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

Abstract

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.IMPORTANCE We are just beginning to understand some of the important interactions that occur between microbes of the human gut microbiota that dictate the composition and abundance of its constituent members. The ability of one member to produce molecules that directly kill a coresident member has been shown among minor gut species and is just starting to be studied in the abundant Bacteroides species. Here, we show that some strains of Bacteroides fragilis have acquired a gene encoding a secreted eukaryotic-like ubiquitin protein with potent inhibitory activity against other B. fragilis stains. This is the first bacterially encoded ubiquitin-like molecule shown to function like a bacterial toxin. This molecule is an example of a gut symbiont acquiring and adapting a eukaryotic molecule likely to increase its competitiveness in the mammalian gut. Understanding antagonistic factors produced by abundant gut symbionts is an important prerequisite to properly engineer strains to colonize the gut for health benefits.

Keywords: Bacteroides; antagonism; microbiota; ubiquitin.

FIG 1

Agar spot assays of eight B. fragilis strains in overlays, showing zones of growth inhibition (dark spots) by secreted molecule(s) from wild-type strain 638R and 638RΔ1646 (BSAP-1 deletion mutant) and by purified, His-tagged BSAP-1. Results for two strains each, named to the right of the panels, are shown as examples of the four different patterns of sensitivity/resistance.

FIG 2

Identification of a gene necessary for antimicrobial activity of 638RΔ1646. (A) Agar spot assays showing results for two transposon mutants of 638RΔ1646 that lost inhibitory activity against four B. fragilis strains. (B) (Top) ORF map of the genetic region where the transposons insertions into the 638R genome resulted in loss of activity. (Bottom) Extent of the BF638R_3923 (ubb) transcript as predicted from analyses of RNA-Seq data. A perfect match with the −7 site of the promoter sequence recognized by the Bacteroides sigma 70 factor is shown (blue letters). (C) Agar spot assays showing the loss of secreted inhibitory activity in a ubb deletion mutant and the resulting phenotypes when the gene is added to the mutant in trans (pubb), as well as results for the vector control.

FIG 3

BfUbb inhibits growth. (A) Alignment of BfUbb with human ubiquitin, showing the extended N-terminal SpI signal sequence of BfUbb. (B) Agar spot assays displaying the sensitivity/resistance profiles of four B. fragilis strains exposed to B. fragilis CM11 with an empty vector or ubb in trans. (C) Agar spot assays showing inhibition activities of dilutions of synthesized 76-aa BfUbb against four sensitive strains and one resistant strain. (D) Agar spot assay showing sensitivity of B. fragilis strain 0878320-1 to BfUbb versus purified bovine ubiquitin.

FIG 4

Heterogeneity of the B. fragilis genome in the ubb genetic region. (A) Gene maps of three representative B. fragilis strains showing the three predominant genetic types in the ubb or corresponding regions. B. fragilis strains each have a similar mutL (orange), and the DNA between each of the three genetic types begins to diverge 39 bp downstream from this gene. The genes colored yellow encode proteins with β-propeller motifs that are divergent even within a genetic type. The green boxes indicate a 52-bp element that is present in each of the three genetic types and is identical between strain 638R and 078320-1, with a few mismatches in strain 3_1_12. The B. fragilis 638R gene map (top) shows that ubb is contained in a genetic region likely acquired with three other small genes. The B. fragilis 3_1_12 gene map (middle) shows that a few B. fragilis genomes have no genes inserted in this region. The B. fragilis 078320-1 gene map (bottom) shows the most predominant B. fragilis genetic type, where higBA genes are present in the divergent region along with an additional copy of the 52-bp element downstream from mutL. (B) Agar spot assay of BfUbb peptide overlaid with B. fragilis CM11 containing an empty vector or the vector expressing higBA. (C) Agar spot assay of the BfUbb peptide overlaid with sensitive strain B. fragilis 078320-1 containing an empty vector or the vector overexpressing the antitoxin-encoding gene higA.