Gut Symbiont Bacteroides fragilis Secretes a Eukaryotic-Like Ubiquitin Protein That Mediates Intraspecies Antagonism

Human gut Bacteroides species produce different types of toxins that antagonize closely related members of the gut microbiota. Some are toxic effectors delivered by type VI secretion systems, and others are non-contact-dependent secreted antimicrobial proteins. Many strains of Bacteroides fragilis secrete antimicrobial molecules, but only one of these toxins has been described to date (Bacteroidales secreted antimicrobial protein 1 [BSAP-1]). In this study, we describe a novel secreted protein produced by B. fragilis strain 638R that mediated intraspecies antagonism. Using transposon mutagenesis and deletion mutation, we identified a gene encoding a eukaryotic-like ubiquitin protein (BfUbb) necessary for toxin activity against a subset of B. fragilis strains. The addition of ubb into a heterologous background strain conferred toxic activity on that strain. We found this gene to be one of the most highly expressed in the B. fragilis genome. The mature protein is 84% similar to human ubiquitin but has an N-terminal signal peptidase I (SpI) signal sequence and is secreted extracellularly. We found that the mature 76-amino-acid synthetic protein has very potent activity, confirming that BfUbb mediates the activity. Analyses of human gut metagenomic data sets revealed that ubb is present in 12% of the metagenomes that have evidence of B. fragilis. As 638R produces both BSAP-1 and BfUbb, we performed a comprehensive analysis of the toxin activity of BSAP-1 and BfUbb against a set of 40 B. fragilis strains, revealing that 75% of B. fragilis strains are targeted by one or the other of these two secreted proteins of strain 638R.