10.7554\/elife.03532.019<\/a><\/p>\nAuthors:<\/h4>\nAbstract:<\/h4>\n
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large \u03b2-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a \u03b2-hairpin and an \u03b1-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the \u03b2-hairpin and\/or the \u03b1-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue ‘communities’) within the translocation domain (especially around \u03b212\u2013\u03b214) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability. https:\/\/doi.org\/10.7554\/eLife.03532.001 eLife digest Escherichia coli is a bacterium that commonly lives in the intestines of mammals, including humans, where it is usually harmless and can even be beneficial to its host. However, some types of E. coli produce hair-like filaments called P pili that allow the bacteria to attach to the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, the filaments have to travel through a protein in the membrane called PapC usher. The PapC usher protein\u2014which is also involved in the assembly of the P pili filaments\u2014contains a tube-like part called a \u03b2-barrel that is usually blocked by another part of the protein called the ‘plug domain’. For the P pili to pass through the \u03b2-barrel, the plug domain has to move. This movement is controlled by two parts of the PapC protein, known as the \u03b1-helix and the \u03b2-hairpin, but it is not clear how. To address this question, Farabella et al. made computer models of the normal PapC protein and versions that lacked the \u03b1-helix and\/or the \u03b2-hairpin. Looking at these structural models and analyzing the evolution of PapC proteins helped to predict that certain regions of the \u03b2-barrel may be involved in controlling the movement of the plug domain, and this was then confirmed experimentally. Farabella et al. propose that these regions\u2014together with the \u03b1-helix and \u03b2-hairpin\u2014control the opening and closing of the \u03b2-barrel. Further work is needed to investigate how other parts of the PapC protein are involved in P pili formation. These new insights could prove useful in the development of alternative treatments to fight bacterial infection. https:\/\/doi.org\/10.7554\/eLife.03532.002 Introduction Gram-negative pathogens commonly express a vast variety of complex surface organelles that are involved in different cellular processes. One of these organelles, known as pili (or fimbriae), forms a class of virulence factors involved in host cell adhesion and recognition, invasion, cell mobility, and biofilm formation. P pili from uropathogenic Escherichia coli are specifically required for the colonization of the human kidney epithelium, a critical event in the kidney infection process (pyelonephritis) (Roberts et al., 1994). P pili are assembled on the bacterial outer membrane (OM) via the chaperone\/usher (CU) pathway (Thanassi et al., 1998), which is often used as a model system to elucidate the mechanism of pilus biogenesis (Waksman and Hultgren, 2009). The biogenesis of pili via the CU pathway is a highly ordered process that comprises sequential steps. The chaperone protein (PapD) brings the pilins to the bacterial OM where they are assembled into a pilus at a transmembrane pore protein known as the usher (PapC). The usher (\u223c800 residues) is composed of five domains (Figure 1A): a periplasmic N-terminal domain (NTD), an OM central translocation domain (TD) that comprises a translocation pore domain (TP), interrupted by a conserved Ig-like plug domain (PD), and two domains at the periplasmic C-terminal end (CTD1 and CTD2) (Thanassi et al., 2002; Ng et al., 2004; Capitani et al., 2006; Phan et al., 2011; Geibel et al., 2013). The structure of the apo TD (Figure 1B,C) consists of a 24-stranded kidney-shaped \u03b2-barrel where the PD is inserted into the loop connecting two \u03b2-strands (\u03b26\u2013\u03b27), occluding the luminal volume of the pore (Remaut et al., 2008; Huang et al., 2009). In the activated form of another archetypal member of the usher family, FimD, the PD is located outside the pore lumen in the periplasm, next to the NTD (Phan et al., 2011; Geibel et al., 2013). In addition to the PD, there are two secondary structure elements that uniquely characterize the large \u03b2-barrel structures of the usher TD (Figure 1B). The first element is a \u03b2-hairpin that creates a large gap in the side of the \u03b2-barrel, a feature unprecedented in previously known OM \u03b2-barrel structures (Remaut et al., 2008). This element (located between strands \u03b25 and \u03b26 of the barrel, Figure 1C) folds into the barrel lumen and constrains the PD laterally inside the barrel pore. Mutants lacking the \u03b2-hairpin show an increased pore permeability suggesting that the \u03b2-hairpin has a role in maintaining the PD in a closed conformation (Volkan et al., 2013). The second element is an \u03b1-helix (located on the loop between \u03b213 and \u03b214, Figure 1B), which caps the \u03b2-hairpin from the extracellular side. Mutants lacking the \u03b1-helix, or in which the interface between the helix and the PD is disrupted, present a remarkable increase in pore permeability, comparable with that of the mutant lacking the PD, suggesting a role for the helix in maintaining the PD in a closed state (Mapingire et al., 2009; Volkan et al., 2013). Figure 1 with 1 supplement see all Download asset Open asset PapC usher organization and detail of its translocation domain. (A) A diagram of the domain organization of PapC usher. NTD (dark-blue) represents the N-terminal domain, CTD1 (light-violet) and CTD2 (dark-violet) represent the C-terminal domains; TD represents the translocation domain, comprising the TP (translocation pore, light-blue) and the PD (plug domain, magenta). (B and C): Ribbon representation of the starting model of the native translocation domain (TD) of PapC with the labels ‘N’ and ‘C’ indicating the N and C termini of the translocation channel. The \u03b2-barrel, PD (including the P-linkers), \u03b2-hairpin, and \u03b1-helix (including the H-linkers) are coloured blue, magenta, orange, and yellow, respectively. The outer membrane position is represented schematically with the labels ‘E’, ‘M’, and ‘P’ indicating the extracellular side, the membrane, and the periplasmic side, respectively. Side view of the TD (B) is shown with the \u03b1-helix, \u03b2-hairpin, H-linker1, H-linker2, P-linker1, P-linker2, and PD, labelled. Extracellular top view of the TD (C) is shown with the barrel \u03b2 strands labelled \u03b21 through \u03b224 and with the PD strands labelled \u03b2A through \u03b2F. The figures were created with Chimera (Pettersen et al., 2004). https:\/\/doi.org\/10.7554\/eLife.03532.003 The mutant lacking both the \u03b2-hairpin and the \u03b1-helix is defective for pilus biogenesis (Mapingire et al., 2009). It has been observed in other OMP \u03b2-barrels that such secondary structure elements (e.g., an \u03b1-helix that protrudes inside the barrel or packs against the transmembrane strands) can use complex allosteric mechanisms to mediate their function (Naveed et al., 2009). These are often combinations of large conformational changes (‘global motions’) dictated by the overall architecture (including movement of secondary structure elements) and smaller changes (‘local motions’, such as the motion of recognition loops and side-chain fluctuations) (Liu and Bahar, 2012). Additionally, it has been shown that important residues in terms of evolution (highly-coevolved or conserved) could have a pivotal role in mediating such allosteric communications (Suel et al., 2003; Tang et al., 2007). In this study, to understand the allosteric mechanism leading to the plug displacement in PapC and the involvement of the \u03b1-helix and \u03b2-hairpin, we used a hybrid computational approach and verified our results experimentally. By combining sequence conservation analysis, mutual information-based coevolution analysis, and all-atom molecular dynamics (AA-MD), we modelled the interaction network within the native PapC TD as well as within different mutants lacking the \u03b1-helix, \u03b2-hairpin, and both. This unique computational approach allowed us to identify residues that are likely to be involved in the transmission of the allosteric signal between the \u03b1-helix, \u03b2-hairpin elements and the plug. These residues were investigated by site-directed mutagenesis, functional studies, and planar lipid bilayer electrophysiology. The results confirmed the involvement of 4 of the 5 distinct communities of residues in modulating the usher’s channel activity and gating, suggesting that they all participate in the allosteric mechanism controlling plug displacement. Results To investigate if the \u03b2-hairpin or \u03b1-helix (or both) of the TD (residues 146\u2013637 in the full length PapC) have a role in the allosteric communication leading to the displacement of the PD (residues 264\u2013324), we performed four independent MD simulations, corresponding to the PapC TD model (sim1, Table 1) and three mutants embedded in a mixed lipid bilayer (Table 1): (i) where the region corresponding to the hairpin between \u03b25 and \u03b26 (residues 233\u2013240) is deleted (sim2); (ii) where the \u03b1-helix between \u03b213 and \u03b214 (residues 447\u2013460) is removed; and where both the regions were removed (sim4). The last 50 ns of simulation were considered for analysis, where the averaged root-mean-square deviation of C\u03b1 atoms (C\u03b1-RMSD) from the averaged structures stabilized around 2.00 \u00b1 0.09 \u00c5, 1.80 \u00b1 0.09 \u00c5, 1.86 \u00b1 0.11 \u00c5, and 2.03 \u00b1 0.10 \u00c5, for the native (sim1), hairpin mutant (sim2), helix mutant (sim3), and helix-hairpin mutant (sim4), respectively (Figure 1\u2014figure supplement 1). This timescale, although limited for a full exploration of the structural changes induced by the mutations, was informative in revealing how local structural perturbations may affect allosteric changes leading to the plug displacement in PapC TD. Table 1 Summary of the simulations. https:\/\/doi.org\/10.7554\/eLife.03532.005 SimulationModel systemsLength (ns)Sim1Native PapC TD72Sim2Hairpin mutant70Sim3Helix mutant70Sim4Helix-hairpin mutant70 Descriptions of the items are: Simulation, the name of the simulation; Model systems, PapC TD model systems simulated; and Length, the length of the simulation. Non-covalent interaction network in the native PapC translocation domain and its perturbation in the absence of the \u03b2-hairpin, \u03b1-helix, or both The changes in the non-covalent interactions (hydrogen bonds and salt bridges) between all residue pairs were analysed within the native TD by calculating their non-covalent interaction score (NCI score) (see ‘Materials and methods’). A non-covalent residue\u2013residue interaction network (RIN) comprising 492 nodes (residues) and 1350 edges (interactions) was then constructed as a weighted undirected graph for the native TD (Figure 2) and the three mutant systems (Figure 2\u2014figure supplement 1A\u2013C), with the weight for each edge given by the corresponding NCI score (Table 2). All four RINs have properties typical of small-world networks (Atilgan et al., 2004; Haiyan and Jihua, 2009; Taylor, 2013), with significant higher clustering coefficient compared to a corresponding random network and a higher mean short path length (Table 2). Within the constructed non-covalent native RIN, we identified 246 weak-to-strong interactions (connecting 362 nodes) with an NCI score of at least 0.3. Among these, 231 nodes connected by 133 edges showed an NCI score greater than 0.6 (i.e., strong interaction) of which 78 involve residues that are part of the barrel strands (58.6%). Figure 2 with 2 supplements see all Download asset Open asset The native TD and its non-covalent interaction network (non-covalent RIN). (A) Ribbon representation of the starting model of the native translocation domain (TD) of PapC with the labels ‘N’ and ‘C’ indicating the N and C termini of the translocation channel. The \u03b2-barrel, PD, P-linker1, P-linker2, \u03b2-hairpin, and \u03b1-helix (including the H-linkers) are coloured grey, magenta, light purple, dark purple, orange, and yellow, respectively. The \u03b1-helix, \u03b2-hairpin, P-linker1, P-linker2, and PD are labelled. (B) Non-covalent RIN representation of the native translocation domain (TD) of PapC visualized with Cytoscape 2.8.2 (Smoot et al., 2011) based on RINalyzer plug-in analysis (Doncheva et al., 2011) (see Figure 2\u2014figure supplement 1 for the RINs of the TD mutants). The nodes (representing residues) are coloured by structural element as in (A) Edges (connecting two residues) are shown in blue, the edge width is proportional to its NCI score from lower to higher values. https:\/\/doi.org\/10.7554\/eLife.03532.006 Table 2 Summary of the residue\u2013residue interaction networks (RINs) parameter. https:\/\/doi.org\/10.7554\/eLife.03532.009 RINFull RINCCrC\/CrLLrL\/LrNative PapC TD1350 (492)0.3840.01232.006.673.781.76Hairpin mutant1196 (485)0.3680.01133.457.203.901.84Helix mutant1225 (476)0.3620.01132.906.673.901.71Helix-hairpin mutant854 (466)0.2620.00832.758.104.701.72 Descriptions of the items are: RIN, residue\u2013residue interaction networks of the different model systems; Full RIN, number of edges in the RIN, in parenthesis the number of node; C, average clustering coefficient; Cr, average clustering coefficient for the random networks with the same size; C\/Cr, average clustering coefficient ratio (as used in Atilgan et al., 2004); L, average shortest path length; Lr, average shortest path length for the random networks with the same size; L\/Lr, average shortest path length ratio (as used in Atilgan et al., 2004). Comparative analysis between the RINs of native and mutants systems revealed slight changes, suggesting a rearrangement in the interaction network. To better understand the mutation-induced changes in network components, we calculated the difference in non-covalent interaction score (\u0394NCI score) between the native TD system and each of the mutant systems (the weakened interactions are shown in Figure 2\u2014figure supplement 2A\u2013C). This information was then added as a weighted undirected edge to the pre-existing native non-covalent RIN (the \u0394NCI edges are shown in Figure 2\u2014figure supplement 2D). Interestingly, 24% of the strong interactions in the native RIN were weakened relative to the RIN of the mutant lacking the \u03b2-hairpin, 22.6% relative to the mutant lacking the \u03b1-helix, and 23.3% relative to the mutant lacking both, suggesting that interactions between nodes that are not part of the deleted secondary structure elements were consistently weakened in the absence of these elements. Evolutionary analysis of PapC TD We first extracted evolutionary information from a multiple sequence alignment of the PapC TD family. The patterns of conservation in the TD using Consurf (Ashkenazy et al., 2010) analysis suggested that the highly conserved residues (score 9) tend to be clustered in two specific regions of the usher (Figure 3A). The first cluster mapped onto the PD and the P-linkers (P-linker1 residues 248\u2013263; P-linker2 residues 325\u2013335) connecting it to the TP. The second cluster (which included the majority of the highly-conserved residues) mapped onto one side of the TP (strand \u03b21\u201314 and \u03b224). It includes residues: (i) near the periplasmic side of the \u03b2-barrel within \u03b21\u20134 strands and \u03b224 strand; (ii) on the extracellular side of the barrel (within \u03b25\u201310); (iii) in the \u03b2-hairpin region (\u03b2-hairpin and \u03b27\u20139); and (iv) in the area of \u03b210\u201314 capped by the \u03b1-helix region, which comprises the \u03b1-helix and its linkers\u2014H-linker1 (residues 445\u2013450) and H-linker2 (461\u2013468, respectively). Surface representation of the TD reveals a continuous patch of conserved residues facing the lipid bilayer, including \u03b213, the extracellular half of \u03b214 and the periplasmic half of \u03b212 (Figure 3B). Intriguingly, this patch (‘\u03b213 conserved patch’) reaches the full height of the pore from the \u03b1-helix region to a functionally important loop located between \u03b212 and \u03b213 strands (Farabella, 2013; Volkan et al., 2013). Figure 3 Download asset Open asset Evolutionary analysis of PapC TD. (A\u2013B) Sequence conservation calculated with Consurf (Ashkenazy et al., 2010) and mapped onto the initial model of the native PapC TD (sim1, t = 0). Amino acid conservation scores are classified into nine levels. The colour scale for residue conservation goes from cyan (non-conserved: grade 1) to maroon (highly conserved: grade 9), unreliable positions are coloured light yellow. (A) Ribbon representation of the model with the highly conserved residues (grade9) shown as spheres and key elements labelled. (B) Molecular surface of the model with \u03b212\u2013\u03b214 labelled. (C\u2013D) Sequence co-evolution calculated with PyCogent (Knight et al., 2007; Caporaso et al., 2008). (C) The co-evolving residues are mapped onto the initial model of the native PapC TD (sim1, t = 0). (D) The co-evolution network as visualized with Cytoscape 2.8.2 Cytoscape 2.8.2 (Smoot et al., 2011) based on RINalyzer plug-in analysis (Doncheva et al., 2011). Edges (connecting two co-evolved residues) are shown in blue, and nodes (representing coevolved residues) are coloured by structural element. The PD, P-linker1, P-linker2, \u03b2-hairpin, and \u03b1-helix are indicated schematically and coloured as in Figure 2. The node size is proportional to its degree of connectivity. https:\/\/doi.org\/10.7554\/eLife.03532.010 In addition to investigating conservation, we performed an analysis to identify the coevolutionary relationships between residues in the structure. Using normalized mutual information (NMI) analysis (Martin et al., 2005) with a Z-score cut-off = 4 (see ‘Materials and methods’) to detect the intra-molecular coevolved residues within PapC TD, a coevolutionary RIN containing 100 coevolved residues (nodes) and 357 connections (edges) was derived (Figure 3D). Mapping the network onto the PapC TD structure showed that many of the residues involved are also connected spatially and are clustered in the same regions where the highly-conserved residues were found (P-linkers, the PD, and the barrel wall capped by the \u03b1-helix, in close proximity to the \u03b2-hairpin) (Figure 3C,D). The obtained coevolutionary RIN showed a significant clustering coefficient compared to a corresponding random network (of 0.493 vs 0.187, respectively) and a comparable mean short path length (3.15 vs 2.57, respectively) (Daily et al., 2008). Identifying allosteric ‘hot spots’ from a hybrid residue interaction network We constructed one hybrid RIN in which the attributes for the nodes and edges are defined by the properties described above (non-covalent networks and evolutionary analysis, see ‘Materials and methods’). Starting from the secondary structure elements (that uniquely characterise the barrel\u2013the \u03b1-helix and \u03b2-hairpin) in this hybrid RIN, we used a multi-step procedure to reconstruct a pathway of communication between them (Figure 4). Figure 4 with 1 supplement see all Download asset Open asset Detection of allosteric hot spots. A flowchart representing the multistep procedure used to identify allosteric hot spots. First, a sub-network of the protein hybrid RIN was generated starting from the \u03b1-helix and \u03b2-hairpin. Then, filters based on the evolutionary information and on the interactions analysis were applied (see Figure 4\u2014figure supplement 1) resulting in a sub-network of ‘hot spot’ residues. https:\/\/doi.org\/10.7554\/eLife.03532.011 This initial large sub-network is formed by 208 nodes (residues) connected by 456 NCI edges (in the native RIN). Applying the dynamic filter (independently) on the edges, based on the difference in non-covalent interaction score between the native TD and each of the mutants (\u0394NCI > 0), revealed that in each case a large part of network has weakened interactions (hairpin mutant: 200 nodes, 438 NCI edges; helix mutant: 199 nodes, 437 NCI edges; helix-hairpin: 202 nodes, 443 NCI edges) (Figure 4\u2014figure supplement 1A). The application of the evolutionary filter revealed that only a small part of the sub-network is made of evolutionary important residues (75 nodes connected by 104 native NCI edges). Combining the filters (Figure 4\u2014figure supplement 1B) resulted in 69 nodes connected by 100 NCI edges (thus representing interacting residues in the native PapC network). The residues of this sub-network (14% of all residues in the TD) were considered ‘hot spots’ in the communication pathway of PapC TD (‘hot spot’ sub-network). Mapping them onto the structure revealed that they are located close together in a continuous area within PapC TD. We analysed the community structure of the hot spot sub-network using the edge-betweenness clustering algorithm (Girvan and Newman, 2002; Morris et al., 2010). This analysis shows that the sub-network has a modular with a of of the is which is typical of based RIN and 2004; et al., 2009). a of communities containing two or residues were from which only five communities are composed by than five residues. For analysis, we to only these five which are between and the P-linkers between the \u03b2-hairpin and the conserved region at the of the \u03b1-helix between and the between the \u03b2-hairpin, P-linker2 and the PD and on the of the PD loop and (Table 3 and Figure Table 3 in the hot spot Descriptions of the items are: the name of the residues that are part of the Figure 5 Download asset Open asset PapC TD communities. The communities of the hot spot sub-network are shown as surface by and indicated schematically to The shows a close of the identified residues located in the the \u03b2-hairpin, the conserved region at the of the \u03b1-helix, in the between The residues are labelled in and to the structure of the apo PapC TD We a number of key residues from the communities hot spot which different elements within each for (Figure These were found in communities in residues linking the P-linkers and the barrel wall that in maintaining the P-linkers in a closed and in residues that the of the \u03b1-helix (the extracellular end of the \u03b213 conserved and \u03b2-hairpin in residues on the interface between and and in residues that are part of the interface with and the periplasmic end of the \u03b213 conserved patch analysis of residues in the hot spot sub-network To if the key hot spot residues identified above elements within each to allosteric within we constructed a set of (Table 4). of the mutants was present at a in the OM compared to the PapC and the not affect the of the usher to form a \u03b2-barrel in the OM not The of the PapC mutants was by to P pili on the bacterial P pili to on human and assembly of functional P pili was using a of the mutants greater than in compared with with 4 of the mutants and activity = (Table 4). The defective mutants were in key residues from communities and roles for these communities in usher Table 4 Analysis of PapC Descriptions of the items are: the PapC the name of the community to which the residue the of bacteria to human Antibiotic the of of around filter with or The sensitivity includes the filter We next used an sensitivity to the PapC mutants for on channel activity of the usher. The OM of Gram-negative bacteria has permeability to such as and to such as and to these In its the usher TP is closed by the PD, of the that channel gating by the PD in the opening of the large TD leading to increased sensitivity of the bacteria to five of the PapC mutants and increased sensitivity to one or of the (Table 4). and not to the allosteric within the same sensitivity and to pili of the native PapC (Table 4). However, the and sensitivity are and as they in sensitivity to smaller changes in the to pili or channel activity of the usher. analysis of mutants In PapC were found to be in their to or in their permeability to or in in in and in We to mutants in view of their channel activity using planar lipid bilayer electrophysiology (which is a only of these mutants protein (as in to the experiments in in in and in the OM and were not to the of the membrane bilayer and to their native conformation in of PapC proteins (see ‘Materials and methods’) was in planar lipid by the membrane to as channel activity was the was to and the to of channel activity at and and at and were The typical of the PapC usher is by at a representing the closed state of the and of These represent of from 50 to (Figure it is not to how many were inserted into the bilayer, the observed of are to represent conformational of a pore. the of the<\/p>\n","protected":false},"excerpt":{"rendered":"
DOI: 10.7554\/elife.03532.019 Authors: Abstract: Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large \u03b2-barrel occluded by […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[11],"tags":[],"class_list":["post-933","post","type-post","status-publish","format-standard","hentry","category-m-470-ac-dc-sds"],"_links":{"self":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/933","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=933"}],"version-history":[{"count":0,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/933\/revisions"}],"wp:attachment":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=933"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=933"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=933"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}