Novel type II toxin-antitoxin systems with VapD-like proteins

Abstract

Type II toxin-antitoxin (TA) systems are widespread in prokaryotes. They consist of neighboring genes encoding two small proteins: a toxin that inhibits a critical cellular process and an antitoxin that binds to and neutralizes the toxin. The VapD nuclease and the VapX antitoxin comprise a type II TA system that contributes to the virulence of the human pathogen Haemophilus influenzae. We analyzed the diversity and evolution of VapD-like proteins. By examining loci adjacent to genes coding for VapD-like proteins, we identified two novel families of antitoxins, which we named VapY and VapW. VapD toxins cognate to novel antitoxins induce the SOS response when overproduced, suggesting they target cellular processes related to genomic DNA integrity, maintenance, or replication. Though VapY has no sequence similarity to VapX, they share the same SH3 fold characterized by the five anti-parallel β sheets that form a barrel. VapW is a homolog of VapD without conserved catalytic residues required for nuclease activity. The crystal structure of the VapD-VapW complex reveals that VapW lacks the dimerization interface essential for the catalytic activity of VapD but retains the second interaction interface that enables VapD hexamerization. This allows VapW to bind VapD in the same manner that VapD dimers bind to each other in hexamers. Thus, though the VapD catalytic cleft remains accessible in the VapD-VapW complex, VapW may disrupt VapD oligomerization. To our knowledge, VapWD provides a unique example of TA systems evolution when a toxin loses its activity and becomes an antitoxin to itself.

Importance: Genes encoding virulence-associated protein D (VapD) homologs are found in many pathogens such as Helicobacter pylori, Haemophilus influenzae, and Xylella fastidiosa. There are many indications that VapD proteins contribute to virulence, even though the exact mechanism is not known. VapD proteins are either encoded by stand-alone genes or form toxin-antitoxin pairs with VapX. We performed a comprehensive census of vapD-like genes and found two new antitoxins, VapW and VapY. The VapW antitoxins are catalytically inactivated variants of VapD, revealing a new evolutionary mechanism for the appearance of toxin-antitoxin pairs.

Keywords: Cas2; SOS-response; VapD; evolution; toxin-antitoxin systems.

Fig 1

Phylogenetic analysis of VapD-like proteins. (A) A maximum-likelihood phylogenetic tree of VapD-like proteins. Transfer bootstrap expectation values are shown with gray circles on branches. Arcs on the outside indicate association with putative antitoxins. The arcs and VapD clades are colored according to a type of putative antitoxin (blue: VapX, green: VapY, and ocher: VapW). Red triangles outside the VapW arc indicate fused VapW-VapD proteins. Proteins are experimentally shown to be toxic and VapD^Hpy are labeled with abbreviated species names. Abbreviations of species harboring TA pairs characterized in this study are shown in bold. (B) Examples of genomic loci containing genes of VapD homologs. Genes are colored according to FlaGs clusterization (39). Genomic loci are labeled with the taxonomic name of the corresponding organism. Loci encoding proteins experimentally characterized in this study are highlighted with labels in bold font. (C) Multiple sequence alignment of VapD-like and VapW proteins. Sequences are labeled with NCBI protein accession numbers and taxonomic names. Labels of proteins experimentally characterized in this study are highlighted with bold font. Unaligned N- and C-terminal regions were omitted for clarity. Putative catalytic residues of VapD are highlighted with a red background. Shading intensity indicates the conservation of the position accounting for the BLOSUM62 score. Secondary structure elements are schematically shown based on the VapD^Cje crystal structure, which is described below.

Fig 2

VapW-VapD and VapY-VapD are novel type II TA systems. (A) Validation of the VapD-VapY TA pair from Muribaculum sp. An289 (Mur). The putative antitoxin (vapY) and toxin (vapD) genes were cloned downstream of tetracycline and ara-inducible promoters, respectively. E. coli BW25113 were transformed with the resulting plasmids pBAD33_VapD and pASK_VapY, and toxin, antitoxin, or both proteins synthesis was induced by the addition of, respectively, 0.2% ara, 0.1 mg/µL of AtC, or both. Cultures were grown for 60 min in the presence or the absence of inducers, and aliquots of serial dilutions of the cultures were deposited on the surface of YT agar plates. Results of overnight growth at 37°C are shown. (B) Validation of VapW-VapD TA pairs from Cje and Seq. Since no effect was observed in a set-up used in panel A (Fig. S4), a different set-up was used to exclude the possibility of antitoxin production due to tetracycline promoter leakage. Putative antitoxin (vapW) and toxin (vapD) genes were cloned downstream of tetracycline and ara-inducible promoters, respectively. The resulting plasmids pBAD33_VapD (Seq or Cje) and pASK_VapW (Seq or Cje) or corresponding empty vectors pBAD33 or pASK_IBA43 were transformed in E. coli BW25113 in various combination. The viability of cells grown in the presence or in the absence of both inducers was next tested as in panel А but after 120 min of incubation. (C) Tandem affinity purification of 6×His-tagged VapD toxins and cognate strep-tagged VapY or VapW antitoxins from extracts of co-overexpressing cells. The protein content of samples corresponding to different steps of purification—before induction (lane 1), after induction (lane 2), material eluted from Talon Co²⁺-resin (lane 3), and material eluted from Strep-Tactin resin (lane 4)—was analyzed by SDS-PAGE and visualized by Coomassie staining. M, protein molecular weight markers.

Fig 3

Expression of VapD toxins leads to the SOS response. (A) E. coli BW25113 was transformed with plasmids bearing wild-type or D7N inactive variant of the vapD^Mur gene under the control of ara-inducible promoter. Cells were grown for 60 min in the presence or the absence of ara, and aliquots of serial dilutions of the cultures were deposited on the surface of YT agar plates. Results of overnight growth at 37°C are shown. (B) Scheme of SOS response measurement assay. E. coli MG1655 Δ10 strain with 10 deleted TA systems was transformed with pBAD33 plasmid containing toxin gene (Tox) under the ara-regulated promoter and reporter plasmid pSulA-RFP containing fluorescent protein TurboRFP gene under the SOS-inducible sulA promoter (55). (С) Time-course measurement of optical density (OD₆₀₀) and TurboRFP fluorescence of ara-induced cells from the experiment described in scheme B. An inactivated D7N VapD^Mur mutant was used as a negative control. The gyrase inhibitor CcdB was used as a positive control for SOS response. RNase RelE was used as a negative control.

Fig 4

Structure of the VapD-VapW complex. (A) Structure of VapD^Cje from VapD-VapW complex 1 (right) and its topology diagram (left). The red rectangle represents the dimerization interface with the C-terminus of another VapD subunit, shown with dashed lines. Two catalytic residues, D11 and S46, are shown on the structure and indicated with two red circles on the diagram. The red area on the structure represents the location of the catalytic cleft that is formed between two VapD^Cje subunits. (B) Structure of VapW^Cje from the VapD-VapW complex 1 (right) and its topology diagram (left). The red rectangle highlights the interaction between the C-terminus and the core region of the protein. Residues V11 and E47, which correspond to catalytic residues D11 and S46 of VapD, are shown on the structure and designed with two gray circles on the diagram. (C) VapW^Cje (orange) and VapD^Cje (gray) structures superimposition. (D) Binding sites of VapD^Cje to two subunits of VapW^Cje. (E) Binding sites of VapW^Cje to two subunits of VapD^Cje. (F) Two kinds of VapD-VapW complexes in the crystal with different distances between VapD subunits. The catalytic cleft is shown in red. (H) Crystal structure of VapD-VapX from H. influenzae (PDB ID: 6ZN8). The figure in the background represents the surface of the VapD-VapW complex 1.