Search

Darobactin A (DAR) is a ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotic, which was initially identified from bacteria belonging to the genus Photorhabdus. In addition, the corresponding biosynthetic gene cluster (BGC) was identified and subsequently detected in several bacteria genera. DAR represents a highly promising lead structure for the development of novel antibacterial therapeutic agents. It targets the outer membrane protein BamA and is therefore specific for Gram-negative bacteria. This, together with the convincing in vivo activities in mouse infection models, makes it a particular promising candidate for further research. To improve compound supply for further investigation of DAR and to enable production of novel derivatives, establishment of an efficient and versatile microbial production platform for these class of RiPP antibiotics is highly desirable. Here we describe design and construction of a heterologous production and engineering platform for DAR, which will ensure production yield and facilitates structure modification approaches. The known Gram-negative workhorses Escherichia coli and Vibrio natriegens were tested as heterologous hosts. In addition to that, DAR producer strains were generated and optimization of the expression constructs yielded production titers of DAR showing around 10-fold increase and 5-fold decrease in fermentation time compared to the original product description. We also report the identification of the minimal DAR BGC, since only two genes were necessary for heterologous production of the RiPP.

We are experiencing an antimicrobial resistance (AMR) crisis, brought on by the drying up of the antibiotic discovery pipeline and the resulting unchecked spread of resistant pathogens. Traditional methods of screening environmental isolates or compound libraries have not produced a new drug in over 30 years. Antibiotic discovery is uniquely difficult due to a highly restrictive penetration barrier and other mechanisms that allow bacteria to survive in the presence of toxic compounds. In this Perspective, we analyze the challenges facing discovery and discuss an emerging new platform for antibiotic discovery. The penetration barrier makes screening conventional synthetic compound libraries largely impractical, and actinomycetes, the main source of natural product compounds, have been overmined. The emerging platform is based on understanding the rules that guide the permeation of molecules into bacteria and on advances in microbiology, which enable us to identify and access attractive groups of secondary metabolite producers. Establishing this platform will enable reliable production of lead compounds to combat AMR.

The spread of resistant organisms is producing a human health crisis, as we are witnessing the emergence of pathogens resistant to all available antibiotics. An increase in chronic infections presents an additional challenge - these diseases are difficult to treat due to antibiotic-tolerant persister cells. Overmining of soil Actinomycetes ended the golden era of antibiotic discovery in the 60s, and efforts to replace this source by screening synthetic compound libraries was not successful. Bacteria have an efficient permeability barrier, preventing penetration of most synthetic compounds. Empirically establishing rules of penetration for antimicrobials will form the knowledge base to produce libraries tailored to antibiotic discovery, and will revive rational drug design. Two untapped sources of natural products hold the promise of reviving natural product discovery. Most bacterial species, over 99%, are uncultured, and methods to grow these organisms have been developed, and the first promising compounds are in development. Genome sequencing shows that known producers harbor many more operons coding for secondary metabolites than we can account for, providing an additional rich source of antibiotics. Revival of natural product discovery will require high-throughput identification of novel compounds within a large background of known substances. This could be achieved by rapid acquisition of transcription profiles from active extracts that will point to potentially novel compounds.

Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure-activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.

SummaryThe majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the laboratory for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.

Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 microM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.

The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or Rh-mediated methodologies, or selective electrophilic substitution. All three methods were based on elaborations from simple indole precursors. SAR studies on these indoles and 2-phenyl-1H-indole in Staphylococcus aureus as NorA efflux pump inhibitors indicated 5-nitro-2-(3-methoxycarbonyl)phenyl-1H-indole was a slightly more potent inhibitor than the lead INF55. A promising new antibacterial lead compound against S. aureus (2-phenyl-1H-indol-5-yl)-methanol, was also found.

The rise of multidrug-resistant pathogens and recalcitrance of biofilm infections present a formidable challenge to combating infectious diseases. There are numerous disinfectants and antiseptics for treating materials in hospitals and community settings, and devices such as catheters impregnated with anti-infectives have been introduced into practice. However, there are many limitations of materials impregnated with a leaching antibacterial agent. Recently, non-leaching, permanent, sterile-surface materials have been developed in which one end of a long-chained hydrophobic polycation containing antimicrobial monomers is attached covalently to the surface of a material, for example, cotton or plastic. The polymeric chain allows the antimicrobial moieties to permeate into, and kill, the cells of the pathogen. These sterile-surface materials kill both air- and waterborne pathogens and are not susceptible to existing resistance mechanisms.

Bioassay-directed fractionation for Staphylococcus aureus multidrug resistance efflux pump inhibitors resulted in isolation of novel acylated neohesperidosides from Geranium caespitosum. The more highly acylated compounds had no direct activity against S. aureus, but potentiated activity of the antibiotics berberine, rhein, ciprofloxacin and norfloxacin. Cellular concentrations of berberine were greatly increased in the presence of active esters.

Unrelated translocases extrude proteins or antimicrobial agents across both membranes of the cell envelope in Gram-negative bacteria. The TolC protein links the translocases to the external environment. The recently determined crystal structure of TolC shows how this universal tunnel operates.

Recently identified small bacterial peptide TisB is a component of a toxin/antitoxin system. TisB toxin induces formation of drug-tolerant persister cells in response to DNA damage. We have found that TisB forms well-defined ion-conductive pores in planar lipid bilayers. Using high-resolution conductance recordings with membranes of varying lipid compositions, we show that bath solution concentrations of TisB higher than 10 µM induce multilevel conductive states, which resemble pore formation by the well-known channel-former antibiotic alamethicin. In 1 M KCl TisB-induced pores usually first appear as stable conductive ohmic states (0.5, 1.5, and 2.6 nS), and, as time progresses, tend to produce various higher conductive states. The transition to these states is also favored by application of higher positive or negative transmembrane voltages. Both low and high conductive states possess close anion selectivity (∼80 % anion current, measured in 1M/0.1M KCl salt gradient). Probing TisB pores in their lowest conductive states with differently-sized polyethylene glycols (PEGs) shows only minute polymer partitioning even for the smallest PEGs of 200 and 300 molecular weight. This finding implies that the lowest conductive states are characterized by relatively small diameter of the aqueous pores. TisB apparently creates a dormant state in persister cells by decreasing the protein motive force across their membranes and reducing ATP production, which leads to antibiotic tolerance.