Methionine-rich domains emerge as facilitators of copper recruitment in detoxification systems.

Copper homeostasis mechanisms are critical for bacterial resistance to copper-induced stress. The Escherichia coli multicopper oxidase copper efflux oxidase (CueO) is part of the copper detoxification system in aerobic conditions. CueO contains a methionine-rich (Met-rich) domain believed to interact with copper, but its exact function and the importance of related copper-binding sites remain unclear. This study investigates these open questions by employing a multimodal and multiscale approach. Through the design of various E. coli CueO (EcCueO) variants with altered copper-coordinating residues and domain deletions, we employ biological, biochemical, and physico-chemical approaches to unravel in vitro CueO catalytic properties and in vivo copper resistance. Strong correlation between the different methods enables evaluation of EcCueO variants' activity as a function of Cu+ availability. Our findings demonstrate the Met-rich domain is not essential for cuprous oxidation, but it facilitates Cu+ recruitment from strongly chelated forms, acting as transient copper binding domain thanks to multiple methionines. They also indicate that the Cu6/7 copper-binding sites previously observed within the Met-rich domain play a negligible role. Meanwhile, Cu5, located at the interface with the Met-rich domain, emerges as the primary and sole substrate-binding active site for cuprous oxidation. The Cu5 coordination sphere strongly affects the enzyme activity and the in vivo copper resistance. This study provides insights into the nuanced role of CueO Met-rich domain, enabling the functions of copper-binding sites and the entire domain itself to be decoupled. This paves the way for a deeper understanding of Met-rich domains in the context of bacterial copper homeostasis.

[Solving the puzzle of persistence in Staphylococcus aureus]. / Résoudre le puzzle de la persistance chez Staphylococcus aureus.

Title: Résoudre le puzzle de la persistance chez Staphylococcus aureus. Abstract: Dans le cadre de l'unité d'enseignement « Rédiger en sciences ¼ proposée par l'université d'Aix-Marseille, les étudiants du master 2 Microbiologie Intégrative et Fondamentale (MIF) ­ en partenariat avec l'Institut de Microbiologie, Bioénergies et Biotechnologie (IM2B) ­ ont été confrontés aux exigences de l'écriture scientifique. Trois thématiques leur ont été proposées : la persistance bactérienne chez Staphylococcus, les approches à l'échelle de la cellule unique en microbiologie et le modèle Dictyostelium pour l'étude de la phagocytose. À partir de trois publications originales, les étudiants ont rédigé une nouvelle soulignant les résultats majeurs et l'impact des articles étudiés. Complété par un entretien avec des chercheurs, l'ensemble offre un éclairage original sur la compréhension du vivant dans le domaine de la microbiologie et de la santé.

[Bacteriophages, new authors of the genome]. / Les bactériophages, nouveaux auteurs du génome.

Title: Les bactériophages, nouveaux auteurs du génome. Abstract: Dans le cadre de l'unité d'enseignement « Rédiger en sciences ¼ proposée par Aix-Marseille université, les étudiants du Master 2 Microbiologie intégrative et fondamentale (MIF) - en partenariat avec l'institut de microbiologie, bioénergies et biotechnologie (IM2B) - ont été confrontés aux exigences de l'écriture scientifique. Trois thématiques leur ont été proposées en relation étroite avec les laboratoires de recherche du tissu local : l'homéostasie des métaux chez les pathogènes, la synthèse de la paroi bactérienne et les systèmes anti-CRISPR des bactériophages. Les étudiants ont ainsi rédigé une nouvelle soulignant les résultats majeurs et l'originalité des articles étudiés. Complété par un entretien avec leurs auteurs, l'ensemble offre un éclairage original sur la compréhension du vivant dans le domaine de la microbiologie et de la santé.

Salmonella Typhimurium uses the Cpx stress response to detect N-chlorotaurine and promote the repair of oxidized proteins.

The cell envelope of gram-negative bacteria constitutes the first protective barrier between a cell and its environment. During host infection, the bacterial envelope is subjected to several stresses, including those induced by reactive oxygen species (ROS) and reactive chlorine species (RCS) produced by immune cells. Among RCS, N-chlorotaurine (N-ChT), which results from the reaction between hypochlorous acid and taurine, is a powerful and less diffusible oxidant. Here, using a genetic approach, we demonstrate that Salmonella Typhimurium uses the CpxRA two-component system to detect N-ChT oxidative stress. Moreover, we show that periplasmic methionine sulfoxide reductase (MsrP) is part of the Cpx regulon. Our findings demonstrate that MsrP is required to cope with N-ChT stress by repairing N-ChT-oxidized proteins in the bacterial envelope. By characterizing the molecular signal that induces Cpx when S. Typhimurium is exposed to N-ChT, we show that N-ChT triggers Cpx in an NlpE-dependent manner. Thus, our work establishes a direct link between N-ChT oxidative stress and the envelope stress response.

Periplasmic oxidized-protein repair during copper stress in E. coli: A focus on the metallochaperone CusF.

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveals that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows conservation of copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins.

[The human gut microbiota to find new antibiotics]. / Du microbiote humain à l'antibiotique de demain.

Title: Du microbiote humain à l'antibiotique de demain. Abstract: Dans le cadre de l'unité d'enseignement « Rédiger en sciences ¼ proposée par Aix-Marseille Université, les étudiants du Master 2 Microbiologie Intégrative et Fondamentale (MIF) - en partenariat avec l'Institut de Microbiologie, Bioénergies et Biotechnologie (IM2B) - ont été confrontés aux exigences de l'écriture scientifique. Trois thématiques leur ont été proposées en relation étroite avec les laboratoires de recherche du tissu local : les bactériophages, la biologie des mycobactéries et les bactériocines. Les étudiants ont ainsi rédigé une nouvelle soulignant les résultats majeurs et l'originalité des articles étudiés. Complétée par un entretien avec leurs auteurs, l'ensemble offre un éclairage original sur la compréhension du vivant dans le domaine de la microbiologie et de la santé.

HprSR Is a Reactive Chlorine Species-Sensing, Two-Component System in Escherichia coli.

Two-component systems (TCS) are signaling pathways that allow bacterial cells to sense, respond to, and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, HprSR has recently been shown to be involved in the regulation of msrPQ, which encodes the periplasmic methionine sulfoxide reductase system. In this study, we demonstrated that hypochlorous acid (HOCl) induces the expression of msrPQ in an HprSR-dependent manner, whereas H2O2, NO, and paraquat (a superoxide generator) do not. Therefore, HprS appears to be an HOCl-sensing histidine kinase. Using a directed mutagenesis approach, we showed that Met residues located in the periplasmic loop of HprS are important for its activity: we provide evidence that as HOCl preferentially oxidizes Met residues, HprS could be activated via the reversible oxidation of its methionine residues, meaning that MsrPQ plays a role in switching HprSR off. We propose that the activation of HprS by HOCl could occur through a Met redox switch. HprSR appears to be the first characterized TCS able to detect reactive chlorine species (RCS) in E. coli. This study represents an important step toward understanding the mechanisms of RCS resistance in prokaryotes. IMPORTANCE Understanding how bacteria respond to oxidative stress at the molecular level is crucial in the fight against pathogens. HOCl is one of the most potent industrial and physiological microbicidal oxidants. Therefore, bacteria have developed counterstrategies to survive HOCl-induced stress. Over the last decade, important insights into these bacterial protection factors have been obtained. Our work establishes HprSR as a reactive chlorine species-sensing, two-component system in Escherichia coli MG1655, which regulates the expression of msrPQ, two genes encoding, a repair system for HOCl-oxidized proteins. Moreover, we provide evidence suggesting that HOCl could activate HprS through a methionine redox switch.

Methionine Redox Homeostasis in Protein Quality Control.

Bacteria live in different environments and are subject to a wide variety of fluctuating conditions. During evolution, they acquired sophisticated systems dedicated to maintaining protein structure and function, especially during oxidative stress. Under such conditions, methionine residues are converted into methionine sulfoxide (Met-O) which can alter protein function. In this review, we focus on the role in protein quality control of methionine sulfoxide reductases (Msr) which repair oxidatively protein-bound Met-O. We discuss our current understanding of the importance of Msr systems in rescuing protein function under oxidative stress and their ability to work in coordination with chaperone networks. Moreover, we highlight that bacterial chaperones, like GroEL or SurA, are also targeted by oxidative stress and under the surveillance of Msr. Therefore, integration of methionine redox homeostasis in protein quality control during oxidative stress gives a complete picture of this bacterial adaptive mechanism.

The Esterase PfeE, the Achilles' Heel in the Battle for Iron between Pseudomonas aeruginosa and Escherichia coli.

Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membrane of P. aeruginosa by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-cultures under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles' heel of P. aeruginosa in communities with bacteria producing ENT.

Bacterial Genetic Approach to the Study of Reactive Oxygen Species Production in Galleria mellonella During Salmonella Infection.

Over the last decade, an increasing number of reports presented Galleria mellonella larvae as an important model to study host-pathogen interactions. Coherently, increasing information became available about molecular mechanisms used by this host to cope with microbial infections but few of them dealt with oxidative stress. In this work, we addressed the role of reactive oxygen species (ROS) produced by the immune system of G. mellonella to resist against Salmonella enterica, an intracellular pathogen responsible for a wide range of infections. We confirmed that Salmonella was pathogen for G. mellonella and showed that it had to reach a minimal bacterial load within the hemolymph to kill the larvae. ROS production by G. mellonella was revealed by the virulence defects of Salmonella mutants lacking catalases/peroxiredoxins or cytoplasmic superoxide dismutases, both strains being highly sensitive to these oxidants. Finally, we used bacterial transcriptional fusions to demonstrate that hydrogen peroxide (H2O2) was produced in the hemolymph of Galleria during infection and sensed by S. enterica. In line with this observation, the H2O2-dependent regulator OxyR was found to be required for bacterial virulence in the larvae. These results led us to conclude that ROS production is an important mechanism used by G. mellonella to counteract bacterial infections and validate this host as a relevant model to study host-pathogen interactions.

[Adjuvants: A second wind for antibiotics]. / Adjuvants : un second souffle pour les antibiotiques.

TITLE: Adjuvants : un second souffle pour les antibiotiques. ABSTRACT: Dans le cadre de l'unité d'enseignement « Rédiger en sciences ¼ proposée par Aix-Marseille Université, les étudiants du master 2 microbiologie - en partenariat avec l'Institut de Microbiologie, bioénergies et biotechnologie - ont été confrontés aux exigences de l'écriture scientifique. Trois thématiques leur ont été proposées : la résistance aux antibiotiques, les chaperons moléculaires et la polymérase des coronavirus. Les étudiants ont rédigé une Nouvelle soulignant les résultats majeurs et l'originalité des articles étudiés. Complété par un entretien avec leurs auteurs, l'ensemble offre un éclairage original sur la compréhension du vivant dans le domaine de la microbiologie et de la santé.

Characterisation of the periplasmic methionine sulfoxide reductase (MsrP) from Salmonella Typhimurium.

The oxidation of free methionine (Met) and Met residues inside proteins leads to the formation of methionine sulfoxide (Met-O). The reduction of Met-O to Met is catalysed by a ubiquitous enzyme family: the methionine sulfoxide reductases (Msr). The importance of Msr systems in bacterial physiology and virulence has been reported in many species. Salmonella Typhimurium, a facultative intracellular pathogen, contains four cytoplasmic Msr. Recently, a periplasmic Msr enzyme (MsrP) has been identified in Escherichia coli. In the present study, the STM14_4072 gene from Salmonella was shown to encode the MsrP protein (StMsrP). We describe the experimental procedure and precautions for the production of this molybdo-enzyme. StMsrP was also demonstrated to reduce free Met-O and to catalyse the complete repair of an oxidized protein. More importantly, this study provides for the first time access to the exhaustive list of the Msr systems of a pathogen, including four cytoplasmic enzymes (MsrA, MsrB, MsrC, BisC) and one periplasmic enzyme (MsrP).

[Bacteria, model organisms to investigate the repair mechanisms of oxidized proteins]. / Les bactéries, organismes de choix pour comprendre les mécanismes de réparation des protéines oxydées.

TITLE: Les bactéries, organismes de choix pour comprendre les mécanismes de réparation des protéines oxydées. ABSTRACT: Dans le cadre de l'unité d'enseignement « Rédiger en sciences ¼ proposée par l'université d'Aix-Marseille, les étudiants du Master 2 de microbiologie se sont confrontés aux exigences de l'écriture scientifique. Quatre thématiques leur ont été proposées : les virus géants, les systèmes de sécrétion, la motilité bactérienne et la réparation des protéines oxydées. Après un travail préparatoire effectué avec l'équipe pédagogique et les auteurs des publications originales, les étudiants, organisés en groupes de trois ou quatre, ont rédigé une Nouvelle soulignant les résultats majeurs et l'originalité des quatre articles étudiés. Complété par un entretien avec les chercheurs auteurs de ces articles, l'ensemble offre un éclairage original sur la compréhension du vivant dans le domaine de la microbiologie.

[The greater wax moth, Galleria mellonella to study host-pathogen interactions]. / La teigne Galleria mellonella pour les études hôte-pathogène.

The massive use of antibiotics in health and agriculture has led to the emergence of pathogenic microorganisms resistant to frequently used treatments. In 2017, the World Health Organization (WHO) published its first ever list of antibiotic-resistant "priority pathogens", a catalogue of twelve families of bacteria that pose the greatest threat to human health. In this context, a new model for the study of host-pathogen interactions is becoming increasingly popular : the greater wax moth, Galleria mellonella. This butterfly larvae, sometimes considered as a new "laboratory rat", has many practical advantages and is an important host in the study of some steps in the pathogenicity of infectious agents and the identification of new treatments. This review presents this alternative model and describes its possible applications.

Author Correction: The MFS efflux pump EmrKY contributes to the survival of Shigella within macrophages.

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