New antibiotics are needed as bacterial infections continue to be a leading cause of death, but efforts to develop compounds with promising antibacterial activity are hindered by a poor understanding ofand limited strategies for elucidatingtheir modes of action. We recently discovered a novel lasso peptide, ubonodin, that is active against opportunistic human lung pathogens from the Burkholderia cepacia complex (Bcc). Ubonodin inhibits RNA polymerase, but only select strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we hypothesized that cellular uptake of ubonodin determines susceptibility. Although Bcc strains harbor numerous nutrient uptake systems, these organisms lack close homologues of the single known lasso peptide membrane receptor, FhuA. Thus, a straightforward homology-driven approach failed to uncover the identity of the ubonodin transporter(s). Here, we used phenotype-guided comparative genomics to identify genes uniquely associated with ubonodin-susceptible Bcc strains, leading to the identification of PupB as the ubonodin outer membrane (OM) receptor in Burkholderia. The loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. We also examine how a conserved iron-regulated transcriptional pathway controls PupB to further tune ubonodin susceptibility. PupB is only the second lasso peptide OM receptor to be uncovered and the first outside of enterobacteria. Finally, we elucidate the full transport pathway for ubonodin by identifying its inner membrane receptor YddA in Burkholderia. Our work provides a complete picture of the mode of action of ubonodin and establishes a general framework for deciphering the transport pathways of other natural products with cytoplasmic targets.

Rifamycins are a class of antibiotics that were first discovered in 1957 and are known for their use in treating tuberculosis (TB). Rifamycins exhibit bactericidal activity against many Gram-positive and Gram-negative bacteria by inhibiting RNA polymerase (RNAP); however, resistance is prevalent and the mechanisms range from primary target modification and antibiotic inactivation to cytoplasmic exclusion. Further, phenotypic resistance, in which only a subpopulation of bacteria grow in concentrations exceeding their minimum inhibitory concentration, and tolerance, which is characterized by reduced rates of bacterial cell death, have been identified as additional causes of rifamycin failure. Here we summarize current understanding and recent developments regarding this critical antibiotic class.

The Burkholderia cepacia complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. We constructed a library of over 90 000 ubonodin variants with 2 amino acid substitutions and used a high-throughput screen and next-generation sequencing to examine the fitness of the entire library, generating the most comprehensive data set on lasso peptide activity so far. This screen revealed information regarding the structure–activity relationship of ubonodin over a large sequence space. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a submicromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.

New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodin’s spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.

TheBurkholderia cepaciacomplex (Bcc) is a group of bacteria including several opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. The natural product ubonodin is a lasso peptide with promising activity against several Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. In this study, we developed a high-throughput screen using next-generation sequencing to examine the fitness of a library of over 90,000 ubonodin variants, generating the most comprehensive dataset on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space, indicating certain residues that can tolerate amino acid substitutions and still retain activity. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a sub-micromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc memberBurkholderia cenocepacia. Ubonodin and several of the variants identified in this study had a lower MIC against certain Bcc strains than many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.