The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin–antitoxin and related phage defense systems

Abstract

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/ .

Multi-conflict islands are a widespread trend withinSerratiaspp

“…This discovery aligns with a growing body of reports documenting domains of diverse origins, including housekeeping proteins, being co-opted for defence against phages 3,4,13,36,52 . Furthermore, it corresponds to observed overlaps between toxin-antitoxin systems and anti-phage defence mechanisms 36,53–55 . It is plausible to speculate that this evolutionary or functional connection may extend to other anti-bacterial effectors or domains of different nature 3,4,36,53,54 .…”

“…Furthermore, it corresponds to observed overlaps between toxin-antitoxin systems and anti-phage defence mechanisms 36,53–55 . It is plausible to speculate that this evolutionary or functional connection may extend to other anti-bacterial effectors or domains of different nature 3,4,36,53,54 .…”

“…A closer look to the group of predicted T6SS-dependent effectors revealed that these are mostly represented by homologues of the orphan VgrG2b toxic domain (Figure 2e). We thus wondered whether this could represent an example of toxins adapted for phage defence 36 . We tested a homologue of the VgrG2b toxic domain and its putative immunity protein (WP_108680118.1 and WP_108680119.1) in E. coli MG1655 and S. marcescens Db10.…”

Diverse Genetic Contexts of HicA Toxin Domains Propose a Role in Anti-Phage Defense

“…Toxinantitoxin (TA) modules, often situated in Defense Islands (1)(2)(3)6), have only recently been associated with anti-phage activity, particularly via an abortive infection mechanism which halts infection at the cost of the host cell, thereby protecting the clonal population (7-9) (Smith, Foster et al NRM 2023, in press). Recent investigations strongly support the notion that TAs commonly function as anti-phage elements (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”

Mechanism of phage sensing and abortion by toxin-antitoxin-chaperone systems

“…In our previous high-throughput analysis of Type II TA systems, the NetFlax algorithm automatically identified TA systems based on their presence in a conserved two-gene architecture 21 . This meant that toxin or antitoxin-like proteins encoded in longer conserved neighbourhoods were not considered.…”