Targeting bacterial outer-membrane remodelling to impact antimicrobial drug resistance

Abstract

How porins are assembled into the bacterial outer membrane is now understood in molecular detail. We also have knowledge of the signals that dictate which porin-encoding genes are activated under specific environmental stimuli, including the presence of antimicrobial drugs. These signals change the protein-specific composition of the outer membrane, a process referred to as outer-membrane remodelling.The general mechanisms by which mutations and/or adaptations confer AMR phenotypes on bacteria are known. One of these mechanisms is outer-membrane remodelling. Its impact on AMR, particularly carbapenem resistance, is a central feature of several of the bacterial pathogens currently rated as being in urgent need of research and new treatments.New therapies are canvassed here and warnings around phage therapy, based on considerations of outer-membrane remodelling, are made clear. The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR). Knowledge of bacterial membrane protein biogenesis and turnover underpins our understanding of bacterial membrane remodelling and the consequences that this process have in the evolution of AMR phenotypes. At the population level, the evolution of phenotypes is a reversible process, and we can use these insights to deploy evolutionary principles to resensitize bacteria to existing antimicrobial drugs. In our opinion, fundamental knowledge is opening a new way of thinking towards sustainable solutions to the mounting crisis in AMR. Here we discuss what is known about outer-membrane remodelling in bacteria and how the process could be targeted as a means to restore sensitivity to antimicrobial drugs. Bacteriophages are highlighted as a powerful means to exert this control over membrane remodelling but they require careful selection so as to reverse, and not exacerbate, AMR phenotypes. The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR). Knowledge of bacterial membrane protein biogenesis and turnover underpins our understanding of bacterial membrane remodelling and the consequences that this process have in the evolution of AMR phenotypes. At the population level, the evolution of phenotypes is a reversible process, and we can use these insights to deploy evolutionary principles to resensitize bacteria to existing antimicrobial drugs. In our opinion, fundamental knowledge is opening a new way of thinking towards sustainable solutions to the mounting crisis in AMR. Here we discuss what is known about outer-membrane remodelling in bacteria and how the process could be targeted as a means to restore sensitivity to antimicrobial drugs. Bacteriophages are highlighted as a powerful means to exert this control over membrane remodelling but they require careful selection so as to reverse, and not exacerbate, AMR phenotypes. The global impact in loss of human life from increases in AMR has distracted many interested parties from the obvious, simple message that a key definition of AMR is that it is a phenotype. As with other phenotypes, it is a transient description of a given population of bacteria (or fungi, or parasites, but here we focus on bacteria) which is subject to evolution, based on selection under a given set of selective pressures. When we push the evolution of a bacterial population by increasing the exposure to antimicrobials we are selecting for an AMR phenotype. Any strategy that would slow or select against the AMR phenotype would be a better idea. For bacteria, four broad mechanisms are recognized by which AMR phenotypes are driven to evolve (Box 1). Firstly, mutations that alter the target of the antimicrobial drug to inhibit drug-binding to said target will generate an AMR phenotype. Secondly, the modification of existing genes or acquisition of new genes encoding efflux pumps (see Glossary) provides an AMR phenotype. Thirdly, the acquisition of new genes encoding enzymes that hydrolyze or modify the drug provides an AMR phenotype, with perhaps the most salient example seen in the multigenerational β-lactam developments towards carbapenems (Box 1). Fourthly, membrane remodelling to prevent drug influx at the cell surface, thereby protecting the internal compartments where most drug targets reside.Box 1Mechanisms for acquiring an AMR phenotypeMutations that alter the target of the antimicrobial drug to decrease (or inhibit) drug-binding to the target will generate an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design drugs that would hit their target in such a way that any change in the target would result in loss-of-function. An example of this is the way in which linezolid binds to bacterial ribosomes: only one other escape conformation is possible in the ribosome and a second 'linezolid-like' drug has been proposed to block this site [55.Belousoff M.J. et al.cryoEM-guided development of antibiotics for drug-resistant bacteria.ChemMedChem. 2019; 14: 527-531Crossref PubMed Scopus (16) Google Scholar].The modification of existing genes, or acquisition of new genes encoding drug-efflux pumps, provides an AMR phenotype. The drug developers’ solution to this scenario involves drugs that would inhibit the efflux pump, to be used in combination with the primary antibiotic. Current inhibitors have issues with toxicity, but recent examples suggest progress both in the types of inhibitors that could be identified and in dosing strategies that could enter future clinical trials [56.Ferrer-Espada R. et al.A permeability-increasing drug synergizes with bacterial efflux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains.Sci. Rep. 2019; 9: 3452Crossref PubMed Scopus (51) Google Scholar, 57.Marshall R.L. et al.New multidrug efflux inhibitors for Gram-negative bacteria.mBio. 2020; 11e01340-20Crossref Scopus (18) Google Scholar, 58.Zwama M. Nishino K. Ever-adapting RND efflux pumps in Gram-negative multidrug-resistant pathogens: a race against time.Antibiotics (Basel). 2021; : 10PubMed Google Scholar].The acquisition of new genes encoding enzymes that hydrolyse the drug provides an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design new-generation drugs that are resistant to existing enzymes. An example of this is provided by the four generations of β-lactam drugs currently in clinical use. The acquisition of genes encoding penicillinases led to the development of cephalosporins. The acquisition of genes encoding cephalosporinases led to the development of later generations of cephems, such as cefotaxime. The acquisition of genes encoding extended-spectrum β-lactamases led to the development of carbapenems. The acquisition of genes encoding carbapenemases is currently without solution.Remodelling of the cell surface to prevent drug influx protects the internal compartments where most drug targets reside. The drug developers’ solution to this scenario is to design new-generation drugs that target surface-exposed features. Examples of this are the recent successes in targeting features exposed on the outer surface of Gram-negative bacteria, such as the β-barrel assembly machinery [59.Steenhuis M. et al.A ban on BAM: an update on inhibitors of the beta-barrel assembly machinery.FEMS Microbiol. Lett. 2021; 368fnab059Crossref PubMed Scopus (10) Google Scholar]. Mutations that alter the target of the antimicrobial drug to decrease (or inhibit) drug-binding to the target will generate an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design drugs that would hit their target in such a way that any change in the target would result in loss-of-function. An example of this is the way in which linezolid binds to bacterial ribosomes: only one other escape conformation is possible in the ribosome and a second 'linezolid-like' drug has been proposed to block this site [55.Belousoff M.J. et al.cryoEM-guided development of antibiotics for drug-resistant bacteria.ChemMedChem. 2019; 14: 527-531Crossref PubMed Scopus (16) Google Scholar]. The modification of existing genes, or acquisition of new genes encoding drug-efflux pumps, provides an AMR phenotype. The drug developers’ solution to this scenario involves drugs that would inhibit the efflux pump, to be used in combination with the primary antibiotic. Current inhibitors have issues with toxicity, but recent examples suggest progress both in the types of inhibitors that could be identified and in dosing strategies that could enter future clinical trials [56.Ferrer-Espada R. et al.A permeability-increasing drug synergizes with bacterial efflux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains.Sci. Rep. 2019; 9: 3452Crossref PubMed Scopus (51) Google Scholar, 57.Marshall R.L. et al.New multidrug efflux inhibitors for Gram-negative bacteria.mBio. 2020; 11e01340-20Crossref Scopus (18) Google Scholar, 58.Zwama M. Nishino K. Ever-adapting RND efflux pumps in Gram-negative multidrug-resistant pathogens: a race against time.Antibiotics (Basel). 2021; : 10PubMed Google Scholar]. The acquisition of new genes encoding enzymes that hydrolyse the drug provides an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design new-generation drugs that are resistant to existing enzymes. An example of this is provided by the four generations of β-lactam drugs currently in clinical use. The acquisition of genes encoding penicillinases led to the development of cephalosporins. The acquisition of genes encoding cephalosporinases led to the development of later generations of cephems, such as cefotaxime. The acquisition of genes encoding extended-spectrum β-lactamases led to the development of carbapenems. The acquisition of genes encoding carbapenemases is currently without solution. Remodelling of the cell surface to prevent drug influx protects the internal compartments where most drug targets reside. The drug developers’ solution to this scenario is to design new-generation drugs that target surface-exposed features. Examples of this are the recent successes in targeting features exposed on the outer surface of Gram-negative bacteria, such as the β-barrel assembly machinery [59.Steenhuis M. et al.A ban on BAM: an update on inhibitors of the beta-barrel assembly machinery.FEMS Microbiol. Lett. 2021; 368fnab059Crossref PubMed Scopus (10) Google Scholar]. In this opinion piece we explore the relationships between bacterial membrane remodelling and AMR. As our awareness grows that many of the worst-case scenarios of bacterial superbug lineages spreading globally are composite phenotypes, addressing the issue of membrane remodelling could be an effective means to resensitize bacteria to existing antibiotics. It is worthy of discussion: the 20th century has taught us that the silver bullet approach of a new antibiotic drug can never be a sustainable solution to AMR, with each new drug failing due to AMR after just a few years in clinical use. This remains true in this first part of the 21st century. Yet we almost know enough about bacterial cell biology to be inventive in how we might push back on evolution to restore susceptibility to some existing drugs within our antibacterial arsenal. It is our opinion that understanding just a few more features of the fundamental biology of bacterial outer membranes will both assist in the development of novel therapies and restore our ability to use existing drugs to treat what would otherwise be AMR infections. All of the antibiotic drugs in widespread clinical use in the 20th century targeted bacterial cell processes via binding to targets in internal compartments of the bacterial cell (Figure 1). In order to do so in Gram-negative bacteria, these drugs have to permeate the bacterial outer membrane [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google Scholar,2.Prajapati J.D. et al.How to enter a bacterium: bacterial porins and the permeation of antibiotics.Chem. Rev. 2021; 121: 5158-5192Crossref PubMed Scopus (62) Google Scholar]. The means by which this otherwise highly impermeable lipopolysaccharide (LPS)–phospholipid barrier can be breached by drugs is via the major porins. Almost all of the Gram-negative bacteria studied to date express a major porin: 'major' in the sense that it is the most abundant protein integrated in the outer membrane, and 'porin' in the sense that it has a central luminal space that serves as an aqueous pore through which nutrients and water-soluble drugs can pass. In the case of Escherichia coli, the major porin represents more than 50% of the total protein integrated in the outer membrane [2.Prajapati J.D. et al.How to enter a bacterium: bacterial porins and the permeation of antibiotics.Chem. 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The outer-membrane is subject to remodelling OmpC or is the major in or OmpC is the major porin in the E. coli outer membrane, under or in is the major porin in the membrane et to of the and OmpC porins in Escherichia Microbiol. PubMed Scopus Google Scholar]. OmpC and are almost with the to a few of the that at the cell surface et of E. coli PubMed Scopus Google Scholar], and we to to these porins with the in E. coli also that is on OmpC in to carbapenems and et of OmpC and in Escherichia coli against of carbapenem 2019; PubMed Scopus Google et of OmpC and by in Escherichia coli exposed to and Proteom. PubMed Scopus Google Scholar]. OmpC is on in to or et of OmpC and by in Escherichia coli exposed to and Proteom. PubMed Scopus Google et of outer membrane in a of in Escherichia coli.J. Proteom. PubMed Scopus (10) Google Scholar]. The major porin in E. coli is a for this outer-membrane remodelling and it is not For K. pneumoniae has four genes and encoding major porins that are et in of the outer membrane antimicrobial in Klebsiella 2020; PubMed Scopus Google Scholar]. It is not which environmental of each of these genes, is to et in clinical of Klebsiella PubMed Scopus Google Scholar]. remodelling of the outer membrane, these porins and to in which drugs they to enter into K. with consequences and for AMR phenotypes [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google et in of the outer membrane antimicrobial in Klebsiella 2020; PubMed Scopus Google Scholar]. it is that many clinical strains of Klebsiella have their outer membrane to express major and this these strains highly resistant to carbapenems [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google et in of the outer membrane antimicrobial in Klebsiella 2020; PubMed Scopus Google et description of antimicrobial resistance in Klebsiella pneumoniae after driven by outer membrane Microbiol. 2020; PubMed Scopus Google J.D. et Klebsiella a key set for global PubMed Scopus Google Scholar]. Remodelling the of the outer membrane on the and of existing membrane and the of of new proteins into the outer membrane (Figure is known about the for membrane protein from the outer membrane, a set of do outer-membrane proteins The of the 2016; PubMed Scopus Google Scholar, et al.The of is for with proteins and the beta-barrel assembly machinery Microbiol. PubMed Scopus (18) Google Scholar, et for in the of essential outer membrane protein PubMed Scopus Google Scholar]. The process, the of porin into the outer membrane, is The process in the with the of porin the membrane, and into the at the outer the porin is integrated into the membrane by the beta-barrel assembly machinery et membrane protein biogenesis in Gram-negative R. Biol. PubMed Scopus Google Scholar, et membrane Rev. Microbiol. PubMed Scopus Google Scholar, to the membrane with a 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, D. et for the assembly of beta-barrel Microbiol. Lett. 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PubMed Scopus Google Scholar], a of way of the to environmental of or or antimicrobial to the porin composition of the outer membrane The of the 2016; PubMed Scopus Google and of porins and efflux pumps in drug Microbiol. Rev. PubMed Scopus Google an that can 2020; PubMed Scopus Google Scholar]. and other of the envelope also into this or antimicrobial drugs et of the envelope is for cell in Escherichia 2016; PubMed Scopus Google is new an update on research on the envelope 2014; PubMed Scopus Google Scholar], and the of increases in the presence of drug with the envelope to the outer membrane in to β-lactam antibiotics et of the and the porin in to antibiotic in Escherichia (Basel). 6: Scopus (18) Google Scholar]. These can be to the outer membrane in several of bacteria, and are this membrane remodelling M. et in PubMed Scopus Google E. coli by PubMed Scopus Google Scholar]. is also the that the remodelling signals to the protein of E. coli in to β-lactam drugs or that of the and are the is to the outer membrane et of outer membrane of Escherichia coli to resistance to and 6: PubMed Scopus Google Scholar]. remodelling it is a target to AMR phenotypes and/or the evolutionary towards AMR. The can the remodelling of bacterial outer membranes be to resensitize bacteria in an site or a to be to existing drugs. that this is a worthy and a issue from the use of this strategy in M. et in PubMed Scopus Google E. coli by PubMed Scopus Google with a awareness that AMR phenotypes are to that are not as as they might have that in the outer-membrane of K. pneumoniae and confer antibiotic resistance, but with an and for these bacteria et of the multidrug efflux pump PubMed Scopus Google Scholar, et a novel porin with carbapenem resistance in Klebsiella PubMed Scopus Google Scholar, et and impact of mutations in Pseudomonas aeruginosa strains in 2021; Full Text Full Text PDF PubMed Scopus Google Scholar, et of the outer membrane protein with and of PubMed Scopus Google Scholar]. in an environmental where drugs are strains that the outer membrane to drug-resistant would have a against bacteria. is is in an where drugs are the outer membrane would on the to it to against bacteria. In mutations AMR are in the of the drug et bacteria for the between Biol. PubMed Scopus Google Scholar]. The of an AMR phenotype could by of a by which the outer membrane is through mutations that (or the outer-membrane to evolution of a phenotype, or a where the to against a that can in a The mechanisms that control membrane remodelling are worthy of for that can on porin would be a means of carbapenem sensitivity in an The need to these is many strains of and Pseudomonas are in to have an but with a that on would for [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google et in of the outer membrane antimicrobial in Klebsiella 2020; PubMed Scopus Google M. et in PubMed Scopus Google M. et and mechanisms carbapenem and resistance in a of 2020; PubMed Scopus Google Scholar, et of in and strains of Pseudomonas aeruginosa from with in a PubMed Scopus Google Scholar, et al.The outer membrane proteins and of confer a in as a human Microbiol. 2020; PubMed Scopus Google Scholar]. The towards phage a powerful means to treat with AMR phenotypes the mechanisms (Box do not impact bacterial susceptibility to phage and bacterial in the Microbiol. Rev. 2019; PubMed Scopus Google Scholar]. we one issue that be into selecting which to use in these In our opinion, that use major porins as their be used in therapy, these would selective on the bacterial to in order to the of is the of the surface F. et are to Microbiol. 2021; 6: PubMed Scopus Google the of phage the key is in the 2021; PubMed Scopus (51) Google Scholar]. The is that that use a major porin as their would be to select for AMR phenotypes in a that can be by of outer-membrane remodelling (Figure are being made in this to antibiotics in the case of phage of multidrug-resistant strains F. et are to Microbiol. 2021; 6: PubMed Scopus Google et resistance mechanisms sensitivity in 2021; Scholar]. The in of AMR is a major to the it is of the global and Gram-negative bacteria of are as urgent by the The to AMR is to be in the and in the The for other are not but will be at as the impact in is to in a to to each by and with a by the M. impact of antibiotic 2019; PubMed Scopus Google and development of effective drug is In the 20th century has taught us that a new antibiotic drug will never be a sustainable solution to AMR. We novel such as phage therapy, to the mechanisms that multidrug-resistant bacteria use to In our opinion, knowledge of bacterial membrane remodelling and the use of evolution and selection for to existing drugs are considerations that need to be in the for development of new antimicrobial drugs and other new therapies (see We that only with will the the urgent of antimicrobial drugs that target essential features at the outer surface of the outer membrane be used to the mechanisms for the evolution of a for of specific outer-membrane such a could have a by with the of be so that the of phage resistance in an site in an in drug This would a to most of the bacteria in the site population by by any with existing antimicrobial drugs. antimicrobial drugs that target essential features at the outer surface of the outer membrane be used to the mechanisms for the evolution of a for of specific outer-membrane such a could have a by with the of porins. be so that the of phage resistance in an site in an in drug This would a to most of the bacteria in the site population by by any with existing antimicrobial drugs. We and for on the by the this is from a population of bacteria in a of and/or and/or The to an or surface and protects bacteria from environmental including antimicrobial drugs. antimicrobial drugs to the β-lactam inhibit cell by binding to the proteins that the of the cell have against both Gram-negative and bacteria. the and outer efflux pumps are that such as and antimicrobial drugs. a of the and of efflux pumps is an by which an AMR phenotype is bacteria with an outer membrane are not to and so are of the pathogens identified for urgent by the and the are including the extended-spectrum and of E. coli, K. and modification or of membrane that specific proteins or to to a new environmental This opinion on the remodelling of membranes by in the membrane protein the is the of proteins by an and the outer-membrane is that of the in the outer the use of that bacteria, that also to treat bacterial in human and outer-membrane proteins that are as a the and a central pore in the β-barrel assist the envelope of Gram-negative bacteria and as to diffusion of including antimicrobial drugs. The is the protein that the outer-membrane surface 50% of the total protein integrated in the outer

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