Revealing AMP mechanisms of action through resistance evolution and quantitative proteomics.

Antimicrobial resistance (AMR) is a significant public health issue that threatens our ability to treat common infections. AMR often emerges in bacteria through upregulation of proteins that allow a subpopulation of resistant bacteria to proliferate through natural selection. Identifying these proteins is crucial for understanding how AMR develops in bacteria and is essential in developing novel therapeutics to combat the threat of widespread AMR. Mass spectrometry-based proteomics is a powerful tool for understanding the biochemical pathways of biological systems, lending remarkable insight into AMR mechanisms in bacteria through measuring the changing protein abundances as a result of antibiotic treatment. Here, we describe a serial passaging method for evolving resistance in bacteria that implements quantitative proteomics to reveal the differential proteomes of resistant bacteria. The focus herein is on antimicrobial peptides (AMPs), but the approach can be generalized for any antimicrobial compound. Comparative proteomics of sensitive vs. resistance strains in response to AMP treatment reveals mechanisms to survive the bioactive compound and points to the mechanism of action for novel AMPs.

Evolution of Polymyxin Resistance Regulates Colibactin Production in Escherichia coli.

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s-1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s-1µM-1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization, structure, and development as a screening platform.

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcatobs , governing interactions with tRNA, were 1.0 µM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 µg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.