Novel therapies are urgently needed to alleviate the current crisis of multiple drug-resistant infections. The bacterial signal transduction mechanisms, known as two-component systems (TCSs), are ideal targets of novel inhibitory molecules. Highly restricted to the bacterial world, TCSs control a diverse set of cellular functions, namely virulence, response to cell envelope stress, and drug efflux. Impaired regulation of any of these aspects could affect the susceptibility of bacterial pathogens to antibiotics, which highlights the potential of TCS as targets of antibiotic adjuvant therapies. Moreover, new high-density transposon mutagenesis methods have revealed the existence of TCSs required for growth and viability. Experimental validation of gene essentiality and phenotypic characterization of knockdown mutants indicate that essential TCSs regulate aspects of the cell envelope homeostasis in coordination with cell division. In this review, we describe essential TCSs, and their potentials as targets for antibacterial molecules. We also discuss methods for the identification of small molecules that inhibit TCSs and possible reasons why antibacterial molecules targeting essential TCSs have not yet reached clinical trials.

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.

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