Distribution of MICs according to the phylum of clinical isolates and reference strains.

<p>Distribution of MICs according to the phylum of clinical isolates and reference strains.</p

Reference strains able to grow at 37°C with high MIC according to source of isolation.

<p>Reference strains able to grow at 37°C with high MIC according to source of isolation.</p

Proportion of reference strains with high MIC.

*<p>Minimal inhibitory concentration.</p

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

<div><p>Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from <i>Pseudomonas aeruginosa</i> (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of <i>Vibrio nigripulchritudo</i> share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of <i>V</i>. <i>nigripulchritudo</i>. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. <i>In vitro</i> in presence of the cation Mg²⁺, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from <i>Bordetella pertussis</i> or <i>Bacillus anthracis</i>. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.</p></div

Rotation of the Vn-ExoY C_B/LID subdomain (red) towards its C_A subdomain (black) upon 3’dATP binding between the nucleotide-free Vn-ExoY^wt-SO42-actin-ATP-LatB:profilin and 3’dATP-bound Vn-ExoY-3’dATP-2*Mg²⁺:actin-ATP-LatB structures.

Side view of both structures superimposed on their black CA subdomain. The axis of rotation (horizontal in S1 Movie) is indicated by cyan spheres in a line. The SO42- ion and 3’dATP-2*Mg2+ are represented by ball-and-stick models and their transparent molecular surfaces. Secondary structures are shown in yellow, except for those of switch A, B, and C, which are shown in blue, cyan, and purple, respectively. Actin:profilin and actin structures are omitted. Domain motion analysis performed with DYNDOM [86] identifies a 25° rotation of the CB/LID subdomain of Vn-ExoY as a rigid body (in red, residues K553-M662VnE-CB/LID) around the hinge regions 532-533VnE and 662-663VnE with respect to the CA subdomain (in black, residues 468-532VnE-CA and 663-849VnE-CA excluding the switch A, B, C, whose conformations vary between the two structures) (see also Fig 4A and 4B). (MP4)</p

S4 Fig -

Overlays of the Vn-ExoY:3’dATP-2Mg2+:actin-ATP-LatB structure (2.1 Å resolution crystal structure, PDB: 8BR1) with (A) the Vv-ExoY:actin-ATP:profilin structure (3.9 Å resolution cryoEM structure, PDB: 7P1H) [28] and (B) Pa-ExoY structure from the Pa-ExoY:3’dGTP-1Mg2+:F-actin-ADP-Pi complex (3.2 Å resolution cryoEM structure, PDB: 7P1G) [28]. The structures are superimposed on their CA subdomain (residues 468-532Vn-ExoY-CA and 663-849Vn-ExoY-CA). Panels (A) and (B-1) show the same side views of the complexes, while (B-2) is a top view of (B-1). (TIF)</p

Switch A region is important for G-/F-actin and actin/calmodulin specificity.

A) Docking of the Vn-ExoY-3’dATP-2Mg2+:actin-ATP structure in complex with profilin and PRM to F-actin (PDB code: 6FHL) shows that the interaction of the Vn-ExoY switch A between actin subdomains 2 and 4 prevents bound actin (see Fig 4C) from assembling at the barbed (+) end of F-actin. It induces important steric clashes of switch A (red explosion symbols) with the penultimate actin subunit (in pink) at the barbed-end. Switch A binding to actin is also the region most incompatible with longitudinal contacts between adjacent actin subunits of the same F-actin strand (white and pink actin). This also most inhibits Vn-ExoY binding along F-actin [5,28]. B) Cartoon representation of the 3’dATP-EF-CaM complex. The NC catalytic domains of EF, CyaA and Vn-ExoY use the same regions, i.e. switch A and C, and a similar orientation to interact with their cofactor. The C-terminal Ca2+-binding globular domain of the 8.4-kDa protein CaM is responsible for most of the interactions with the EF or CyaA switch A and C [24,25,27]. However, it is much smaller than 42-kDa actin. It therefore only overlaps with actin subdomains 1 and 3. C) Structural comparison of G-actin- and CaM-activated Vn-ExoY and EF conformations, respectively. The common catalytic core, CA (yellow) and CB (orange) domains are shown in a cartoon tube representation, and the regions determinant for cofactor specificity, switch A (blue and cyan) and C (pink and purple) regions, are shown in a cartoon representation. The positioning of switch A in EF or CyaA bound to CaM and in Vn-ExoY or Pa-ExoY bound to G or F-actin, respectively, is the most divergent region at the cofactor-toxin binding interface. (TIF)</p

Crystallographic data-collection and refinement statistics.

The first and last residues indicated in the Vn-ExoY constructs correspond to those of the Uniprot sequence A0A6N3LUE9_9VIBR from the MARTX toxin of Vibrio nigripulchritudo, retaining the full numbering. In the text and figures, however, the thousands, i.e. 3000, have been omitted for ease of reading. (DOCX)</p

Interactions with non-cyclisable ATP or GTP analogues and metal ions, and position of the LID/C_B relative to the C_A subdomain in CaM-bound EF/CyaA and F-actin-bound Pa-ExoY structures.

The active site of (A) CaM-activated EF with 3′dATP and two Mg2+ metal ions (3.35 Å resolution crystal structure, PDB: 1XFV) [25], (B) CaM-activated CyaA structure with adefovir diphosphate (9-(2-(phosphonomethoxy)ethyl)adenine diphosphate, or PMEAPP) and two Mg2+ metal ions (2.20 Å resolution crystal structure, PDB: 1ZOT) [27], (C) F-actin-activated Pa-ExoY with 3′dGTP and a single Mg2+ metal ion (cryoEM structure at an average 3.20 Å resolution, PDB: 7P1G) [28], (D) CaM-activated EF structure with 3′dATP and a single Yb3+ metal ion (2.75 Å resolution crystal structure, PDB: 1K90) [24], and (E) G-actin-activated Vn-ExoY with 3′dATP and two Mg2+ metal ions (2.04 Å resolution crystal structure presented in this article, PDB: 8BR1). Thresholds for interaction detection are those of the PLIP (protein-ligand interaction profiler) [88] and Arpeggio [89] web servers, and those from the PoseView [99] tool available on the ProteinsPlus web server [90,100]. S8 Fig shows the position of the LID/CB relative to the CA subdomain in PDBs 1XFV (A), 1ZOT (B) and 1K90 (D). The use of an unconventional metal ion such as Yb3+ for CaM-bound EF crystallisation is expected to alter the coordination of EF NBP with ATP only slightly compared to the putative physiological metal-ligand Mg2+, as the metal Yb3+ only slightly reduces the AC catalytic activity of EF [24]. (TIF)</p

S6 Fig -

The structures of EF bound to CaM and ATP analogue (A, B) or Pa-ExoY bound to F-actin and GTP analogue (C) show strong active-site structural heterogeneity. They show either 1 or 2 metal ions associated with the substrate analogue, variable conformation or positioning of the different moieties of the purine nucleotide substrate analogues, and different positions of the LID/CB subdomain relative to the CA subdomain. The protein structures are shown in cartoon and coloured in cyan (PDB: 1XFV [27]), green (PDB: 1K90 [24]), light magenta (PDB: 1S26 [29]) and yellow (PDB: 7P1G [28]). The catalytic domains of the EF and Pa-ExoY NC toxins are superimposed on their CA subdomain. Metal ions are shown as spheres and the ligands and side chains of important catalytic residues (K346EF-CA/K81Pa-ExoY-CA, H353EF-LID/K88Pa-ExoY-LID and H577EF-Switch-B/H292Pa-ExoY-Switch-B) are shown as sticks. (A) Overlay of the CaM-activated structures of EF bound to 3’dATP containing 1 or 2 metal cofactors. (B) Overlay of the CaM-activated structures of EF bound to ATP analogues containing 1 metal cofactor. (C) Overlay of EF CaM-activated structure bound to 3’dATP with 1 Yb3+ metal ion from (A and B) and Pa-ExoY F-actin-activated structure bound to 3’dGTP with 1 Mg2+ metal ion. S7 Fig complements S6 Fig, offering together unambiguous evidence of the variations in the number of ions, the position and coordination of metal ion(s), and the position, conformation, and coordination of nucleotide substrate analogues. This includes variations in the coordination between the enzyme and nucleotide, as well as between the nucleotide and the metal ion(s). For instance, panel S6-B illustrates that the adenine ring of the AMPCPP bound to CaM-bound EF (PDB 1S26) is rotated approximately 180° compared to 3’dATP bound to CaM-bound EF (PDB 1XFV, 1K90) concerning the nucleotide conformation. Moreover, panels S6-A and S6-C demonstrate that 3’dATP (S6A Fig) or 3’dGTP (S6B Fig) can be bound with either 1 or 2 metal ions in previous structures. Additionally, the conserved K353EF-LID (S6A Fig) or K88PaE-LID (S6C Fig), equivalent to K535VnE-LID in Vn-ExoY (Figs 6A and 7A), can either be away from the γ-phosphate oxygens or coordinate with them. (TIF)</p