Targeted killing of Pseudomonas aeruginosa by pyocin G occurs via the hemin transporter hur

Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because multi-drug-resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognizes bacterial cells by binding to Hur, an outer-membrane TonB-dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB-ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane. Inner-membrane translocation of PyoG is dependent on the conserved inner-membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition

Intermembrane crosstalk drives inner membrane protein organization in Escherichia coli

Gram-negative bacteria depend on energised protein complexes that connect the two membranes of the cell envelope. However, β-barrel outer-membrane proteins (OMPs) and α-helical inner-membrane proteins (IMPs) display quite different organisation. OMPs cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Here, using live cell imaging of Escherichia coli, we demonstrate that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs and that such connections impact IMP function. We show that while establishing a translocon for import, the colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilising role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology

Bacterial competition systems share a domain required for inner membrane transport of the bacteriocin Pyocin G from Pseudomonas aeruginosa.

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa-specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics

Structural constraints of pyocin S2 import through the ferripyoverdine receptor FpvAI

TonB-dependent transporters (TBDTs) mediate energized transport of essential nutrients into gram-negative bacteria. TBDTs are increasingly being exploited for the delivery of antibiotics to drug-resistant bacteria. While much is known about ground state complexes of TBDTs, few details have emerged about the transport process itself. In this study, we exploit bacteriocin parasitization of a TBDT to probe the mechanics of transport. Previous work has shown that the N-terminal domain of Pseudomonas aeruginosa–specific bacteriocin pyocin S2 (PyoS2NTD) is imported through the pyoverdine receptor FpvAI. PyoS2NTD transport follows the opening of a proton-motive force-dependent pore through FpvAI and the delivery of its own TonB box that engages TonB. We use molecular models and simulations to formulate a complete translocation pathway for PyoS2NTD that we validate using protein engineering and cytotoxicity measurements. We show that following partial removal of the FpvAI plug domain which occludes the channel, the pyocin's N-terminus enters the channel by electrostatic steering and ratchets to the periplasm. Application of force, mimicking that exerted by TonB, leads to unraveling of PyoS2NTD as it squeezes through the channel. Remarkably, while some parts of PyoS2NTD must unfold, complete unfolding is not required for transport, a result we confirmed by disulfide bond engineering. Moreover, the section of the FpvAI plug that remains embedded in the channel appears to serve as a buttress against which PyoS2NTD is pushed to destabilize the domain. Our study reveals the limits of structural deformation that accompanies import through a TBDT and the role the TBDT itself plays in accommodating transport

Nuclear decommissioning: project management and leadership

Management and leadership for safety relate to managerial competencies necessary to develop, promote and sustain a safety culture and to set goals, lead others and manage knowledge and projects to enhance safety performance. The development of these competencies is needed to enrich and complement the predominant technical background and skills of engineers and/or managers involved in the nuclear sector and particularly in decommissioning and dismantling (D&D) projects. The recent recognition of the importance of managing for safety led the International Atomic Energy Agency (IAEA) to develop formal safety requirements that are now implemented by its member states. This includes the need to develop training and education for beginning- and mid-career managers with nuclear safety responsibilities and, considering the time frame of D&D projects, for future generations of managers. Training and education challenges are acute in all Instrument for Nuclear Safety Cooperation (INSC) and European countries, where managers need to develop knowledge and comprehensive safety-related competencies to run the D&D projects of nuclear facilities in a context where the generational change of managers in the nuclear field is happening fast. In 2016/2017 the IAEA and the European Commission (EC) developed a cooperative framework to jointly address a similar challenge related to operation and regulatory oversight of nuclear installations. The development of these projects was possible by funding from the European Union (EU) through its INSC instrument. The first project, led by the IAEA in 2017, was the development of a pilot school for safety leadership at the University Côte d\u27Azur (UCA), France. Encouraged by this success, the agency has since then developed the syllabus into a 2-week programme, still based on experiential learning, which is offered to IAEA member states who wish to organise sessions for their managers (regulatory bodies or industry). The second project, named ELSE, was operated by UCA and aimed to develop training to help managers acquire leadership for safety capabilities, which are key professional requirements in complex, high-risk and highly regulated sectors such as the nuclear sector. The originality of the ELSE project stemmed from its science-based approach, integrating the most recent findings of management and other social sciences. The dedicated ELSE training programme is composed of a massive online open course (MOOC), a 10 d of face-to-face training and an individually tutored project. Based on the success of these experiences, the EU decided to prolong these actions in the field of nuclear D&D, leading to the start-up, in 2023, of the Decommissioning Management and Leadership for Safety Education (DMaLSE) project. This project has also been entrusted to UCA, in partnership with SKEMA Business School, the Karlsruhe Institute of Technology (KIT) and Jacques Repussard Conseil. DMaLSE has two main objectives, namely to develop a science-based training programme for future D&D project managers and to extend the impact of the project through bachelor-degree-level on-site training for operators involved in D&D projects

Pyocin S5 import into Pseudomonas aeruginosa reveals a generic mode of bacteriocin transport

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria

Exploitation of an iron transporter for bacterial protein antibiotic import

Unlike their descendants, mitochondria and plastids, bacteria do not have dedicated protein import systems. However, paradoxically, import of protein bacteriocins, the mechanisms of which are poorly understood, underpins competition among pathogenic and commensal bacteria alike. Here, using X-ray crystallography, isothermal titration calorimetry, confocal fluorescence microscopy, and in vivo photoactivatable cross-linking of stalled translocation intermediates, we demonstrate how the iron transporter FpvAI in the opportunistic pathogen Pseudomonas aeruginosa is hijacked to translocate the bacteriocin pyocin S2 (pyoS2) across the outer membrane (OM). FpvAI is a TonB-dependent transporter (TBDT) that actively imports the small siderophore ferripyoverdine (Fe-Pvd) by coupling to the proton motive force (PMF) via the inner membrane (IM) protein TonB1. The crystal structure of the N-terminal domain of pyoS2 (pyoS2NTD) bound to FpvAI (Kd = 240 pM) reveals that the pyocin mimics Fe-Pvd, inducing the same conformational changes in the receptor. Mimicry leads to fluorescently labeled pyoS2NTD being imported into FpvAI-expressing P. aeruginosa cells by a process analogous to that used by bona fide TBDT ligands. PyoS2NTD induces unfolding by TonB1 of a force-labile portion of the plug domain that normally occludes the central channel of FpvAI. The pyocin is then dragged through this narrow channel following delivery of its own TonB1-binding epitope to the periplasm. Hence, energized nutrient transporters in bacteria also serve as rudimentary protein import systems, which, in the case of FpvAI, results in a protein antibiotic 60-fold bigger than the transporter’s natural substrate being translocated across the OM

Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (Kd ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years