ABSTRACT
BACKGROUND: Lysins (cell wall hydrolases) targeting gram-negative organisms require engineering to permeabilize the outer membrane and access subjacent peptidoglycan to facilitate killing. In the current study, the potential clinical utility for the engineered lysin CF-370 was examined in vitro and in vivo against gram-negative pathogens important in human infections. METHODS: Minimum inhibitory concentration (MICs) and bactericidal activity were determined using standard methods. An in vivo proof-of-concept efficacy study was conducted using a rabbit acute pneumonia model caused by Pseudomonas aeruginosa. RESULTS: CF-370 exhibited potent antimicrobial activity, with MIC50/90 values (in µg/mL) for: P aeruginosa, 1/2; Acinetobacter baumannii, 1/1; Escherichia coli, 0.25/1; Klebsiella pneumoniae, 2/4; Enterobacter cloacae 1/4; and Stenotrophomonas maltophilia 2/8. CF-370 furthermore demonstrated bactericidal activity, activity in serum, a low propensity for resistance, anti-biofilm activity, and synergy with antibiotics. In the pneumonia model, CF-370 alone decreased bacterial densities in lungs, kidneys, and spleen versus vehicle control, and demonstrated significantly increased efficacy when combined with meropenem (vs either agent alone). CONCLUSIONS: CF-370 is the first engineered lysin described with potent broad-spectrum in vitro activity against multiple clinically relevant gram-negative pathogens, as well as potent in vivo efficacy in an animal model of severe invasive multisystem infection.
Subject(s)
Anti-Bacterial Agents , Drug Synergism , Meropenem , Microbial Sensitivity Tests , Pneumonia, Bacterial , Pseudomonas Infections , Pseudomonas aeruginosa , Animals , Meropenem/pharmacology , Meropenem/therapeutic use , Meropenem/administration & dosage , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Rabbits , Pseudomonas aeruginosa/drug effects , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pneumonia, Bacterial/drug therapy , Pneumonia, Bacterial/microbiology , Disease Models, Animal , Gram-Negative Bacteria/drug effects , Biofilms/drug effectsABSTRACT
This corrects the article DOI: 10.1038/nature23874.
ABSTRACT
Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).
Subject(s)
Amides/metabolism , Bacteria/metabolism , Biological Mimicry , Gastrointestinal Tract/microbiology , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Symbiosis , Amides/chemistry , Animals , Bacteria/enzymology , Bacteria/genetics , Blood Glucose/metabolism , Female , Gastrointestinal Microbiome/genetics , Gastrointestinal Microbiome/physiology , Gastrointestinal Tract/metabolism , HEK293 Cells , Homeostasis , Humans , Ligands , Male , MiceABSTRACT
Bacterial natural products have inspired the development of numerous antibiotics in use today. As resistance to existing antibiotics has become more prevalent, new antibiotic lead structures and activities are desperately needed. An increasing number of natural product biosynthetic gene clusters, to which no known molecules can be assigned, are found in genome and metagenome sequencing data. Here we access structural information encoded in this underexploited resource using a synthetic-bioinformatic natural product (syn-BNP) approach, which relies on bioinformatic algorithms followed by chemical synthesis to predict and then produce small molecules inspired by biosynthetic gene clusters. In total, 157 syn-BNP cyclic peptides inspired by 96 nonribosomal peptide synthetase gene clusters were synthesized and screened for antibacterial activity. This yielded nine antibiotics with activities against ESKAPE pathogens as well as Mycobacterium tuberculosis. Not only are antibiotic-resistant pathogens susceptible to many of these syn-BNP antibiotics, but they were also unable to develop resistance to these antibiotics in laboratory experiments. Characterized modes of action for these antibiotics include cell lysis, membrane depolarization, inhibition of cell wall biosynthesis, and ClpP protease dysregulation. Increasingly refined syn-BNP-based explorations of biosynthetic gene clusters should allow for more rapid identification of evolutionarily inspired bioactive small molecules, in particular antibiotics with diverse mechanism of actions that could help confront the imminent crisis of antimicrobial resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biological Products/pharmacology , Computational Biology , Mycobacterium tuberculosis/drug effects , Algorithms , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Microbial Sensitivity Tests , Molecular StructureABSTRACT
Bioinformatic analysis of sequenced bacterial genomes has uncovered an increasing number of natural product biosynthetic gene clusters (BGCs) to which no known bacterial metabolite can be ascribed. One emerging method we have investigated for studying these BGCs is the synthetic-Bioinformatic Natural Product (syn-BNP) approach. The syn-BNP approach replaces transcription, translation, and in vivo enzymatic biosynthesis of natural products with bioinformatic algorithms to predict the output of a BGC and in vitro chemical synthesis to produce the predicted structure. Here we report on expanding the syn-BNP approach to the design and synthesis of cyclic peptides inspired by nonribosomal peptide synthetase BGCs associated with the human microbiota. While no syn-BNPs we tested inhibited the growth of bacteria or yeast, five were found to be active in the human cell-based MTT metabolic activity assay. Interestingly, active peptides were mostly inspired by BGCs found in the genomes of opportunistic pathogens that are often more commonly associated with environments outside the human microbiome. The cyclic syn-BNP studies presented here provide further evidence of its potential for identifying bioactive small molecules directly from the instructions encoded in the primary sequences of natural product BGCs.
Subject(s)
Biological Products/chemistry , Biosynthetic Pathways/genetics , Microbiota/genetics , Multigene Family , Peptide Synthases/genetics , Peptides, Cyclic/chemistry , Computational Biology/methods , Genome, Bacterial , Humans , Molecular StructureABSTRACT
Objectives: Preventing bacterial contact with host cells can provide an additional approach to tackling MDR Acinetobacter baumannii. Recently, we identified AOA-2 as a potential blocker of A. baumannii outer membrane protein A without presenting bactericidal activity. Here, we aimed to study whether AOA-2 can increase the activity of colistin against colistin-resistant A. baumannii in vitro and in vivo. Methods: Reference and clinical A. baumannii strains susceptible and resistant to colistin (CST-S and CST-R) were used. Microdilution and time-kill curve assays were performed to determine the synergy between AOA-2 and colistin. SDS-PAGE assays with CST-S and CST-R outer membrane proteins and MALDI-TOF-TOF (MS-MS/MS) analysis were performed to determine the AOA-2 and colistin synergy mechanism. In a murine peritoneal sepsis model, the therapeutic efficacy of AOA-2 (10 mg/kg/24 h) in combination with a sub-optimal dose of colistin (10 mg/kg/24 h) against CST-R was evaluated by determining the bacterial load in tissues and blood, and mouse survival. Results: We showed that AOA-2 increased the in vitro colistin susceptibility of reference and clinical CST-S and CST-R strains. This combination also enhanced their killing activity after 24 h of drug exposure. This synergy is mediated by the overexpression of Omp25. In vivo, the combination of AOA-2 with colistin significantly reduced the bacterial load in tissues and blood, and increased mouse survival, compared with colistin monotherapy. Conclusions: We identified a novel class of antimicrobial agents that has proven to be effective in combination with colistin in an experimental model of severe infection by CST-R A. baumannii.
Subject(s)
Acinetobacter Infections/microbiology , Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Colistin/pharmacology , Drug Synergism , Enzyme Inhibitors/pharmacology , Acinetobacter Infections/drug therapy , Animals , Anti-Bacterial Agents/administration & dosage , Colistin/administration & dosage , Disease Models, Animal , Enzyme Inhibitors/administration & dosage , Female , Mice, Inbred C57BL , Microbial Sensitivity Tests , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Treatment OutcomeABSTRACT
Here we present a natural product discovery approach, whereby structures are bioinformatically predicted from primary sequence and produced by chemical synthesis (synthetic-bioinformatic natural products, syn-BNPs), circumventing the need for bacterial culture and gene expression. When we applied the approach to nonribosomal peptide synthetase gene clusters from human-associated bacteria, we identified the humimycins. These antibiotics inhibit lipid II flippase and potentiate ß-lactam activity against methicillin-resistant Staphylococcus aureus in mice, potentially providing a new treatment regimen.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Biological Products/isolation & purification , Biological Products/pharmacology , Drug Discovery/methods , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbiota/genetics , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Humans , Lipopeptides/chemical synthesis , Lipopeptides/chemistry , Lipopeptides/genetics , Lipopeptides/pharmacology , Methicillin-Resistant Staphylococcus aureus/enzymology , Microbial Sensitivity Tests , Molecular Conformation , Peptide Synthases/genetics , beta-Lactams/agonists , beta-Lactams/metabolismABSTRACT
Bacterial culture broth extracts have been the starting point for the development of numerous therapeutics. However, only a small fraction of bacterial biosynthetic diversity is accessible using this strategy. Here, we apply a discovery approach that bypasses the culturing step entirely by bioinformatically predicting small molecule structures from the primary sequences of the biosynthetic gene clusters. These structures are then chemically synthesized to give synthetic-bioinformatic natural products (syn-BNPs). Using this approach, we screened syn-BNPs inspired by nonribosomal peptide synthetases against microbial pathogens, and discovered an antibiotic for which no resistance could be identified and an antifungal agent with activity against diverse fungal pathogens.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Bacteria/drug effects , Biological Products/pharmacology , Fungi/drug effects , Peptide Synthases/genetics , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Antifungal Agents/chemistry , Antifungal Agents/metabolism , Biological Products/chemistry , Biological Products/metabolism , Computational Biology , Microbial Sensitivity Tests , Multigene Family , Peptide Synthases/metabolismABSTRACT
OBJECTIVES: Acinetobacter nosocomialis has increasingly been reported as an opportunistic pathogen causing nosocomial infections. Although it is more susceptible to all antimicrobial agents than Acinetobacter baumannii, MDR clinical isolates have also been described. In addition, several studies have shown a high percentage of resistance to colistin. Therefore, in the present study we investigated the mechanism of resistance to colistin in this microorganism. METHODS: Colistin-resistant strains were selected from the original colistin-susceptible A. nosocomialis strain following multi-step mutant selection. Comparative genomic and proteomic analyses of both colistin-susceptible and colistin-resistant A. nosocomialis strains were performed. In addition, virulence was investigated using the Caenorhabditis elegans assay. RESULTS: The colistin-resistant mutants selected showed a lower resistance profile for other types of antibacterial agents together with a significant decrease in virulence. The LT50 (i.e. time required to kill 50% of the nematodes) for the colistin-susceptible strain (WT) was 7 days compared with 9 days for the colistin-resistant strain (256) (Pâ<â0.0001). In the genomic studies, several mutations were observed in the lpxD genes, leading to the loss of LPS in the colistin-resistant strains. The proteomic studies showed several up- and down-regulated proteins that may be involved in colistin resistance or in a decrease in the resistance profile for several antibiotics. CONCLUSIONS: This study shows that the mechanism of resistance to colistin by A. nosocomialis is mainly associated with the loss of LPS due to mutations in the lpxD gene, although changes in the expression of some proteins cannot be ruled out. In addition, the acquisition of colistin resistance is related to a decrease in virulence.
Subject(s)
Acinetobacter/drug effects , Acinetobacter/pathogenicity , Anti-Bacterial Agents/pharmacology , Colistin/pharmacology , Drug Resistance, Bacterial , Lipopolysaccharides/metabolism , Virulence Factors/metabolism , Acinetobacter/isolation & purification , Acinetobacter Infections/microbiology , Animals , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Caenorhabditis elegans , DNA Mutational Analysis , Disease Models, Animal , Genome, Bacterial , Mutation , Proteome , Serial Passage , VirulenceABSTRACT
Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide.
Subject(s)
Acid Sensing Ion Channels/metabolism , Elapid Venoms/pharmacology , Peptides/pharmacology , Acid Sensing Ion Channels/genetics , Animals , Binding Sites/drug effects , Dose-Response Relationship, Drug , Elapid Venoms/chemistry , Models, Molecular , Molecular Structure , Peptides/chemistry , Rats , Structure-Activity RelationshipABSTRACT
The development of high-level daptomycin resistance (HLDR; MIC of ≥ 256 mg/liter) after exposure to daptomycin has recently been reported in viridans group streptococcus (VGS) isolates. Our study objectives were as follows: to know whether in vitro development of HLDR after exposure to daptomycin was common among clinical isolates of VGS and Streptococcus bovis; to determine whether HLDR also developed during the administration of daptomycin to treat experimental endocarditis caused by the daptomycin-susceptible, penicillin-resistant Streptococcus mitis strain S. mitis 351; and to establish whether combination with gentamicin prevented the development of HLDR in vitro and in vivo. In vitro studies were performed with 114 VGS strains (mitis group, 92; anginosus group, 10; mutans group, 8; and salivarius group, 4) and 54 Streptococcus bovis strains isolated from 168 consecutive patients with infective endocarditis diagnosed between 1995 and 2010. HLDR was only observed after 24 h of exposure to daptomycin in 27% of the mitis group, including 27% of S. mitis isolates, 47% of S. oralis isolates, and 13% of S. sanguis isolates. In our experimental model, HLDR was detected in 7/11 (63%) and 8/12 (67%) isolates recovered from vegetations after 48 h of daptomycin administered at 6 mg/kg of body weight/24 h and 10 mg/kg/24 h, respectively. In vitro, time-kill experiments showed that daptomycin plus gentamicin was bactericidal against S. mitis 351 at tested concentrations of 0.5 and 1 times the MIC and prevented the development of HLDR. In vivo, the addition of gentamicin at 1 mg/kg/8 h to both daptomycin arms prevented HLDR in 21 out of 23 (91%) rabbits. Daptomycin plus gentamicin was at least as effective as vancomycin plus gentamicin. In conclusion, HLDR develops rapidly and frequently in vitro and in vivo among mitis group streptococci. Combining daptomycin with gentamicin enhanced its activity and prevented the development of HLDR in most cases.
Subject(s)
Anti-Bacterial Agents/pharmacology , Daptomycin/pharmacology , Drug Resistance, Bacterial/drug effects , Endocarditis, Bacterial/drug therapy , Gentamicins/pharmacology , Streptococcal Infections/drug therapy , Streptococcus mitis/drug effects , Animals , Drug Synergism , Drug Therapy, Combination , Endocarditis, Bacterial/complications , Endocarditis, Bacterial/microbiology , Humans , Microbial Sensitivity Tests , Rabbits , Species Specificity , Streptococcal Infections/complications , Streptococcal Infections/microbiology , Streptococcus anginosus/drug effects , Streptococcus anginosus/physiology , Streptococcus bovis/drug effects , Streptococcus bovis/physiology , Streptococcus mitis/physiology , Streptococcus mutans/drug effects , Streptococcus mutans/physiology , Vancomycin/pharmacologyABSTRACT
The synthesis of proteins by native chemical ligation greatly enhances the application of chemistry to complex molecules such as proteins. The essential building blocks for this approach traditionally have been peptide-thioester segments that are linked chemoselectively in consecutive reactions. By using peptide selenoesters instead of thioesters, the ligation rate can be significantly accelerated permitting couplings at difficult sites and potentially enabling new ligation strategies. To facilitate the routine synthesis of selenoester peptides, a general and straightforward procedure has been developed that generates a suitably functionalized resin from which the desired selenoester peptide can be readily synthesized. This simple approach utilizes readily available and cheap chemical agents and enables production of peptide selenoesters of excellent quality in short time and with high recovery. In addition, the stability of peptide selenoesters was examined under different native chemical ligation conditions and compared to thioesters. Selenoesters are slightly more reactive and more susceptible to hydrolysis and aminolysis than thioesters but sufficiently stable under mildly acidic conditions (pH 6.5). Under these conditions, rapid selenoester-mediated ligation is kinetically favoured.
Subject(s)
Esters/chemical synthesis , Organoselenium Compounds/chemical synthesis , Peptides/chemical synthesis , Esters/chemistry , Organoselenium Compounds/chemistry , Peptides/chemistryABSTRACT
Lysins (peptidoglycan hydrolases) are promising new protein-based antimicrobial candidates under development to address rising antibiotic resistance encountered among pathogenic bacteria. Exebacase is an antistaphylococcal lysin and the first member of the lysin class to have entered clinical trials in the United States. In this study, the bacteriolytic activity of exebacase was characterized with time-kill assays, turbidity reduction assays, and microscopy. Three methicillin-susceptible Staphylococcus aureus and three methicillin-resistant S. aureus isolates were tested in time-kill assays over a range of concentrations from 0.25 to 8 × MIC. Exebacase demonstrated a concentration-dependent killing and showed bactericidal activity (≥3 log10 kill achieved relative to the starting inoculum) within 3 h at 1 × MIC against all strains tested. Dose-dependent lysis by exebacase was, furthermore, observed in the turbidity reduction assay, wherein decreases in initial OD600 of 50% were observed within ~15 min at concentrations as low as 4 µg/mL. Membrane dissolution, loss of cytoplasmic material, and lysis were confirmed by video and electron microscopy. The demonstrated rapid bacteriolytic effect of exebacase is an important distinguishing feature of this novel modality. IMPORTANCE To guide the development of an investigational new antibacterial entity, microbiological data are required to evaluate the killing kinetics against target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus. Killing by exebacase was determined in multiple assay formats including time-kill assays, wherein reductions of viability of ≥3 log10 colony-forming units/mL were observed within 3 h for multiple different isolates tested, consistent with very rapid bactericidal activity. Rapid reductions in optical density were likewise observed in exebacase-treated cultures, which were visually consistent with microscopic observations of rapid lysis. Overall, exebacase provides a novel antimicrobial modality against S. aureus, characterized by a rapid cidal and lytic activity.
ABSTRACT
Direct lytic agents (DLAs) are novel antimicrobial compounds with unique mechanisms of action based on rapid cell wall destabilization and bacteriolysis. DLAs include two classes of purified polypeptides-lysins (peptidoglycan hydrolase enzymes) and amurins (outer membrane targeting peptides). Their intended use is to kill bacteria in a manner that is complimentary to and synergistic with traditional antibiotics without selection for DLA resistance. Lysins were originally described as having activity against Gram-positive pathogens and of those, exebacase, is the first to have advanced into Phase 3 of clinical development. Recently, both engineered and native DLAs have now been described with potent bactericidal activity against a range of Gram-negative pathogens, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Importantly, novel DLAs targeting Gram-negatives, including the lysin CF-370 and the amurin peptides, are active in biological matrices (blood/serum) and, as such, offer promise for therapeutic use as systemically administered agents for the treatment of life-threatening invasive infections. In this review, DLAs are discussed as potential new classes of antimicrobial biologics that can be used to treat serious systemic infections.
ABSTRACT
The antibiotic paenimucillin A was originally identified using a culture-independent synthetic-bioinformatic natural product (syn-BNP) discovery approach. Here we report on a bioinformatics-guided survey of paenimucillin A analogs that led to the discovery of paenimucillin C. Paenimucillin C inhibits the growth of multidrug-resistant (MDR) Acinetobacter baumannii clinical isolates, as well as other Gram-negative bacterial pathogens. In a rat cutaneous wound model, it completely sterilized MDR A. baumannii wound infections with no sign of rebound. Mechanistic studies point to a membrane-associated mode of action that results in leakage of intracellular contents. IMPORTANCE Natural product-inspired antibiotics have saved millions of lives and played a critical role in modern medicine. However, the emergence of drug-resistant pathogens is outpacing the rate at which new clinically useful antibiotics are being discovered. The lack of a means to combat infections caused by multidrug-resistant (MDR) Acinetobacter baumannii is of particular concern. The sharp increase in cases of MDR A. baumannii infections in recent years prompted the CDC (https://www.cdc.gov/drugresistance/biggest_threats.html) and WHO (http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/) to list this pathogen as a "serious threat" and "critical pathogen," respectively. Here we report a new antibiotic, paenimucillin C, active against Gram-negative bacterial pathogens, including many clinical isolates of MDR A. baumannii strains. Mechanistic studies point to membrane disruption leading to leakage of intracellular contents as its antibacterial mode of action. Paenimucillin C sterilizes MDR A. baumannii infections in a rat cutaneous wound model with no sign of rebound infection, providing a potential new therapeutic regimen.
ABSTRACT
The flippase MurJ is responsible for transporting the cell wall intermediate lipid II from the cytoplasm to the outside of the cell. While essential for the survival of bacteria, it remains an underexploited target for antibacterial therapy. The humimycin antibiotics are lipid II flippase (MurJ) inhibitors that were synthesized on the basis of bioinformatic predictions derived from secondary metabolite gene clusters found in the human microbiome. Here, we describe an SAR campaign around humimycin A that produced humimycin 17S. Compared to humimycin A, 17S is a more potent ß-lactam potentiator, has a broader spectrum of activity, which now includes both methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE), and did not lead to any detectable resistance when used in combination with a ß-lactam. Combinations of ß-lactam and humimycin 17S provide a potentially useful long-term MRSA regimen.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Discovery , Drug Resistance, Multiple, Bacterial/drug effects , Microbiota , Staphylococcus aureus/drug effects , beta-Lactams/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Drug Synergism , Humans , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/genetics , Microbial Sensitivity Tests , Models, Molecular , Mutation , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/genetics , Staphylococcus aureus/genetics , Structure-Activity RelationshipABSTRACT
Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identification of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of biofilm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA offers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli.
Subject(s)
Acinetobacter Infections/immunology , Acinetobacter baumannii/physiology , Alveolar Epithelial Cells/physiology , Bacterial Adhesion/drug effects , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Escherichia coli Infections/immunology , Escherichia coli/physiology , Peptides/metabolism , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/physiology , RNA Helicases/metabolism , Sepsis/immunology , Virulence Factors/antagonists & inhibitors , Acinetobacter Infections/drug therapy , Animals , Anti-Bacterial Agents/therapeutic use , Biofilms , Cell Line , DNA Helicases , Disease Models, Animal , Escherichia coli Infections/drug therapy , Female , Humans , Mice , Mice, Inbred C57BL , Multifunctional Enzymes , Peptides/genetics , Peptides/therapeutic use , Pseudomonas Infections/drug therapy , RNA Helicases/genetics , RNA Helicases/therapeutic useABSTRACT
This study was undertaken to characterize functions of the outer membrane protein OmpW, which potentially contributes to the development of colistin- and imipenem-resistance in Acinetobacter baumannii. Reconstitution of OmpW in artificial lipid bilayers showed that it forms small channels (23 pS in 1 m KCl) and markedly interacts with iron and colistin, but not with imipenem. In vivo, (55) Fe uptake assays comparing the behaviours of ΔompW mutant and wild-type strains confirmed a role for OmpW in A. baumannii iron homeostasis. However, the loss of OmpW expression did not have an impact on A. baumannii susceptibilities to colistin or imipenem.
Subject(s)
Acinetobacter baumannii/metabolism , Bacterial Outer Membrane Proteins/metabolism , Colistin/metabolism , Iron/metabolism , Porins/metabolism , Amino Acid Sequence , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/isolation & purification , Molecular Sequence Data , Porins/chemistry , Porins/isolation & purification , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
In the last decade the number of Acinetobacter baumannii and Pseudomonas aeruginosa isolates showing extended drug resistance and pandrug resistance has steadily increased, thereby limiting or eliminating the antibiotics that can be used to treat infections by these micro-organisms. In addition, few antibiotics have been launched in the last decade. The objective of this study was to investigate the in vitro activity of several ceragenins against A. baumannii and P. aeruginosa. Four ceragenins (CSA-138, -13, -131 and -44) were tested both against colistin-susceptible and colistin-resistant A. baumannii and P. aeruginosa clinical isolates using the microdilution method. Time-kill curves of CSA-131 were performed against colistin-resistant A. baumannii and P. aeruginosa strains. The ceragenin CSA-131 showed the best activity against A. baumannii and P. aeruginosa, with minimum inhibitory concentrations (MICs) of 2 mg/L and <0.5 mg/L, respectively. MIC(50) and MIC(90) values were determined using 15 epidemiologically unrelated A. baumannii and P. aeruginosa strains, with MIC(50) and MIC(90) values for CSA-131 being 2 mg/L for A. baumannii and 1 mg/L and 2 mg/L, respectively, for P. aeruginosa. The killing curves of CSA-131 showed bactericidal behaviour at all of the concentrations tested, with re-growth at the lowest concentrations both in A. baumannii and P. aeruginosa. The good MICs of CSA-131 both against A. baumannii and P. aeruginosa and its high bactericidal activity may make this ceragenin a potential future agent to treat infections caused by these two pathogens even when the strain is resistant to colistin.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Colistin/pharmacology , Drug Resistance, Bacterial , Pseudomonas aeruginosa/drug effects , Steroids/pharmacology , Humans , Microbial Sensitivity Tests , Microbial Viability/drug effects , Time FactorsABSTRACT
Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.