Characterization of TelE, an LXG effector ofStreptococcus gallolyticus, antagonized by a non-canonical immunity protein

AbstractStreptococcus gallolyticussubsp.gallolyticus(Sgg) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the Type VIIb Secretion System (T7SSb) machinery is uniquely present inSggin two different arrangements.SggUCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by T7SSb, we inactivated theessCgene encoding the motor of this machinery. Comparison of the proteins secreted by Sgg UCN34 WT and its isogenicΔessCmutant revealed six T7SSb effector proteins, including an LXG-family toxin named herein TelE displaying pore-forming activity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C-terminus were identified inSggisolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE pore-forming activity. Unlike other pore-forming toxins commonly antagonized by a membrane protein, TelE activity was antagonized by a cytosolic protein TipE belonging to the DUF5085 family. Finally, we demonstrated that purified TelE can permeabilizeE. colicells rapidly when introduced extracellularly. Overall, we report herein a uniqueSggT7SSb effector exhibiting a pore-forming activity against non-immune bacteria.Author summaryIn this study, 38 clinical isolates ofStreptococcus gallolyticussubsp. gallolyticus(Sgg) were sequenced and a genetic locus encoding the Type VIIb secretion system (T7SSb) was found conserved in all 38 genomes. This locus absent from the closely relatedS. gallolyticussubsp.pasteurianus, constitutes a pathogenicity island. T7SSb is a specialized machinery involved in the secretion of multiple effectors that have been implicated in bacterial virulence, interbacterial killing or host immunity modulation. Here, we report that the model organismSggstrain UCN34 secretes six T7SSb effectors. One of the six effectors named TelE displayed a strong toxicity when expressed in Gram-negative bacteria. Our results indicate that TelE is a pore forming toxin whose activity can be antagonized by a non-canonical immunity protein named TipE. Overall, we report a unique toxin-immunity protein pair and our data expand the range of effectors secreted through T7SSb. As shown previously for gallocin, a bacteriocin giving a selective advantage toSggin the tumoral microenvironment, we propose that T7SSb effectors including TelE may helpSggto alter the host microbiota contributing to colon colonization.