The P. aeruginosa effector Tse5 forms membrane pores disrupting the membrane potential of intoxicated bacteria

The type VI secretion system (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate that Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions; therefore, membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.We gratefully acknowledge the Laboratories of Dr. Daniel Ladant (Institut Pasteur, Paris) and Dr. Victor de Lorenzo (Centro Nacional de Biotecnologia, Madrid) for the plasmids received (pKTop and pSEVA plasmids, respectively). Also, we would like to acknowledge the Laboratory of Dr. Joseph Mougous for the P. aeruginosa strains received. The technical assistance from Cristina Civantos and Adrian Ruiz is also very much appreciated. We acknowledge the FGCZ for the mass spectrometry analyses and the technical support (Functional Genomics Center Zurich (FGCZ), University/ETH Zurich). D.A.-J. acknowledges support by the MINECO Contracts CTQ2016-76941-R and PID2021-127816NB-I00, Fundacion Biofisica Bizkaia, the Basque Excellence Research Centre (BERC) programme, and IT709-13 and IT1745-22 of the Basque Government, and Fundacion BBVA. A.G.-M. acknowledges the financial support received from the Spanish Ministry of Universities and the Grants for the requalification of the Spanish university system for 2021-2023, financed by the European Union-Next Generation EU-Margarita Salas Modality. A.A. acknowledges support from the Spanish Ministry of Science and Innovation (Project 2019-108434GB-I00 funded by MCIN/AEI/10.13039/501100011033), Generalitat Valenciana (project AICO/2020/066) and Universitat Jaume I (project UJI-B2018-53). M.Q.-M. acknowledges support from the Spanish Ministry of Science and Innovation (Project IJC2018-035283-I funded by MCIN/AEI/10.13039/501100011033) and Universitat Jaume I (project UJI-A2020-21). P.B acknowledges the financial support received from the Spanish Ministry of Science and Innovation through the Ramon y Cajal Programme (contract RYC2019-026551-I)

Solid-state supramolecular organization, established directly from powder diffraction data, and photoluminescence efficiency of rigid-core oligothiophene-S,S-dioxides.

The "rigid-core" material 3,5-dimethyl-2,3'-bis(3-methylthiophene)-dithieno[3,2-b:',3'−d]thiophene-4,4-dioxide (DTTOMe4) has the highest photoluminescence ever reported for thiophene-based molecule..

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an alpha/beta architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.This work was supported by the European Commission Contract HEALTH-F3-2011-260872, the Spanish Ministry of Economy and Competitiveness Contract BIO2013-49022-C2-2-R, and the Basque Government (to M.E.G.); Slovak Research and Development Agency Contract No. DO7RP-0015-11 (to K.M.) and the NIH/NIAID grant AI064798 (to M.J.). D.A.-J. acknowledges the support from Fundacion Biofisica Bizkaia. We gratefully acknowledge Sonia Lopez-Fernandez (Unit of Biophysics, CSIC, UPV/EHU, Spain), Drs E. Ogando and T. Mercero (Scientific Computing Service UPV/EHU, Spain) for technical assistance. We thank the Swiss Light Source (SLS), and the Diamond Light Source (DLS) for granting access to synchrotron radiation facilities and their staff for the onsite assistance. We specially thank the BioStruct-X project to support access to structural biology facilities. We also acknowledge all members of the Structural Glycobiology Group (Spain) for valuable scientific discussions. The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, Strain H37Rv, Purified Phosphatidylinositol Mannosides 1 and 2 (PIM1,2), NR-14846

Type VI Secretion System in Pseudomonas aeruginosa: Secretion and Multimerization of VgrG Proteins

Pseudomonas aeruginosa is a Gram-negative bacterium causing chronic infections in cystic fibrosis patients. Such infections are associated with an active type VI secretion system (T6SS), which consists of about 15 conserved components, including the AAA+ ATPase, ClpV. The T6SS secretes two categories of proteins, VgrG and Hcp. Hcp is structurally similar to a phage tail tube component, whereas VgrG proteins show similarity to the puncturing device at the tip of the phage tube. In P. aeruginosa, three T6SSs are known. The expression of H1-T6SS genes is controlled by the RetS sensor. Here, 10 vgrG genes were identified in the PAO1 genome, among which three are co-regulated with H1-T6SS, namely vgrG1a/b/c. Whereas VgrG1a and VgrG1c were secreted in a ClpV1-dependent manner, secretion of VgrG1b was ClpV1-independent. We show that VgrG1a and VgrG1c form multimers, which confirmed the VgrG model predicting trimers similar to the tail spike. We demonstrate that Hcp1 secretion requires either VgrG1a or VgrG1c, which may act independently to puncture the bacterial envelope and give Hcp1 access to the surface. VgrG1b is not required for Hcp1 secretion. Thus, VgrG1b does not require H1-T6SS for secretion nor does H1-T6SS require VgrG1b for its function. Finally, we show that VgrG proteins are required for secretion of a genuine H1-T6SS substrate, Tse3. Our results demonstrate that VgrG proteins are not only secreted components but are essential for secretion of other T6SS substrates. Overall, we emphasize variability in behavior of three P. aeruginosa VgrGs, suggesting that, although very similar, distinct VgrGs achieve specific functions

Dissecting the structural and chemical determinants of the "open-to-closed" motion in the mannosyltransferase PimA from Mycobacteria

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-To-β-strand and β-strand-To-α-helix transitions and (ii) an "open-To-closed"motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-To-closed"motion, with the β-phosphate group providing the high-Affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-To-closed"motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors

TssA forms a gp6-like ring attached to the type VI secretion sheath

The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS

A native ternary complex trapped in crystal reveals the catalytic mechanism of a retaining glycosyltransferase

et al.Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes including cell signaling, cell development and host-pathogen interactions. Glycosyl transfer can proceed with either ‘inversion’ or ‘retention’ of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. We report the first native ternary complex of a GT, that of the retaining glucosyl-3-phosphoglycerate synthase GpgS from Mycobacterium tuberculosis, in the presence of the sugar donor UDP-Glc, the acceptor substrate phosphoglycerate and the divalent cation cofactor, in a productive mode for catalysis. In combination with structural, chemical, enzymatic, molecular dynamics and quantum-mechanics/molecular-mechanics (QM/MM) calculations, we unravel its catalytic mechanism, providing a strong experimental support for a front-side, substrate assisted SNi-type reaction.This work was supported by the EU Contract HEALTH-F3-2011-260872, MINECO Contract BIO2013-49022-C2-2-R, and the Basque Government (to M.E.G.); MINECO Contracts CTQ2011-24292 and CTQ2014-53144-P (to J.M.LL.) and “UAB - Banco Santander Program” (to L.M.); CTQ2013-44367-C2-1-P (to P.M.) and MINECO Contract BIO2013-49022-C2-1-R (to A.P). F.G.-B. and F.M. acknowledge support from the JAE Predoc Program (CSIC) and “Becas de Doctorado en el Extranjero - Becas Chile - CONICYT” Program, respectively.Peer reviewe

The P. aeruginosa type VI secretion system effector Tse5 forms ion-selective membrane pores that disrupt the membrane potential of intoxicated cells

Póster presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.Pseudomonas aeruginosa is an opportunistic pathogen of great clinical impact, being the main cause of acute and chronic infections in immunosuppressed and cystic fibrosis patients. This virulence is largely due to the secretion systems possessed by the bacteria, among which the Type 6 Secretion System (T6SS) in P. aeruginosa stands out 1. The T6SS is a nanomachine that assembles inside the bacteria and injects severa effectors/toxins into target cells 2. The large number of effectors and their functional diversity play a crucial role in the virulence of the infection caused by this gram-negative bacterium. One of these effectors is the Type VI secretion system exported effector 5 (Tse5) which was described to have bacteriolytic activity although its molecular activity is still unknown 3,4. In this work we studied the molecular function of Tse5 effector. To do so, we performed growth inhibition curves to determine if it has a bacteriolytic or bacteriostatic effect. We hypothesised that the toxic domain of Tse5 (Tse5-CT) exerts its toxic activity on the membrane since its immunity protein (Tsi5) inserts into the inner bacterial membrane to neutralise the toxicity 3. To define if this toxin can increase cell permeability or disrupt membrane potential of the target bacteria, we carried out flow cytometry experiments in Pseudomonas putida. Furthermore, we designed a Tse5-CT deletion mutants with a dual reporter localize at the C-terminus that allowed to identify transmembrane regions. Our data indicate that Tse5-CT expression has a bacteriolytic effect on Pseudomonas putida cells that can be reversed by co-expression of the cognate immunity protein Tsi5. Moreover, Tse5 toxin inserts into the membrane through transmembrane and amphipathic domains and disrupts membrane potential of target cells. The results obtained in this study help to gain insight regarding the function and the mechanisms of action Tse5 effector and eventually could help to develop drugs that mimic its behaviour

The synthesis, structure, reactivity and electrochemical properties of ruthenium complexes featuring cyanoacetylide ligands

The complex [Ru(CCCN)(dppe)Cp*] (1) is readily obtained (ca. 70%) from the sequential reaction of [Ru(CCH2)(dppe)Cp*]PF6 with nBuLi and phenyl cyanate. The complex behaves as a typical transition metal acetylide upon reaction with tetracyanoethene, affording a metallated pentacyanobutadiene. Complex 1 is a useful metalloligand, and its reactions with [W(thf)(CO)5], [RuCl(PPh3)2Cp], [RuCl(dppe)Cp*] or cis-[RuCl2(dppe)2] all afforded products featuring the M–CCCN–M′ motif, for which ground state structures indicate a degree of polarisation. Electrochemical and spectroelectrochemical studies reveal moderate interactions between the metal centres in the 35-electron dications [{Cp*(dppe)Ru}(μ-CCCN){RuL2Cp′}]2+ (RuL2Cp′ = Ru(PPh3)2Cp, Ru(dppe)Cp*)