RESUMO
The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.
Assuntos
Bactérias Gram-Negativas/efeitos dos fármacos , Pirróis/metabolismo , Pirróis/farmacologia , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Animais , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Feminino , Ácido Fólico/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Ovariectomia , Proteômica , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation ex vivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.
Assuntos
Plaquetas/efeitos dos fármacos , Fatores de Transcrição NFATC/antagonistas & inibidores , Agregação Plaquetária/efeitos dos fármacos , Sepse/patologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Comunicação Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Humanos , Inflamação , Camundongos , Fatores de Transcrição NFATC/metabolismo , Neutrófilos/metabolismo , Receptores de Trombina/metabolismo , Sepse/metabolismoRESUMO
Lipopolysaccharide molecules represent a unique family of glycolipids based on a highly conserved lipid moiety known as lipid A. These molecules are produced by most gram-negative bacteria, in which they play important roles in the integrity of the outer-membrane permeability barrier and participate extensively in host-pathogen interplay. Few bacteria contain lipopolysaccharide molecules composed only of lipid A. In most forms, lipid A is glycosylated by addition of the core oligosaccharide that, in some bacteria, provides an attachment site for a long-chain O-antigenic polysaccharide. The complexity of lipopolysaccharide structures is reflected in the processes used for their biosynthesis and export. Rapid growth and cell division depend on the bacterial cell's capacity to synthesize and export lipopolysaccharide efficiently and in large amounts. We review recent advances in those processes, emphasizing the reactions that are essential for viability.
Assuntos
Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Bactérias , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Glicolipídeos/metabolismo , Glicosilação , Bactérias Gram-Negativas/metabolismo , Antígenos O/metabolismo , Permeabilidade , Polissacarídeos/metabolismoRESUMO
The cell envelope is a multilayered structure that insulates the interior of bacterial cells from an often chaotic outside world. Common features define the envelope across the bacterial kingdom, but the molecular mechanisms by which cells build and regulate this critical barrier are diverse and reflect the evolutionary histories of bacterial lineages. Intracellular pathogens of the genus Brucella exhibit marked differences in cell envelope structure, regulation, and biogenesis when compared to more commonly studied gram-negative bacteria and therefore provide an excellent comparative model for study of the gram-negative envelope. We review distinct features of the Brucella envelope, highlighting a conserved regulatory system that links cell cycle progression to envelope biogenesis and cell division. We further discuss recently discovered structural features of the Brucella envelope that ensure envelope integrity and that facilitate cell survival in the face of host immune stressors.
Assuntos
Brucella , Parede Celular , Membrana Celular , Evolução Biológica , Brucella/genética , Divisão CelularRESUMO
All bacterial cells must expand their envelopes during growth. The main load-bearing and shape-determining component of the bacterial envelope is the peptidoglycan cell wall. Bacterial envelope growth and shape changes are often thought to be controlled through enzymatic cell wall insertion. We investigated the role of cell wall insertion for cell shape changes during cell elongation in Gram-negative bacteria. We found that both global and local rates of envelope growth of Escherichia coli remain nearly unperturbed upon arrest of cell wall insertion-up to the point of sudden cell lysis. Specifically, cells continue to expand their surface areas in proportion to biomass growth rate, even if the rate of mass growth changes. Other Gram-negative bacteria behave similarly. Furthermore, cells plastically change cell shape in response to differential mechanical forces. Overall, we conclude that cell wall-cleaving enzymes can control envelope growth independently of synthesis. Accordingly, the strong overexpression of an endopeptidase leads to transiently accelerated bacterial cell elongation. Our study demonstrates that biomass growth and envelope forces can guide cell envelope expansion through mechanisms that are independent of cell wall insertion.
Assuntos
Parede Celular , Escherichia coli , Parede Celular/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Ciclo Celular , Bactérias Gram-Negativas/metabolismo , Peptidoglicano/metabolismoRESUMO
Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.
Assuntos
Antibacterianos , Lipopolissacarídeos , Humanos , Antibacterianos/química , Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , beta-Lactamases/genética , Testes de Sensibilidade MicrobianaRESUMO
The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Membrana/metabolismo , Transporte Biológico , Lipoproteínas/metabolismoRESUMO
Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.
Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli/genética , beta-Lactamases/genética , beta-Lactamases/farmacologia , Proteômica , Farmacorresistência Bacteriana , Farmacorresistência Bacteriana Múltipla/genética , Klebsiella pneumoniae , Testes de Sensibilidade MicrobianaRESUMO
The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the Escherichia coli TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia Crioeletrônica , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismoRESUMO
SUMMARYDespite the early recognition of their therapeutic potential and the current escalation of multidrug-resistant (MDR) pathogens, the adoption of bacteriophages into mainstream clinical practice is hindered by unfamiliarity with their basic pharmacokinetic (PK) and pharmacodynamic (PD) properties, among others. Given the self-replicative nature of bacteriophages in the presence of host bacteria, the adsorption rate, and the clearance by the host's immunity, their PK/PD characteristics cannot be estimated by conventional approaches, and thus, the introduction of new considerations is required. Furthermore, the multitude of different bacteriophage types, preparations, and treatment schedules impedes drawing general conclusions on their in vivo PK/PD features. Additionally, the drawback of acquired bacteriophage resistance of MDR pathogens with clinical and environmental implications should be taken into consideration. Here, we provide an overview of the current state of the field of PK and PD of bacteriophage therapy with a focus on its application against MDR Gram-negative infections, highlighting the potential knowledge gaps and the challenges in translation from the bench to the bedside. After reviewing the in vitro PKs and PDs of bacteriophages against the four major MDR Gram-negative pathogens, Klebsiella pneumoniae, Acinetobacter baumannii complex, Pseudomonas aeruginosa, and Escherichia coli, specific data on in vivo PKs (tissue distribution, route of administration, and basic PK parameters in animals and humans) and PDs (survival and reduction of bacterial burden in relation to the route of administration, timing of therapy, dosing regimens, and resistance) are summarized. Currently available data merit close scrutiny, and optimization of bacteriophage therapy in the context of a better understanding of the underlying PK/PD principles is urgent to improve its therapeutic effect and to minimize the occurrence of bacteriophage resistance.
RESUMO
Uropathogenic Escherichia coli (UPEC) secrete multiple siderophore types to scavenge extracellular iron(III) ions during clinical urinary tract infections, despite the metabolic costs of biosynthesis. Here, we find the siderophore enterobactin (Ent) and its related products to be prominent components of the iron-responsive extracellular metabolome of a model UPEC strain. Using defined Ent biosynthesis and import mutants, we identify lower molecular weight dimeric exometabolites as products of incomplete siderophore catabolism, rather than prematurely released biosynthetic intermediates. In E. coli, iron acquisition from iron(III)-Ent complexes requires intracellular esterases that hydrolyze the siderophore. Although UPEC are equipped to consume the products of completely hydrolyzed Ent, we find that Ent and its derivatives may be incompletely hydrolyzed to yield products with retained siderophore activity. These results are consistent with catabolic inefficiency as means to obtain more than one iron ion per siderophore molecule. This is compatible with an evolved UPEC strategy to maximize the nutritional returns from metabolic investments in siderophore biosynthesis.
Assuntos
Sideróforos , Escherichia coli Uropatogênica , Enterobactina/metabolismo , Compostos Férricos/metabolismo , Ferro/metabolismo , Sideróforos/metabolismo , Escherichia coli Uropatogênica/metabolismoRESUMO
The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Cobre , Salmonella , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Homeostase , Oxirredução , Oxirredutases/metabolismo , Salmonella/metabolismo , Compostos de Sulfidrila , Proteínas de Transporte/metabolismoRESUMO
Gram-negative bacteria use TonB-dependent transport to take up nutrients from the external environment, employing the Ton complex to import a variety of nutrients that are either scarce or too large to cross the outer membrane unaided. The Ton complex contains an inner-membrane motor (ExbBD) that generates force, as well as nutrient-specific transport proteins on the outer membrane. These two components are coupled by TonB, which transmits the force from the inner to the outer membrane. TonB contains an N-terminus anchored in the inner membrane, a C-terminal domain that binds the outer-membrane transporter, and a proline-rich linker connecting the two. While much is known about the interaction between TonB and outer-membrane transporters, the critical interface between TonB and ExbBD is less well understood. Here, we identify a conserved motif within TonB that we term the D-box, which serves as an attachment point for ExbD. We characterize the interaction between ExbD and the D-box both functionally and structurally, showing that a homodimer of ExbD captures one copy of the D-box peptide via beta-strand recruitment. We additionally show that both the D-box motif and ExbD are conserved in a range of Gram-negative bacteria, including members of the ESKAPE group of pathogens. The ExbD:D-box interaction is likely to represent an important aspect of force transduction between the inner and outer membranes. Given that TonB-dependent transport is an important contributor to virulence, this interaction is an intriguing potential target for novel antibacterial therapies.
Assuntos
Proteínas de Bactérias , Proteínas de Membrana , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ligação ProteicaRESUMO
As species diverge, a wide range of evolutionary processes lead to changes in protein-protein interaction (PPI) networks and metabolic networks. The rate at which molecular networks evolve is an important question in evolutionary biology. Previous empirical work has focused on interactomes from model organisms to calculate rewiring rates, but this is limited by the relatively small number of species and sparse nature of network data across species. We present a proxy for variation in network topology: variation in drug-drug interactions (DDIs), obtained by studying drug combinations (DCs) across taxa. Here, we propose the rate at which DDIs change across species as an estimate of the rate at which the underlying molecular network changes as species diverge. We computed the evolutionary rates of DDIs using previously published data from a high-throughput study in gram-negative bacteria. Using phylogenetic comparative methods, we found that DDIs diverge rapidly over short evolutionary time periods, but that divergence saturates over longer time periods. In parallel, we mapped drugs with known targets in PPI and cofunctional networks. We found that the targets of synergistic DDIs are closer in these networks than other types of DCs and that synergistic interactions have a higher evolutionary rate, meaning that nodes that are closer evolve at a faster rate. Future studies of network evolution may use DC data to gain larger-scale perspectives on the details of network evolution within and between species.
Assuntos
Filogenia , Evolução Molecular , Mapas de Interação de Proteínas , Interações Medicamentosas , Bactérias Gram-Negativas/genética , Evolução Biológica , Redes e Vias MetabólicasRESUMO
Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.
Assuntos
Bacteriocinas/química , Colicinas/química , Escherichia coli/metabolismo , Porinas/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Sítios de Ligação , Colicinas/genética , Colicinas/metabolismo , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Porinas/genética , Porinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , TermodinâmicaRESUMO
Antibacterial drug resistance of gram-negative bacteria (GNB) results in high morbidity and mortality of GNB infection, seriously threaten human health globally. Developing new antibiotics has become the critical need for dealing with drug-resistant bacterial infections. Cefiderocol is an iron carrier cephalosporin that achieves drug accumulation through a unique "Trojan horse" strategy into the bacterial periplasm. It shows high antibacterial activity against multidrug-resistant (MDR) Enterobacteriaceae and MDR non-fermentative bacteria. The application of cefiderocol offers new hope for treating clinical drug-resistant bacterial infections. However, limited clinical data and uncertainties about its resistance mechanisms constrain the choice of its therapeutic use. This review aimed to summarize the clinical applications, drug resistance mechanisms, and co-administration of cefiderocol.
Assuntos
Cefiderocol , Infecções por Bactérias Gram-Negativas , Humanos , Sideróforos/farmacologia , Sideróforos/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefalosporinas/farmacologia , Cefalosporinas/uso terapêutico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Bactérias Gram-Negativas , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade MicrobianaRESUMO
In this article, we describe the development of the plant immunity field, starting with efforts to understand the genetic basis for disease resistance, which â¼30 y ago led to the discovery of diverse classes of immune receptors that recognize and respond to infectious microbes. We focus on knowledge gained from studies of the rice XA21 immune receptor that recognizes RaxX (required for activation of XA21 mediated immunity X), a sulfated microbial peptide secreted by the gram-negative bacterium Xanthomonas oryzae pv. oryzae. XA21 is representative of a large class of plant and animal immune receptors that recognize and respond to conserved microbial molecules. We highlight the complexity of this large class of receptors in plants, discuss a possible role for RaxX in Xanthomonas biology, and draw attention to the important role of sulfotyrosine in mediating receptor-ligand interactions.
Assuntos
Resistência à Doença/imunologia , Oryza/imunologia , Proteínas de Plantas/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Agricultura/história , Alergia e Imunologia/história , Alergia e Imunologia/tendências , Infecções Bacterianas/genética , Proteínas de Bactérias/genética , Resistência à Doença/genética , História do Século XIX , História do Século XX , História do Século XXI , Peptídeos/química , Doenças das Plantas/microbiologia , Imunidade Vegetal/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
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 ß-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 â¼200 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.
Assuntos
Antibacterianos/metabolismo , Farmacorresistência Bacteriana/fisiologia , Porinas/metabolismo , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Bactérias Gram-Negativas/metabolismo , Testes de Sensibilidade Microbiana , Modelos Teóricos , Simulação de Dinâmica Molecular , Permeabilidade , Porinas/fisiologia , Porinas/ultraestruturaRESUMO
Gram-negative (G-) bacterial infections remain one of the most urgent global health threats, because the distinctive envelope structure hinders the penetration of therapeutics. Here, we showed that a perfluorooctyl bromide nanoemulsion (PFOB NE) uniquely interacts with G- bacteria. After cell envelope attachment, the PFOB can infiltrate the cell and was diffused throughout. In this process, it impaired the membranes by disintegrating phospholipid molecules, enhancing the consequent ultrasonic cavitation to break the envelope. We identified through ultrasound that the NE had remarkable bactericidal effects against various antibiotic-resistant pathogens. Using in situ sterilization, this approach accelerated the recovery of bacteria-infected murine skin wounds. Thus, combining PFOB and ultrasound might be an alternative tool for conquering the growing threat of G- pathogens.
Assuntos
Fluorocarbonos , Hidrocarbonetos Bromados , Camundongos , Animais , Bactérias Gram-Negativas , Fluorocarbonos/química , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
Pseudomonas aeruginosa is a challenging opportunistic pathogen due to its intrinsic and acquired mechanisms of antibiotic resistance. A large repertoire of efflux transporters actively expels antibiotics, toxins, and metabolites from cells and enables growth of P. aeruginosa in diverse environments. In this study, we analyzed the roles of representative efflux pumps from the Resistance-Nodulation-Division (RND), Major Facilitator Superfamily (MFS), and Small Multidrug Resistance (SMR) families of proteins in the susceptibility of P. aeruginosa to antibiotics and bacterial growth under stresses imposed by human hosts during bacterial infections: an elevated temperature, osmotic stress, low iron, bile salts, and acidic pH. We selected five RND pumps MexAB-OprM, MexEF-OprN, MexCD-OprJ, MuxABC-OpmB, and TriABC-OpmH that differ in their substrate specificities and expression profiles, two MFS efflux pumps PA3136-3137 and PA5158-5160 renamed here into MfsAB and MfsCD-OpmG, respectively, and an SMR efflux transporter PA1540-1541 (MdtJI). We found that the most promiscuous RND pumps such as MexEF-OprN and MexAB-OprM are integrated into diverse survival mechanisms and enable P. aeruginosa growth under various stresses. MuxABC-OpmB and TriABC-OpmH pumps with narrower substrate spectra are beneficial only in the presence of the iron chelator 2,2'-dipyridyl and bile salts, respectively. MFS pumps do not contribute to antibiotic efflux but play orthogonal roles in acidic pH, low iron, and in the presence of bile salts. In contrast, MdtJI protects against polycationic antibiotics but does not contribute to survival under stress. Thus, efflux pumps play specific, non-interchangeable functions in P. aeruginosa cell physiology and bacterial survival under stresses. IMPORTANCE: The role of multidrug efflux pumps in the intrinsic and clinical levels of antibiotic resistance in Pseudomonas aeruginosa and other gram-negative bacteria is well-established. Their functions in bacterial physiology, however, remain unclear. The P. aeruginosa genome comprises an arsenal of efflux pumps from different protein families, the substrate specificities of which are typically assessed by measuring their impact on susceptibility to antibiotics. In this study, we analyzed how deletions and overproductions of efflux pumps affect P. aeruginosa growth under human-infection-induced stresses. Our results show that the physiological functions of multidrug efflux pumps are non-redundant and essential for the survival of this important human pathogen under stress.