Defining the secondary metabolites in the Pseudomonas protegens PBL3 secretome with antagonistic activity against Burkholderia glumae.

Rice production worldwide is threatened by the disease Bacterial Panicle Blight (BPB) caused by Burkholderia glumae. Despite the threat, resources to control this disease such as completely resistant cultivars or effective chemical methods are still lacking. However, the need to control this disease has paved the way to explore biologically based approaches harnessing the antimicrobial activities of environmental bacteria. Previously, the bacterium Pseudomonas protegens PBL3 was identified as a potential biological control agent against B. glumae due to its antimicrobial activity against B. glumae. Such antimicrobial activity in vitro and in planta was associated with the P. protegens PBL3 bacteria-free secreted fraction (secretome), although the specific molecules responsible for this activity have remained elusive. In this work, we advance the characterization of the P. protegens PBL3 secretome, by evaluating the antimicrobial activity in vitro of selected secondary metabolites predicted by the P. protegens PBL3 genomic sequence against B. glumae. In addition, using Reversed Phase Liquid Chromatography Tandem Mass Spectrometry (RPLC-MS/MS), of the P. protegens PBL3 secretome, enabled us to successfully detect and quantify Pyoluteorin, 2,4-diacetylphloroglucinol (2,4-DAPG) and Pyochelin. Among those, Pyoluteorin and 2,4-DAPG reduced the growth of B. glumae in vitro along with reducing the symptoms of BPB and bacterial growth in planta, suggesting that these compounds could be effective as biopesticides to mitigate BPB.

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.