Characterization of TelE, a T7SS LXG effector exhibiting a conserved C-terminal glycine zipper motif required for toxicity

Abstract

Streptococcus gallolyticus subsp. 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 in Sgg in two different arrangements. Sgg UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by T7SSb, we inactivated the essC gene encoding the motor of this machinery. Comparison of the proteins secreted by Sgg UCN34 WT and its isogenic ∆essC mutant revealed six T7SSb effector proteins, including an LXG-family toxin named herein TelD displaying pore-forming activity. Seven homologs of TelD harboring a conserved glycine zipper motif at the C-terminus were identified in Sgg isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelD pore-forming activity. Unlike other pore-forming toxins commonly antagonized by a membrane protein, TelD activity was antagonized by a cytosolic protein TipD belonging to the DUF5085 family. Finally, we demonstrated that purified TelD can permeabilize E. coli cells rapidly when introduced extracellularly. Overall, we report herein a unique Sgg T7SSb effector exhibiting a pore-forming activity against non-immune bacteria.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract

“…For example, LXG toxin TspA is secreted by the T7SSb in S. aureus but is not encoded in the T7SS locus (Ulhuq et al ., 2020). Further, L. monocytogenes, B. subtilis, S. gallolyticus, S. intermedius and S. suis encode multiple full-length LXG toxins, not all of which are associated with the T7SS locus (Bowran & Palmer, 2021, Whitney et al ., 2017, Kobayashi, 2021, Teh et al ., 2022, Liang et al ., 2022). While we did not identify any orphaned full-length GBS LXG proteins based on presence of an LXG motif with the first 100 amino acids of the protein, N-terminal homology to other proteins, or by searching specifically in genomic regions that encode orphaned DUF4176 or WXG100 proteins, it is possible that orphaned C-terminal toxin fragments may exist in GBS strains.…”

“…Some T7SS toxins function in interbacterial competition and are frequently co-transcribed with chaperones that facilitate their secretion and immunity factors that prevent self-toxicity. These functions have been described in Staphylococcus aureus, Streptococcus intermedius, Bacillus subtilis, Enterococcus faecalis , and recently Streptococcus gallolyticus (Cao et al ., 2016, Whitney et al ., 2017, Klein et al ., 2018, Ulhuq et al ., 2020, Kobayashi, 2021, Chatterjee et al ., 2021, Teh et al ., 2022, Klein et al ., 2022). Interestingly, in some cases, these toxins also promote virulence and modulate immune responses within the host (Dai et al ., 2017, Ohr et al ., 2017, Ulhuq et al ., 2020).…”

The oncogenic role ofStreptococcus gallolyticus subsp.gallolyticusis linked to activation of multiple cancer-related signaling pathways

“…S8A-B ). Interestingly, the genetic island encoding the type VII secretion system of SGG TX20005, shown to contribute to tumor development, exhibits significant differences in term of organization, expression, and secreted effectors with the majority of other SGG isolates exemplified by SGG UCN34 [52].…”

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract

“…Many T7SS + Gram‐positive species encode orphaned LXG proteins elsewhere in the genome (e.g., S. aureus TspA [Ulhuq et al, 2020]). L. monocytogenes , B. subtilis , S. gallolyticus , S. intermedius , and S. suis also encode multiple full‐length LXG toxins, not all of which are associated with the T7SS locus (Bowran & Palmer, 2021; Kobayashi, 2021; Liang et al, 2022; Teh et al, 2022; Whitney et al, 2017). While we did not identify any orphaned full‐length GBS LXG proteins in our representative strains based on presence of an LXG motif with the first 100 amino acids of the protein, N‐terminal homology to other proteins, or by searching specifically in genomic regions that encode orphaned DUF4176 or WXG100 proteins, it is possible that orphaned C‐terminal toxin fragments may exist in GBS strains.…”

“…Some T7SS toxins function in interbacterial competition and are frequently co-transcribed with chaperones that facilitate their secretion and immunity factors that prevent self-toxicity. These functions have been described in Staphylococcus aureus, Streptococcus intermedius, Bacillus subtilis, Enterococcus faecalis, and recently Streptococcus gallolyticus (Cao et al, 2016;Chatterjee et al, 2021;Klein et al, 2018Klein et al, , 2022Kobayashi, 2021;Teh et al, 2022;Ulhuq et al, 2020;Whitney et al, 2017). Interestingly, in some cases, these toxins also promote virulence and modulate immune responses within the host (Dai et al, 2017;Ohr et al, 2017;Ulhuq et al, 2020).…”