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)

Formoterol reduces muscle wasting in mice undergoing doxorubicin chemotherapy

BackgroundEven though doxorubicin (DOX) chemotherapy promotes intense muscle wasting, this drug is still widely used in clinical practice due to its remarkable efficiency in managing cancer. On the other hand, intense muscle loss during the oncological treatment is considered a bad prognosis for the disease’s evolution and the patient’s quality of life. In this sense, strategies that can counteract the muscle wasting induced by DOX are essential. In this study, we evaluated the effectiveness of formoterol (FOR), a β2-adrenoceptor agonist, in managing muscle wasting caused by DOX.Methods and resultsTo evaluate the effect of FOR on DOX-induced muscle wasting, mice were treated with DOX (2.5 mg/kg b.w., i.p. administration, twice a week), associated or not to FOR treatment (1 mg/kg b.w., s.c. administration, daily). Control mice received vehicle solution. A combination of FOR treatment with DOX protected against the loss of body weight (p<0.05), muscle mass (p<0.001), and grip force (p<0.001) promoted by chemotherapy. FOR also attenuated muscle wasting (p<0.01) in tumor-bearing mice on chemotherapy. The potential mechanism by which FOR prevented further DOX-induced muscle wasting occurred by regulating Akt/FoxO3a signaling and gene expression of atrogenes in skeletal muscle.ConclusionsCollectively, our results suggest that FOR can be used as a pharmacological strategy for managing muscle wasting induced by DOX. This study provides new insights into the potential therapeutic use of FOR to improve the overall wellbeing of cancer patients undergoing DOX chemotherapy

Jardins per a la salut

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia. Assignatura: Botànica farmacèutica. Curs: 2014-2015. Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són el recull de les fitxes botàniques de 128 espècies presents en el Jardí Ferran Soldevila de l’Edifici Històric de la UB. Els treballs han estat realitzats manera individual per part dels estudiants dels grups M-3 i T-1 de l’assignatura Botànica Farmacèutica durant els mesos de febrer a maig del curs 2014-15 com a resultat final del Projecte d’Innovació Docent «Jardins per a la salut: aprenentatge servei a Botànica farmacèutica» (codi 2014PID-UB/054). Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pels professors de l’assignatura. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica. També s’ha pretès motivar els estudiants a través del retorn de part del seu esforç a la societat a través d’una experiència d’Aprenentatge-Servei, deixant disponible finalment el treball dels estudiants per a poder ser consultable a través d’una Web pública amb la possibilitat de poder-ho fer in-situ en el propi jardí mitjançant codis QR amb un smartphone

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

Effects of S‐pindolol in mouse pancreatic and lung cancer cachexia models

Abstract Background It is known that S‐pindolol attenuates muscle loss in animal models of cancer cachexia and sarcopenia. In cancer cachexia, it also significantly reduced mortality and improved cardiac function, which is strongly compromised in cachectic animals. Methods Here, we tested 3 mg/kg/day of S‐pindolol in two murine cancer cachexia models: pancreatic cancer cachexia (KPC) and Lewis lung carcinoma (LLC). Results Treatment of mice with 3 mg/kg/day of S‐pindolol in KPC or LLC cancer cachexia models significantly attenuated the loss of body weight, including lean mass and muscle weights, leading to improved grip strength compared with placebo‐treated mice. In the KPC model, treated mice lost less than half of the total weight lost by placebo (−0.9 ± 1.0 vs. −2.2 ± 1.4 g for S‐pindolol and placebo, respectively, P < 0.05) and around a third of the lean mass lost by tumour‐bearing controls (−0.4 ± 1.0 vs. −1.5 ± 1.5 g for S‐pindolol and placebo, respectively, P < 0.05), whereas loss of fat mass was similar. In the LLC model, the gastrocnemius weight was higher in sham (108 ± 16 mg) and S‐pindolol tumour‐bearing (94 ± 15 mg) mice than that in placebo (83 ± 12 mg), whereas the soleus weight was only significantly higher in the S‐pindolol‐treated group (7.9 ± 1.7 mg) than that in placebo (6.5 ± 0.9). Grip strength was significantly improved by S‐pindolol treatment (110.8 ± 16.2 vs. 93.9 ± 17.1 g for S‐pindolol and placebo, respectively). A higher grip strength was observed in all groups; whereas S‐pindolol‐treated mice improved by 32.7 ± 18.5 g, tumour‐bearing mice only show minimal improvements (7.3 ± 19.4 g, P < 0.01). Conclusions S‐pindolol is an important candidate for clinical development in the treatment of cancer cachexia that strongly attenuates loss of body weight and lean body mass. This was also seen in the weight of individual muscles and resulted in higher grip strength

Structural and functional insights into the delivery of a bacterial Rhs pore-forming toxin to the membrane

Abstract Bacterial competition is a significant driver of toxin polymorphism, which allows continual compensatory evolution between toxins and the resistance developed to overcome their activity. Bacterial Rearrangement hot spot (Rhs) proteins represent a widespread example of toxin polymorphism. Here, we present the 2.45 Å cryo-electron microscopy structure of Tse5, an Rhs protein central to Pseudomonas aeruginosa type VI secretion system-mediated bacterial competition. This structural insight, coupled with an extensive array of biophysical and genetic investigations, unravels the multifaceted functional mechanisms of Tse5. The data suggest that interfacial Tse5-membrane binding delivers its encapsulated pore-forming toxin fragment to the target bacterial membrane, where it assembles pores that cause cell depolarisation and, ultimately, bacterial death

The atypical β‐blocker S‐oxprenolol reduces cachexia and improves survival in a rat cancer cachexia model

Background: Beta‐blockers and selected stereoisomers of beta‐blockers, like bisoprolol and S‐pindolol (ACM‐001), have been shown to be effective in preclinical cancer cachexia models. Here, we tested the efficacy of stereoisomers of oxprenolol in two preclinical models of cancer cachexia—the Yoshida AH‐130 rat model and the Lewis lung carcinoma (LLC) mouse model. Methods and Results: In the Yoshida AH130 hepatoma rat cancer cachexia model and compared with placebo, 50 mg/kg/d S‐oxprenolol (HR: 0.49, 95% CI: 0.28–0.85, P = 0.012) was superior to 50 mg/kg/d R‐oxprenolol (HR: 0.83, 95% CI 0.38–1.45, P = 0.51) in reducing mortality (= reaching ethical endpoints). Combination of the three doses (12.5, 25 and 50 mg/kg/d) that had a significant effect on body weight loss in the S‐oxprenolol groups vs the same combination of the R‐oxprenolol groups lead to a significantly improved survival of S‐oxprenolol vs R‐oxprenolol (HR: 1.61, 95% CI: 1.08–2.39, P = 0.0185). Interestingly, there is a clear dose dependency in S‐oxprenolol‐treated (5, 12.5, 25 and 50 mg/kg/d) groups, which was not observed in groups treated with R‐oxprenolol. A dose‐dependent attenuation of weight and lean mass loss by S‐oxprenolol was seen in the Yoshida rat model, whereas R‐oxprenolol had only had a significant effect on fat mass. S‐oxprenolol also non‐significantly reduced weight loss in the LLC model and also improved muscle function (grip strength 428 ± 25 and 539 ± 37 g/100 g body weight for placebo and S‐oxprenolol, respectively). However, there was only a minor effect on quality of life indicators food intake and spontaneous activity in the Yoshida model (25 mg/kg/S‐oxprenolol: 11.9 ± 2.5 g vs placebo: 4.9 ± 0.8 g, P = 0.013 and also vs 25 mg/kg/d R‐oxprenolol: 7.5 ± 2.6 g, P = 0.025). Both enantiomers had no effects on cardiac dimensions and function at the doses used in this study. Western blotting of proteins involved in the anabolic/catabolic homoeostasis suggest that anabolic signalling is persevered (IGF‐1 receptor, Akt) and catabolic signalling is inhibited (FXBO‐10, TRAF‐6) by S‐pindolol, but not he R‐enantiomer. Expression of glucose transporters Glut1 and Glut 4 was similar in all groups, as was AMPK. Conclusions: S‐oxprenolol is superior to R‐oxprenolol in cancer cachexia animal models and shows promise for a human application in cancer cachexia