Vibrio microcins. A hidden field of targeted anti-cholerae natural products.

Project Summary Cholera is a deadly diarrheal disease caused by the bacterial pathogen Vibrio cholerae. It remains poorly controlled in many parts of the world and outbreaks have been surging despite global efforts to reduce infection. New antibacterials for Gram-negative bacteria like V. cholerae are greatly needed but are also the most challenging to make because the outer membrane of these bacteria excludes nearly all molecules from entering the cell. An innovative strategy to overcome this permeability barrier is shown by the new clinical antibiotic cefiderocol, which binds outer membrane siderophore receptors and uses active transport systems to enter the periplasm. Unfortunately, few molecules are known that are capable of this feat, and we have been largely limited to using siderophore conjugates to develop these innovative Trojan horse antibiotics. Our work on the understudied class of bacteriocins called microcins provides a rare opportunity to investigate a broader class of antibacterials with this membrane translocation ability. Microcins are small antibacterial proteins (<10kDa) that selectively bind Gram-negative outer membrane proteins and hijack active transport processes to enter the periplasm. Microcins have are effective at controlling bacterial pathogen growth in vivo and have many characteristics that could make them attractive antibiotic scaffolds. Despite their potential value, advances in microcin biology have been impeded by the challenges of their identification and the limited characterization of the only 15 known examples. To overcome the discovery bottleneck, we developed an approach for systematic identification and validation of new microcins. Coupling an in silico pipeline with a new method for microcin activity screening, we are identifying microcins across phylogenetically diverse bacteria, including phylogroups that have never been examined for microcin activity, such as the Vibrionaceae. We have identified potent Vibrio microcins active against all clinical strains of V. cholerae tested and which we show can be delivered by bacteria vectors to reduce V. cholerae colonization in mice. Our new appreciation for class II microcin diversity and their potential to treat V. cholerae infections has made plain the critical need to develop detailed knowledge of their sequence-activity relationships and ability to prevent and treat gut infections using cell-based delivery vectors to empower their use in antibiotic development. To pursue these critical gaps in knowledge, we will take advantage of our exclusive repertoire of unrecognized V. cholerae class II microcins and will (Aim 1) provide the first in- depth microcin sequence-activity study to uncover domains import for receptor binding, cell entry, and antibacterial activity, (Aim 2) investigate the use of different delivery bacteria, dosing regime, and microcin expression systems on microcin efficacy in mouse models, and (Aim 3) expand our understanding of the range of outer membrane receptors that can be targeted by Vibrio microcins for cell entry.