Membrane-targeting Antimicrobials Research Articles

High Throughput Screen of Group A Streptococcus Clinical Isolates to Identify Conserved Strain-Specific Polymorphisms in Two-Component Systems Associated with Susceptibility to Membrane-Targeting Antimicrobials

Abstract Background Group A Streptococcus (GAS) is a common human pathogen that causes various diseases including pharyngitis, impetigo, and rheumatic fever. Although penicillin remains an effective first-line treatment for GAS infections, GAS strains are becoming increasingly resistant to second-line treatments used when patients have beta-lactam allergies. Two-component systems (TCS) in bacteria sense and respond to environmental cues like cell envelope stresses, which include oxidative and antibiotic stress. The TCS known as LiaFSR is specific to Gram-positive bacteria and associated with daptomycin resistance in Enterococcus spp. A major target of LiaFSR regulation is SpxA2, a known regulator of GAS virulence associated with tolerance to oxidative stress. LiaFSR single nucleotide polymorphisms (SNPs) that potentially influence GAS cell envelope stress response are present in a strain-specific pattern. We sought to define SNPs in LiaFSR that affect the ability of GAS to resist and respond to cell envelope stress. Hypothesis We hypothesized that conserved strain-specific polymorphisms in the LiaFSR two-component system influence GAS response to cell envelope stress. Methods We identified conserved strain-specific polymorphisms in LiaFSR in a collection of pediatric GAS isolates from Texas Children’s Hospital and Children’s Memorial Hermann Hospital in Houston, TX. 55 clinical isolates were chosen from eight emm (M protein gene) types for testing. Isolates were screened for susceptibility to oxidative stress on plates containing diamide, a thiol oxidizing agent. Survival of serially diluted isolate cultures was assessed relative to an emm3 wild-type strain. Representative isolates from emm types showing differences in diamide tolerance were subjected to varying concentrations of bacitracin, polymyxin B, LL-37, nisin, or daptomycin and compared to the emm3 wild-type strain. Survival in the presence of antimicrobials was measured on a 96-well plate reader and compared to calibration growth curves to obtain virtual colony counts (CFUV). Differences in isolate CFUV following antimicrobial exposure relative to the emm3 wild-type strain were validated by colony counts (CFU) on blood agar plates of selected isolate cultures following exposure to inhibitory concentrations of antimicrobials. Results A P170S mutation in LiaF (emm25 and emm81) appeared to increase susceptibility to diamide-induced oxidative stress independent of genomic background. In the emm75 background, an H210N mutation in LiaS showed decreased diamide susceptibility relative to wild-type LiaS emm75 isolates. The emm75 isolate analyzed showed reduced susceptibility to nisin relative to an emm3 wild-type strain. Conclusion Strain-specific polymorphisms in LiaF and LiaS are worth pursuing to further our understanding of the influence LiaFSR has on cell envelope stress phenotypes. The findings of this screen provide a starting point for future research that will enable the production of targeted therapies against LiaFSR to optimize clinical outcomes and reduce the burden of multidrug resistance in GAS.

Facially Amphiphilic Bile Acid-Functionalized Antimicrobials: Combating Pathogenic Bacteria, Fungi, and Their Biofilms.

We report facially amphiphilic bile acid-based antimicrobials with a broad spectrum of activity against both bacterial and fungal pathogens and negligible detrimental effects on mammalian cells. Two lead compounds eliminated dormant subpopulations of various bacterial species, unlike conventional antibiotics. The lead compounds were also effective in eradicating biofilms of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. Additionally, these compounds substantially inhibited the formation of fungal biofilms (C. albicans). Mechanistic investigations revealed the membrane-active nature and endogenous reactive oxygen species (ROS) induction ability of these compounds. Finally, no detectable resistance was developed by the bacterial strains against this class of membrane-targeting antimicrobials.

Discovery of Amphiphilic Xanthohumol Derivatives as Membrane-Targeting Antimicrobials against Methicillin-Resistant Staphylococcus aureus.

Infections caused by multidrug-resistant (MDR) bacteria are increasing worldwide, and with limited clinically available antibiotics, it is urgent to develop new antimicrobials to combat these MDR bacteria. Here, a class of novel amphiphilic xanthohumol derivatives were prepared using a building-block approach. Bioactivity assays showed that the molecule IV15 not only exhibited a remarkable antibacterial effect against clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates (MICs: 1-2 μg/mL) but also had the advantages of rapid bactericidal properties, low toxicity, good plasma stability, and not readily inducing bacterial resistance. Mechanistic studies indicated that IV15 has good membrane-targeting ability and can bind to phosphatidylglycerol and cardiolipin in bacterial membranes, thus disrupting the bacterial cell membranes and causing increased intracellular reactive oxygen species and leakage of proteins and DNA, eventually resulting in bacterial death. Notably, IV15 exhibited remarkable in vivo anti-MRSA efficacy, superior to vancomycin, making it a potential candidate to combat MRSA infections.

Design, synthesis, and biological evaluation of tetrahydroquinoline amphiphiles as membrane-targeting antimicrobials against pathogenic bacteria and fungi

The rising prevalence of drug-resistant pathogens is one of the biggest threats to human health. The development of new antibiotics that can overcome drug resistance is in urgent need. Herein, we designed and synthesized a series of amphiphilic tetrahydroquinoline derivatives as small-molecule-based antimicrobial peptidomimetics. Two lead compounds 36 and 52 which contained the tetrahydroquinoline core, hydrophobic alkyl chains (n-nonyl or isoprenyl group), different spacer lengths (n = 4 or 8), and cationic guanidine moiety, showed poor hemolytic activity, low cytotoxicity, and potent broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungi. The further biological evaluation revealed that compounds 36 and 52 can kill bacteria and fungi rapidly via membrane-targeting action and avoid drug resistance development. More importantly, compounds 36 and 52 exhibited similarly potent in vivo antimicrobial activities in a murine corneal infection caused by Staphylococcus aureus ATCC29213 or Pseudomonas aeruginosa ATCC9027, as compared to vancomycin or gatifloxacin. These results suggest that compounds 36 and 52 have great potential as new broad-spectrum antimicrobial agents to combat microbial resistance.

Unlocking the bacterial membrane as a therapeutic target for next-generation antimicrobial amphiphiles

Gram-positive bacteria like Enterococcus faecium and Staphylococcus aureus, and Gram-negative bacteria like Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Spp. are responsible for most of fatal bacterial infections. Bacteria present a handful of targets like ribosome, RNA polymerase, cell wall biosynthesis, and dihydrofolate reductase. Antibiotics targeting the protein synthesis like aminoglycosides and tetracyclines, inhibitors of RNA/DNA synthesis like fluoroquinolones, inhibitors of cell wall biosynthesis like glycopeptides and β-lactams, and membrane-targeting polymyxins and lipopeptides have shown very good success in combating the bacterial infections. Ability of the bacteria to develop drug resistance is a serious public health challenge as bacteria can develop antimicrobial resistance against newly introduced antibiotics that enhances the challenge for antibiotic drug discovery. Therefore, bacterial membranes present a suitable therapeutic target for development of antimicrobials as bacteria can find it difficult to develop resistance against membrane-targeting antimicrobials. In this review, we present the recent advances in engineering of membrane-targeting antimicrobial amphiphiles that can be effective alternatives to existing antibiotics in combating bacterial infections.

Development of Highly Potent Carbazole Amphiphiles as Membrane-Targeting Antimicrobials for Treating Gram-Positive Bacterial Infections.

The development of new antimicrobial agents capable of curing drug-resistant bacteria-induced infections is becoming a major challenge to the global healthcare system. To develop antimicrobials with new molecular entities, a series of novel carbazole-based compounds were designed and synthesized by biomimicking the structural properties and biological function of antimicrobial peptides. Compound 29 was selected as a lead compound from the structure-activity relationship analyses and biological activity evaluation. Compound 29 showed excellent antimicrobial activity against Gram-positive bacteria (MICs = 0.78-1.56 μg/mL), poor hemolytic activity (HC50 > 200 μg/mL), and low cytotoxicity to mammalian cells. Compound 29 had fast bactericidal properties and effectively prevented bacterial resistance in laboratory simulations. Antibacterial mechanism studies revealed that compound 29 directly destroyed bacterial cell membranes, leading to bacterial deaths. Importantly, compound 29 displayed an excellent efficacy in a murine bacterial keratitis model caused by Staphylococcus aureus ATCC29213.

Lipidomic and Ultrastructural Characterization of the Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum.

Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When S. aureus was grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus require a living or nondenatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed that CEs and TGs were the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host.IMPORTANCE Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests that human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

Cholic Acid-Derived Amphiphile which Combats Gram-Positive Bacteria-Mediated Infections via Disintegration of Lipid Clusters.

Inappropriate and uncontrolled use of antibiotics results in the emergence of antibiotic resistance, thereby threatening the present clinical regimens to treat infectious diseases. Therefore, new antimicrobial agents that can prevent bacteria from developing drug resistance are urgently needed. Selective disruption of bacterial membranes is the most effective strategy for combating microbial infections as accumulation of genetic mutations will not allow for the emergence of drug resistance against these antimicrobials. In this work, we tested cholic acid (CA) derived amphiphiles tethered with different alkyl chains for their ability to combat Gram-positive bacterial infections. In-depth biophysical and biomolecular simulation studies suggested that the amphiphile with a hexyl chain (6) executes more effective interactions with Gram-positive bacterial membranes as compared to other hydrophobic counterparts. Amphiphile 6 is effective against multidrug resistant Gram-positive bacterial strains as well and does not allow the adherence of S. aureus on amphiphile 6 coated catheters implanted in mice. Further, treatment of wound infections with amphiphile 6 clears the bacterial infections. Therefore, the current study presents strategic guidelines in design and development of CA-derived membrane-targeting antimicrobials for Gram-positive bacterial infections.

Membrane-targeting antibiotics: recent developments outside the peptide space.

The rise of antibiotic resistant bacteria requires unconventional strategies toward efficient chemotherapeutic agents, preferably with alternative mechanisms of action. The bacterial cell membrane has become an appealing target since its essential and highly conservative structure are key challenges to resistance mechanisms. Inspired by natural antimicrobial peptides, research on membrane-targeting antimicrobials has been growing out of the peptide space. The pursuit of more druggable molecules led to the discovery that the pharmacophore of antimicrobial peptides is smaller than anticipated. Several promising classes of membrane-targeting antimicrobials have been discovered, such as ceragenins, reutericyclines, carbohydrate amphiphiles - among others. This review will discuss the most recent findings on membrane-targeting antibiotics, focusing on small molecules outside the antimicrobial peptide molecular space.

Distinct Intramolecular Hydrogen Bonding Dictates Antimicrobial Action of Membrane-Targeting Amphiphiles.

As mechanisms underpinning the molecular interactions between membrane-targeting antimicrobials and Gram-negative bacterial membranes at atomistic scale remain elusive, we used cholic acid (CA)-derived amphiphiles with different hydrophobicities as model antimicrobials and assessed the effect of their conformational flexibility on antimicrobial activity. Relative to other hydrophobic counterparts, a compound with a hexyl chain (6) showed the strongest binding with the lipopolysaccharide (LPS) of Gram-negative bacterial membranes and acted as an effective antimicrobial. Biomolecular simulations, validated by complementary approaches, revealed that specific intramolecular hydrogen bonding imparts conformationally rigid character to compound 6. This conformational stability of compound 6 allows minimum but specific interactions of the amphiphile with LPS that are a sum of exothermic processes like electrostatic interactions, membrane insertion, and endothermic contributions from disaggregation of LPS. Therefore, our study reveals that a membrane-targeting mechanism with the help of conformationally selective molecules offers a roadmap for developing future therapeutics against bacterial infections.

Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating Salmonella-Mediated Gut Infection and Inflammation

A major impediment to developing effective antimicrobials against Gram-negative bacteria like Salmonella is the ability of the bacteria to develop resistance against existing antibiotics and the inability of the antimicrobials to clear the intracellular bacteria residing in the gastrointestinal tract. As the critical balance of charge and hydrophobicity is required for effective membrane-targeting antimicrobials without causing any toxicity to mammalian cells, herein we report the synthesis and antibacterial properties of cholic acid-derived amphiphiles conjugated with alkyl chains of varied hydrophobicity. Relative to other hydrophobic counterparts, a compound with hexyl chain (6) acted as an effective antimicrobial against different Gram-negative bacteria. Apart from its ability to permeate the outer and inner membranes of bacteria; compound 6 can cross the cellular and lysosomal barriers of epithelial cells and macrophages and kill the facultative intracellular bacteria without disrupting the mammalian cell membranes. Oral delivery of compound 6 was able to clear the Salmonella-mediated gut infection and inflammation, and was able to combat persistent, stationary, and multi-drug-resistant clinical strains. Therefore, our study reveals the ability of cholic acid-derived amphiphiles to clear intracellular bacteria and Salmonella-mediated gut infection and inflammation.

Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes

Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.

The Gram-positive model organism Bacillus subtilis does not form microscopically detectable cardiolipin-specific lipid domains.

Rather than being homogenous diffusion-dominated structures, biological membranes can exhibit areas with distinct composition and characteristics, commonly termed as lipid domains. Arguably the most comprehensively studied examples in bacteria are domains formed by cardiolipin, which have been functionally linked to protein targeting, the cell division process and the mode of action of membrane-targeting antimicrobials. Cardiolipin domains were originally identified in the Gram-negative model organism Escherichia coli based on preferential staining by the fluorescent membrane dye nonylacridine orange (NAO), and later reported to also exist in other Gram-negative and -positive bacteria. Recently, the lipid-specificity of NAO has been questioned based on studies conducted in E. coli. This prompted us to reanalyse cardiolipin domains in the Gram-positive model organism Bacillus subtilis. Here we show that logarithmically growing B. subtilis does not form microscopically detectable cardiolipin-specific lipid domains, and that NAO is not a specific stain for cardiolipin in this organism.

Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

Amino acid modified xanthone derivatives: novel, highly promising membrane-active antimicrobials for multidrug-resistant Gram-positive bacterial infections.

Antibiotic resistance is a critical global health care crisis requiring urgent action to develop more effective antibiotics. Utilizing the hydrophobic scaffold of xanthone, we identified three components that mimicked the action of an antimicrobial cationic peptide to produce membrane-targeting antimicrobials. Compounds 5c and 6, which contain a hydrophobic xanthone core, lipophilic chains, and cationic amino acids, displayed very promising antimicrobial activity against multidrug-resistant Gram-positive bacteria, including MRSA and VRE, rapid time-kill, avoidance of antibiotic resistance, and low toxicity. The bacterial membrane selectivity of these molecules was comparable to that of several membrane-targeting antibiotics in clinical trials. 5c and 6 were effective in a mouse model of corneal infection by S. aureus and MRSA. Evidence is presented indicating that 5c and 6 target the negatively charged bacterial membrane via a combination of electrostatic and hydrophobic interactions. These results suggest that 5c and 6 have significant promise for combating life-threatening infections.

Correction to Design and Synthesis of Amphiphilic Xanthone-Based, Membrane-Targeting Antimicrobials with Improved Membrane Selectivity

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionCorrection to Design and Synthesis of Amphiphilic Xanthone-Based, Membrane-Targeting Antimicrobials with Improved Membrane SelectivityHanxun Zou, Jun-Jie Koh, Jianguo Li, Shengxiang Qiu, Thet Tun Aung, Huifen Lin, Rajamani Lakshminarayanan, Xiaoping Dai, Charles Tang, Fang Hui Lim, Lei Zhou, Ai Ling Tan, Chandra Verma, Donald T. H. Tan, Hardy Sze On Chan, Padmanabhan Saraswathi, Derong Cao*, Shouping Liu*, and Roger W. Beuerman*Cite this: J. Med. Chem. 2013, 56, 9, 3747Publication Date (Web):April 26, 2013Publication History Published online26 April 2013Published inissue 9 May 2013https://doi.org/10.1021/jm400514xCopyright © 2013 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views736Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (114 KB) Get e-Alerts Get e-Alerts

Design and Synthesis of Amphiphilic Xanthone-Based, Membrane-Targeting Antimicrobials with Improved Membrane Selectivity

This work describes how to tune the amphiphilic conformation of α-mangostin, a natural compound that contains a hydrophobic xanthone scaffold, to improve its antimicrobial activity and selectivity for Gram-positive bacteria. A series of xanthone derivatives was obtained by cationic modification of the free C3 and C6 hydroxyl groups of α-mangostin with amine groups of different pKa values. Modified structures using moieties with high pKa values, such as AM-0016 (3b), exhibited potent antimicrobial properties against Gram-positive bacteria. Compound 3b also killed bacteria rapidly without inducing drug resistance and was nontoxic when applied topically. Biophysical studies and molecular dynamics simulations revealed that 3b targets the bacterial inner membrane, forming an amphiphilic conformation at the hydrophobic-water interface. In contrast, moieties with low pKa values reduced the antimicrobial activity of the parent compound when conjugated to the xanthone scaffold. This strategy provides a new way to improve "hits" for the development of membrane-active antibiotics that target drug-resistant pathogens.

The metabolically active subpopulation inPseudomonas aeruginosabiofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms

Biofilms are reported to be inherently refractory toward antimicrobial attack and, therefore, cause problems in industrial and medical settings. Pseudomonas aeruginosa biofilms contain subpopulations that exhibit high metabolic activity and subpopulations that exhibit low metabolic activity. We have found that membrane-targeting antimicrobials such as colistin, EDTA, SDS, and chlorhexidine specifically kill the inactive subpopulation in P.aeruginosa biofilms, whereas the active subpopulation survives exposure to these compounds. Because treatment of P.aeruginosa biofilms with the membrane-targeting compounds colistin, EDTA, SDS, and chlorhexidine resulted in the same spatial distribution of live and dead bacteria, we investigated whether tolerance to these compounds originated from the same molecular mechanisms. Development of colistin-tolerant subpopulations was found to depend on the pmr genes encoding lipopolysaccharide modification enzymes, as well as on the mexAB-oprM, mexCD-oprJ, and muxABC-opmB genes encoding antimicrobial efflux pumps, but does not depend on the mexPQ-opmE efflux pump genes. Development of chlorhexidine-tolerant subpopulations was found to depend on the mexCD-oprJ genes, but does not depend on the pmr, mexAB-oprM, mexPQ-opmE, or muxABC-opmB genes. Tolerance to SDS and EDTA in P. aeruginosa biofilms is linked to metabolically active cells, but does not depend on the pmr, mexAB, mexCD, mexPQ, or muxABC genes. Our data suggest that the active subpopulation in P.aeruginosa biofilms is able to adapt to exposure to membrane-targeting agents through the use of different genetic determinants, dependent on the specific membrane-targeting compound.