Discovery of aPseudomonas aeruginosaType VI secretion system toxin targeting bacterial protein synthesis using a global genomics approach

Abstract

19The Type VI secretion system (T6SS) is a bacterial weapon which delivers toxic 20 effectors to kill competitors or subvert some of their key functions. Here we use transposon 21 directed insertion-site sequencing (TraDIS) to identify T6SS toxins associated with the H1-22 T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach 23 identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of 24 this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey 25 cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis 26 in bacteria lacking either one or both of the asparagine or glutamine tRNA synthases. Our data 27 suggests that Tse8 combines as a non-cognate component of the transamidosome complex, 28 reducing fitness by limiting the ability of the cell to synthesize proteins. This is the first 29 demonstration of a T6SS toxin affecting protein synthesis, expanding the range of cellular 30 components targeted by this bacterial weapon. The success of the current study validates the 31 use of our TraDIS approach as a tool to drastically expand the repertoire of T6SS toxins in any 32 T6SS-encoding bacterium. 33 34 3 Bacteria rarely exist in a single-species planktonic state and instead form complex 35 polymicrobial structures, called biofilms 1,2 . Within this context bacteria often compete with 36 other microorganisms to secure space and nutrients. The Type VI secretion system (T6SS) is a 37 Gram-negative bacterial weapon which delivers toxins into neighbouring competitors to either 38 kill or subvert their key functions in order to attain dominance within a given niche 3-5 . The 39 T6SS is composed of 13 core components, several of which are structurally related to proteins 40 from the T4 bacteriophage tail 6 . The Hcp tube-like structure is capped by a VgrG-PAAR tip 41 complex, or spike, and encapsulated within a TssBC, or VipAB, contractile sheath. Upon 42 extension of the sheath within the cytoplasm and subsequent contraction, the spike is thought 43 to facilitate the puncturing of the cell membranes of both the producing and target cells, 44 allowing delivery of the attached toxins 7,8 . T6SS toxins have been shown to be secreted in 45 association with the VgrG tip complex, the Hcp tube, or as extension domains of the VgrG, 46 PAAR or Hcp proteins 9-12 . Importantly, neighbouring bacterial sister cells are protected from 47 the effects of the toxins by production of cognate immunity proteins, which are usually encoded 48 adjacent to the toxin gene in the genome 13 . The major identified targets of T6SS toxins to date 49 are components of the cell wall, as well as the cell membrane and nucleic acids 14 . These T6SS 50 toxins have mainly been identified by searching in the genomic proximity of known T6SS 51 components, or by detection of toxins in the secretome 9,12,15 . 52 Pseudomonas aeruginosa is a highly antibiotic-resistant Gram-negative pathogen and 53 ranked second by the World...

Pseudomonas fluorescens F113 type VI secretion systems mediate bacterial killing and adaption to the rhizosphere microbiome

“…This effector could replace a functional methyltransferase and only be toxic in those strains where this enzyme is necessary for bacterial viability. A similar case has been described in P. aeruginosa PAO1, in which the effector Tse8 is not linked to an immunity pair and replaces a functional component of the transamidosome complex only present in some strains 74 . Tfe8 may represent a new type of effector identified for the first time in this work and contains a MatE domain.…”

Bacterial injection machines: Evolutionary diverse but functionally convergent

“…By comparing the number of transposon insertions in each gene, they identified putative immunity proteins (which are essential genes), allowing the search for adjacent toxins. Following this approach, they discovered Tse8, an antibacterial toxin with an original mechanism of action, the inhibition of protein synthesis, and its cognate immunity protein Tsi8 (Nolan et al, ).…”

Causalities of war: The connection between type VI secretion system and microbiota

“…See text for additional details 2019; Hachani, Allsopp, Oduko, & Filloux, 2014;Hood et al, 2010;LaCourse et al, 2018;Nolan et al, 2019;Pissaridou et al, 2018;Russell et al, 2011;Whitney et al, 2014;Whitney et al, 2015). See text for additional details 2019; Hachani, Allsopp, Oduko, & Filloux, 2014;Hood et al, 2010;LaCourse et al, 2018;Nolan et al, 2019;Pissaridou et al, 2018;Russell et al, 2011;Whitney et al, 2014;Whitney et al, 2015).…”

“…The Pseudomonas aeruginosa H1-T6SS has nine experimentally tested effectors that target nucleic acids, peptidoglycan, the inner membrane, protein synthesis, NADP+, and the ATP pools (Ahmad et al, F I G U R E 2 Key functional impact and roles of the T6SS. See text for additional details 2019; Hachani, Allsopp, Oduko, & Filloux, 2014;Hood et al, 2010;LaCourse et al, 2018;Nolan et al, 2019;Pissaridou et al, 2018;Russell et al, 2011;Whitney et al, 2014;Whitney et al, 2015). The requirement for many distinct T6SS effectors is debatable.…”

“…Bioinformatic approaches focusing on conserved motifs, proximity to known T6SS components, or global genomic and proteomic approaches continue to uncover additional effectors, indicating that we are underestimating their number and thus their functions/cellular targets (Coulthurst, 2019;Jana, Fridman, Bosis, & Salomon, 2019;Nolan et al, 2019). T6SS-harbouring microorganisms also have multiple independent T6SS clusters; for example, P. aeruginosa encodes three and Burkholderia up to six (Schell et al, 2007).…”