A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors

Abstract

Gram-negative bacteria deliver effectors via the type VI secretion system (T6SS) to outcompete their rivals. Each bacterial strain carries a different arsenal of effectors; the identities of many remain unknown. Here, we present an approach to identify T6SS effectors encoded in bacterial genomes of interest, without prior knowledge of the effectors' domain content or genetic neighborhood. Our pipeline comprises a comparative genomics analysis followed by screening using a surrogate T6SS + strain. Using this approach, we identify an antibacterial effector belonging to the T6SS1 of Vibrio parahaemolyticus, representing a widespread family of T6SS effectors sharing a C-terminal domain that we name Tme (Type VI membranedisrupting effector). Tme effectors function in the periplasm where they intoxicate bacteria by disrupting membrane integrity. We believe our approach can be scaled up to identify additional T6SS effectors in various bacterial genera.

Activity, delivery, and diversity of Type VI secretion effectors

“…T6SS membrane disrupting effectors (Tme) or pore‐forming toxins exert their toxicity from the periplasm of the target cells, by inserting into the inner membrane and causing membrane depolarization. Unlike other effectors, Tme effectors do not possess catalytic activity and induce growth inhibition rather than cell lysis (Fridman et al, 2020; LaCourse et al, 2018; Mariano et al, 2019; Miyata et al, 2013). These effectors share homologies with pore‐forming colicins or have predicted transmembrane helices (TMH) in their C‐terminal region (Fridman et al, 2020; LaCourse et al, 2018; Miyata et al, 2011).…”

“…Unlike other effectors, Tme effectors do not possess catalytic activity and induce growth inhibition rather than cell lysis (Fridman et al, 2020; LaCourse et al, 2018; Mariano et al, 2019; Miyata et al, 2013). These effectors share homologies with pore‐forming colicins or have predicted transmembrane helices (TMH) in their C‐terminal region (Fridman et al, 2020; LaCourse et al, 2018; Miyata et al, 2011). These TMH can bear glycine zipper motifs (LaCourse et al, 2018), a motif commonly found in channel proteins or amyloid proteins and known to promote homo‐dimerization (Kim et al, 2005; LaCourse et al, 2018).…”

“…P. aeruginosa Tse4 and Serratia marcescens Ssp6 effectors form ion‐selective pores, but are not permeable to larger compounds (LaCourse et al, 2018; Mariano et al, 2019). In contrast, the activities of VasX and Vibrio parahaemolyticus Tme1 and Tme2 induce permeability to larger compounds such as propidium iodide or ONPG (Fridman et al, 2020; Miyata et al, 2013). However, the precise molecular mechanisms underlying pore formation and/or membrane disruption induced by these different toxins remain to be determined.…”

“…To protect themselves from intoxication T6SS + bacteria possess immunity genes adjacent to the cognate toxin genes. As a rule, the immunity proteins protecting from periplasm‐active toxins are themselves located in the periplasm, being membrane‐anchored lipoproteins, periplasmic, or inner membrane proteins (Flaugnatti et al, 2016; Fridman et al, 2020; Russell et al, 2013; Wood et al, 2019a). Effectors can also be encoded without adjacent immunity genes when they are active against targets not found in the effector‐producing bacterium, for instance, targets only found in eukaryotes (Zhang et al, 2012).…”

Virulence genes and previously unexplored gene clusters in four commensal Neisseria spp. isolated from the human throat expand the neisserial gene repertoire

“…One possible intervention, as suggested by Unterweger et al [128], would allow non-pathogenic commensal bacteria such as these N. subflava biovar perflava possessing the T6SS to outcompete pathogens such as N. meningitidis and N. gonorrhoeae within a specific niche. The T6SS effector proteins, which are antibacterial to competitor species, have also been proposed to be developed as therapeutic agents against multidrug-resistant bacterial pathogens [129].…”