapeutic strategies. In this review, we focus on the specific role of porins in carbapenem-resistance in Enterobacteriaceae and Pseudomonas aeruginosa , which are major human pathogens. Porins are outer membrane proteins, which play a key role in the bacterial permeability to allow nutrients to enter and toxic waste to leave. However, these channels are also “Achilles’ heel” of bacteria as antibiotics can also pass through them to reach their target and kill the bacteria. After describing normal structures and pathways regulating the expression of porins, we discuss strategies implemented by bacteria to limit the access of carbapenems to their cytoplasmic target. We further examine the real impact of changes in porins on carbapenems susceptibility. Finally, we decipher what is the effect of such changes on bacterial fitness and virulence. Our goal is to integrate all these findings to give a global overview of how bacteria modify their porins to face antibiotic selective pressure trying to not induce fitness cost.

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion.

Outer membrane proteins (OMPs) define the surface biology of Gram-negative bacteria, with roles in adhesion, transport, catalysis and signalling. Specifically, porin beta-barrels are common diffusion channels, predominantly monomeric/trimeric in nature. Here we show that the major OMP of the bacterial predator Bdellovibrio bacteriovorus , PopA, differs from this architecture, forming a pentameric porin-like superstructure. Our X-ray and cryo-EM structures reveal a bowl-shape composite outer β-wall, which houses a central chamber that encloses a section of the lipid bilayer. We demonstrate that PopA, reported to insert into prey inner membrane, causes defects when directed into Escherichia coli membranes. We discover widespread PopA homologues, including likely tetramers and hexamers, that retain the lipid chamber; a similar chamber is formed by an unrelated smaller closed-barrel family, implicating this as a general feature. Our work thus defines oligomeric OMP superfamilies, whose deviation from prior structures requires us to revisit existing membrane-interaction motifs and folding models. This study reveals that an outer membrane protein from the predator Bdellovibrio bacteriovorus forms a pentameric assembly that traps a lipid monolayer within. This allows the discovery of two superfamilies, distributed across a wide range of bacteria, likely to adopt a similar architecture.