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Bacteria are often exposed to nitrosative stress from their environment, from atmospheric pollution or from the defense mechanisms of other organisms. Reactive nitrogen species (RNS), which mediate nitrosative stress, are notably involved in the mammalian immune response through the production of nitric oxide (NO) by the inducible NO synthase iNOS. RNS are highly reactive and can alter various biomolecules such as lipids, proteins and DNA, making them toxic for biological organisms. Resistance to RNS is therefore important for the survival of bacteria in various environments, and notably to successfully infect their host. The fuel combustion processes used in industries and transports are responsible for the emission of important quantities of two major RNS, NO and the more toxic nitrogen dioxide (NO

Anthropogenic atmospheric pollution and immune response regularly expose bacteria to toxic nitrogen oxides such as NO

Legionella pneumophila, the causative agent of Legionnaires’ disease, is mostly found in man-made water systems and is one of the most closely monitored waterborne pathogens. With the aim of finding natural ways to control waterborne pathogens and thus further reduce the impact of disinfection by-products on human health, some studies have demonstrated the ability of bacteria to kill Legionella through the production of secondary metabolites or antimicrobial compounds. Here, we describe an unexpected growth inhibition of L. pneumophila when exposed to a physically separated strain of Pseudomonas fluorescens, designated as MFE01. Most of the members of the Legionellaceae family are sensitive to the volatile substances emitted by MFE01, unlike other bacteria tested. Using headspace solid-phase microextraction GC-MS strategy, a volatilome comparison revealed that emission of 1-undecene, 2-undecanone, and 2-tridecanone were mainly reduced in a Tn5-transposon mutant unable to inhibit at distance the growth of L. pneumophila strain Lens. We showed that 1-undecene was mainly responsible for the inhibition at distance in vitro, and led to cell lysis in small amounts, as determined by gas chromatography-mass spectrometry (GC-MS). Collectively, our results provide new insights into the mode of action of bacterial volatiles and highlight them as potent anti-Legionella agents to focus research on novel strategies to fight legionellosis. IMPORTANCE Microbial volatile compounds are molecules whose activities are increasingly attracting the attention of researchers. Indeed, they can act as key compounds in long-distance intrakingdom and interkingdom communication, but also as antimicrobials in competition and predation. In fact, most studies to date have focused on their antifungal activities and only a few have reported on their antibacterial properties. Here, we describe that 1-undecene, naturally produced by P. fluorescens, is a volatile with potent activity against bacteria of the genus Legionella. In small amounts, it is capable of inducing cell lysis even when the producing strain is physically separated from the target. This is the first time that such activity is described. This molecule could therefore constitute an efficient compound to counter bacterial pathogens whose treatment may fail, particularly in pulmonary diseases. Indeed, inhalation of these volatiles should be considered as a possible route of therapy in addition to antibiotic treatment.

In Pseudomonas aeruginosa, SigX is an extra-cytoplasmic function σ factor that belongs to the cell wall stress response network. In previous studies, we made the puzzling observation that sigX mutant growth was severely affected in rich lysogeny broth (LB) but not in minimal medium. Here, through comparative transcriptomic and proteomic analysis, we show that the absence of SigX results in dysregulation of genes, whose products are mainly involved in transport, carbon and energy metabolisms. Production of most of these genes is controlled by carbon catabolite repression (CCR), a key regulatory system than ensures preferential carbon source uptake and utilization, substrate prioritization and metabolism. The strong CCR response elicited in LB was lowered in a sigX mutant, suggesting altered nutrient uptake. Since the absence of SigX affects membrane composition and fluidity, we suspected membrane changes to cause such phenotype. The detergent polysorbate 80 (PS80) can moderately destabilize the envelope resulting in non-specific increased nutrient intake. Remarkably, growth, membrane fluidity and expression of dysregulated genes in the sigX mutant strain were restored in LB supplemented with PS80. Altogether, these data suggest that SigX is indirectly involved in CCR regulation, possibly via its effects on membrane integrity and fluidity.

The striking feature of the ubiquitous protein EfTu (Thermo unstable ribosomal Elongation factor) is its moonlighting (multifunctional) activity. Beyond its function at the ribosomal level it should be exported to the bacterial surface and act as an environmental sensor. In Bacillus cereus, and other cutaneous bacteria, it serves as a Substance P (SP) receptor and is essential for bacterial adaptation to the host. However, the modus operandi of EfTu as a bacterial sensor remains to be investigated. Studies realized by confocal and transmission electron microscopy revealed that, in the absence of an exogenous signal, EfTu is not exposed on the bacterial surface but is recruited under the effect of SP. In addition, SP acts as a transcriptional regulator of the tuf gene encoding for EfTu. As observed using gadolinium chloride, an inhibitor of membrane mechanosensitive channels (Msc), Msc control EfTu export and subsequently the bacterial response to SP both in terms of cytotoxicity and biofilm formation activity. Microscale thermophoresis revealed that in response to SP, EfTu can form homopolymers. This event should occur after EfTu export and, as shown by proteo-liposome reconstruction studies, SP appears to promote EfTu polymers association to the membrane, leading subsequently to the bacterial response. Molecular modeling suggests that this mechanism should involve EfTu unfolding and insertion into the bacterial cytoplasmic membrane, presumably through formation of homopolymers. This study is unraveling the original mechanism action of EfTu as a bacterial sensor but also reveals that this protein should have a broader role, including in eukaryotes.

International audience; Les canaux ioniques mécanosensibles sont des protéines ubiquitaires dont l’ouverture sous l’effet de contraintes membranaires permet le passage d’ions ou de molécules selon la sélectivité de celui-ci. Chez Escherichia coli, le canal de faible conductance MscS est l’un des plus étudiés. Il est impliqué dans la régulation de la pression de turgescence lors d’un abaissement brusque de l’osmolarité du milieu et son gène est régulé par RpoS, le facteur sigma de phase stationnaire. Chez Pseudomonas aeruginosa, CmpX, qui possède 52% de similarité de séquence avec MscS, est une protéine de fonction inconnue. Nous avons montré que son gène fait partie avec cfrX d’une unité transcriptionnelle localisée en amont de sigX et d’oprF. SigX est un facteur sigma à fonction extracytoplasmique (ECF) de réponse au stress d’enveloppe, qui contrôle de manière directe l’expression de cfrX-cmpX, et dont l’activité est régulée négativement par CfrX à partir de la fin de phase exponentielle. OprF, la protéine majoritaire de la membrane externe est impliquée dans l’intégrité de l’enveloppe et la perception de l’environnement. Dans le but de préciser rôle de CmpX, un mutant de délétion a été construit chez la souche H103 et caractérisé aux niveaux moléculaire (qRT-PCR) et phénotypique pour (i) sa résistance à des chocs thermiques, (ii) sa fluidité (polarisation généralisée et de fluorescence) et sa composition membranaire (FAME), (iii) sa production de facteurs de virulence. Nous montrons que l’absence de CmpX provoque en phase stationnaire une diminution de la fluidité des régions profondes du core hydrophobe des membranes, l’activation de la réponse au stress d’enveloppe et une augmentation de l’expression de relA et spoT dont les produits sont impliqués dans le métabolisme du ppGpp. Ces altérations membranaires sont associées à une diminution de la production de rhamnolipides, un facteur de virulence sous le contrôle du système de communication Rhl. Ces travaux suggèrent que CmpX joue un rôle primordial dans l’homéostasie de l’enveloppe et la pathogénicité chez P. aeruginosa dans des conditions de stress telles que la phase stationnaire

Legionella pneumophila, the causative agent of Legionnaires' disease, is a waterborne bacterium mainly found in man-made water systems in close association with free-living amoebae and multispecies biofilms. Pseudomonas strains, originating from various environments including freshwater systems or isolated from hospitalized patients, were tested for their antagonistic activity towards L. pneumophila. A high amount of tested strains was thus found to be active. This antibacterial activity was correlated to the presence of tensioactive agents in culture supernatants. As Pseudomonas strains were known to produce biosurfactants, these compounds were specifically extracted and purified from active strains and further characterized using reverse-phase HPLC and mass spectrometry methods. Finally, all biosurfactants tested (lipopeptides and rhamnolipids) were found active and this activity was shown to be higher towards Legionella strains compared to various other bacteria. Therefore, described biosurfactants are potent anti-Legionella agents that could be used in the water treatment industry although tests are needed to evaluate how effective they would be under field conditions.

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International audience; Nowadays air pollution is increasing due to anthropogenic activity. Among all air pollutants, nitrogen oxides (NOx) such as NO are predominant. It is well known that those compounds exhibit direct toxic effects on human health. However, microorganisms are also exposed to them, but the effect of NOx on the virulence of air microbiota is still poorly understood. In this study, we evaluated the impact of NO on the adaptability and virulence of an airborne strain of P. fluorescens, MFA76a, by exposition of this strain to 45 ppm of NO2. The growth kinetics and cultivability were analysed. A decrease of cultivability coupled with an increase of the lag phase was observed suggesting a potential toxicity of NO2. Since NOx particularly target lipids, the membrane permeability was assessed thanks to Live Dead tests and confocal microscopy. A significant alteration of membrane permeability was observed. Furthermore, more abundant bacterial aggregates were detected compared to the control. Thus, a lipidomic study was performed using MALDI-TOF MS Imaging coupled to HPTLC. Interestingly, bacteria exposed to NO were lacking one putative glycerophospholipid molecule. In agreement with a previous study from Kondakova et al., these data demonstrate the adaptation potential of P. fluorescens MFAF76a to an air pollutant such as NO.

International audience; Antimicrobial peptides (AMPs) are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, there are currently 15 distinct AMP families published so far in the literature, mainly isolated from members of the Decapoda order. Up to now, armadillidin is the sole non-decapod AMP isolated from the haemocytes of Armadillidium vulgare, a crustacean isopod. Its first description demonstrated that armadillidin is a linear glycine-rich (47%) cationic peptide with an antimicrobial activity directed toward Bacillus megaterium. In the present work, we report identification of armadillidin Q, a variant of armadillidin H (earlier known as armadillidin), from crude haemocyte extracts of A. vulgare using LC-MS approach. We demonstrated that both armadillidins displayed broad spectrum antimicrobial activity against several Gram-positive and Gram-negative bacteria, fungi, but were totally inactive against yeasts. Membrane permeabilization assays, only performed with armadillidin H, showed that the peptide is membrane active against bacterial and fungal strains leading to deep changes in cell morphology. This damaging activity visualized by electronic microscopy correlates with a rapid decrease of cell viability leading to highly blebbed cells. In contrast, armadillidin H does not reveal cytotoxicity toward human erythrocytes. Furthermore, no secondary structure could be defined in this study [by circular dichroism (CD) and nuclear magnetic resonance (NMR)] even in a membrane mimicking environment. Therefore, armadillidins represent interesting candidates to gain insight into the biology of glycine-rich AMPs.