Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia.

Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

Evaluation of the electron transfer flavoprotein as an antibacterial target in Burkholderia cenocepacia.

There are hundreds of essential genes in multidrug-resistant bacterial genomes, but only a few of their products are exploited as antibacterial targets. An example is the electron transfer flavoprotein (ETF), which is required for growth and viability in Burkholderia cenocepacia. Here, we evaluated ETF as an antibiotic target for Burkholderia cepacia complex (Bcc). Depletion of the bacterial ETF during infection of Caenorhabditis elegans significantly extended survival of the nematodes, proving that ETF is essential for survival of B. cenocepacia in this host model. In spite of the arrest in respiration in ETF mutants, the inhibition of etf expression did not increase the formation of persister cells, when treated with high doses of ciprofloxacin or meropenem. To test if etf translation could be inhibited by RNA interference, antisense oligonucleotides that target the etfBA operon were synthesized. One antisense oligonucleotide was effective in inhibiting etfB translation in vitro but not in vivo, highlighting the challenge of reduced membrane permeability for the design of drugs against B. cenocepacia. This work contributes to the validation of ETF of B. cenocepacia as a target for antibacterial therapy and demonstrates the utility of a C. elegans liquid killing assay to validate gene essentiality in an in vivo infection model.

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.