Bacteroidetocins Target the Essential Outer Membrane Protein BamA of <i>Bacteroidales</i> Symbionts and Pathogens

Abstract

Bacteroidetocins are a family of antibacterial peptide toxins that are produced by and target members of the phylum <i>Bacteroidetes</i>. To date, 19 bacteroidetocins have been identified, and four have been tested and shown to kill diverse <i>Bacteroidales</i> species (M. J. Coyne, N. Béchon, L. M. Matano, V. L. McEneany, et al., Nat Commun 10:3460, 2019, https://doi.org/10.1038/s41467-019-11494-1). Here, we identify the target and likely mechanism of action of the bacteroidetocins. We selected seven spontaneous mutants of four different genera, all resistant to bacteroidetocin A (Bd-A) and found that all contained mutations in a single gene, <i>bamA</i>. Construction of three of these <i>bamA</i> mutants in the wild-type (WT) strains confirmed they confer resistance to Bd-A as well as to other bacteroidetocins. We identified an aspartate residue of BamA at the beginning of exterior loop 3 (eL3) that, when altered, renders strains resistant to Bd-A. Analysis of a panel of diverse <i>Bacteroidales</i> strains showed a correlation between the presence of this aspartate residue and Bd-A sensitivity. Fluorescence microscopy and transmission electron microscopy (TEM) analysis of Bd-A-treated cells showed cellular morphological changes consistent with a BamA defect. Transcriptomic analysis of Bd-A-treated cells revealed gene expression changes indicative of cell envelope stress. Studies in mice revealed that bacteroidetocin-resistant mutants are outcompeted by their WT strain <i>in vivo</i>. Analyses of longitudinal human gut isolates showed that <i>bamA</i> mutations leading to bacteroidetocin resistance do not become fixed in the human gut, even in bacteroidetocin-producing strains and nonproducing coresident strains. Together, these data lend further support to the applicability of the bacteroidetocins as therapeutic peptides in the treatment of maladies involving <i>Bacteroidales</i> species. <b>IMPORTANCE</b> The bacteroidetocins are a newly discovered class of bacteriocins specific to <i>Bacteroidetes</i> with a spectrum of targets extending from symbiotic gut <i>Bacteroides</i>, <i>Parabacteroides</i>, and <i>Prevotella</i> species to pathogenic oral and vaginal <i>Prevotella</i> species. We previously showed that one such bacteroidetocin, Bd-A, is active at nanomolar concentrations, is water soluble, and is bactericidal, all desirable features in a therapeutic antibacterial peptide. Here, we identify the target of several of the bacteroidetocins as the essential outer membrane protein BamA. Although mutations in <i>bamA</i> can be selected in bacteria grown <i>in vitro</i>, we show both in a mouse model and in human gut ecosystems that <i>bamA</i> mutants leading to Bd-A resistance are fitness attenuated and are not selected. These features further support the potential usefulness of the bacteroidetocins as therapeutics for maladies associated with pathogenic <i>Prevotella</i> species, such as recurrent bacterial vaginosis, for which there are few effective treatments.