Antibacterial T6SS effectors with a VRR-Nuc domain are structure-specific nucleases

Abstract

The type VI secretion system (T6SS) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here we characterize the function of the SPI-22 T6SS of Salmonella bongori showing that it has antibacterial activity and identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins with a DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV3 recognizes specific DNA structures and preferentially cleave splayed arms, generating DNA double-strand breaks and inducing the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanisms regulating the activity of these toxins.

Intercepting biological messages: Antibacterial molecules targeting nucleic acids during interbacterial conflicts

“…parahaemolyticus ( Jana et al ., 2019 ), IdrD from Proteus mirabilis ( Sirias et al ., 2020 ), RhsB from Acidovorax citrulli ( Pei et al ., 2022 ), and TseV from P . aeruginosa and Salmonella bongori ( Wang et al ., 2021 ; Hespanhol et al ., 2022 ). In addition, SitA3 involved in interbacterial antagonism via outer membrane exchange contains the conserved αβββαβ fold ( Vassallo et al ., 2017 ).…”

“…The VRR-Nuc (virus-type replication repair nuclease) domain is found in enzymes involved in interstrand DNA crosslink repair ( Kratz et al ., 2010 ; Liu et al ., 2010 ; MacKay et al ., 2010 ; Smogorzewska et al ., 2010 ; Gwon et al ., 2014 ; Wang et al ., 2014 ; Zhao et al ., 2014 ), but recent studies identified effectors containing this domain - named TseVs (type VI effector VRR-Nuc) ( Wang et al ., 2021 ; Hespanhol et al ., 2022 ). TseV2 and TseV3 from S .…”

“…bongori were shown to participate in interbacterial competition in a T6SS-dependent manner ( Hespanhol et al ., 2022 ). TseV3 is a structure-specific nuclease that cleaves DNA substrates with a Y shape (named splayed arm), which resemble replication forks or transcription bubbles ( Hespanhol et al ., 2022 ). TseV2 and TseV3 induce DNA double-strand breaks and activate the SOS response in vivo ( Hespanhol et al ., 2022 ).…”

“…TseV3 is a structure-specific nuclease that cleaves DNA substrates with a Y shape (named splayed arm), which resemble replication forks or transcription bubbles ( Hespanhol et al ., 2022 ). TseV2 and TseV3 induce DNA double-strand breaks and activate the SOS response in vivo ( Hespanhol et al ., 2022 ).…”

2nd Workshop in Microbial Molecular Biology

“…Next, were two oral presentations from submitted abstracts. Julia Takuno Hespanhol, a Master's student from Ethel Bayer‐Santos laboratory (ICB/USP) presented their recent work describing antibacterial toxins secreted by a T6SS from Salmonella bongori that function as endonucleases, generating double‐strand breaks in the DNA of target cells (Hespanhol et al, 2022). Letícia Franco, a post‐doc from Mario Henrique Barros group (ICB/USP) discussed work performed with Alexander Tzagoloff (Columbia University, USA) describing the mechanisms of assembly of cytochrome oxidase and ATP synthase in Saccharomyces cerevisiae .…”