Cross-regulation and cross-talk of conserved and accessory two-component regulatory systems orchestrate Pseudomonas copper resistance.

Bacteria use diverse strategies and molecular machinery to maintain copper homeostasis and to cope with its toxic effects. Some genetic elements providing copper resistance are acquired by horizontal gene transfer; however, little is known about how they are controlled and integrated into the central regulatory network. Here, we studied two copper-responsive systems in a clinical isolate of Pseudomonas paraeruginosa and deciphered the regulatory and cross-regulation mechanisms. To do so, we combined mutagenesis, transcriptional fusion analyses and copper sensitivity phenotypes. Our results showed that the accessory CusRS two-component system (TCS) responds to copper and activates both its own expression and that of the adjacent nine-gene operon (the pcoA2 operon) to provide resistance to elevated levels of extracellular copper. The same locus was also found to be regulated by two core-genome-encoded TCSs-the copper-responsive CopRS and the zinc-responsive CzcRS. Although the target palindromic sequence-ATTCATnnATGTAAT-is the same for the three response regulators, transcriptional outcomes differ. Thus, depending on the operon/regulator pair, binding can result in different activation levels (from none to high), with the systems demonstrating considerable plasticity. Unexpectedly, although the classical CusRS and the noncanonical CopRS TCSs rely on distinct signaling mechanisms (kinase-based vs. phosphatase-based), we discovered cross-talk in the absence of the cognate sensory kinases. This cross-talk occurred between the proteins of these two otherwise independent systems. The cusRS-pcoA2 locus is part of an Integrative and Conjugative Element and was found in other Pseudomonas strains where its expression could provide copper resistance under appropriate conditions. The results presented here illustrate how acquired genetic elements can become part of endogenous regulatory networks, providing a physiological advantage. They also highlight the potential for broader effects of accessory regulatory proteins through interference with core regulatory proteins.

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

The regulation of bacterial two-partner secretion systems.

Two-partner secretion (TPS) systems, also known as Type Vb secretion systems, allow the translocation of effector proteins across the outer membrane of Gram-negative bacteria. By secreting different classes of effectors, including cytolysins and adhesins, TPS systems play important roles in bacterial pathogenesis and host interactions. Here, we review the current knowledge on TPS systems regulation and highlight specific and common regulatory mechanisms across TPS functional classes. We discuss in detail the specific regulatory networks identified in various bacterial species and emphasize the importance of understanding the context-dependent regulation of TPS systems. Several regulatory cues reflecting host environment during infection, such as temperature and iron availability, are common determinants of expression for TPS systems, even across relatively distant species. These common regulatory pathways often affect TPS systems across subfamilies with different effector functions, representing conserved global infection-related regulatory mechanisms.

Determination of the two-component systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages.

Pseudomonas aeruginosa possesses one of the most complex bacterial regulatory networks, which largely contributes to its success as a pathogen. However, most of its transcription factors (TFs) are still uncharacterized and the potential intra-species variability in regulatory networks has been mostly ignored so far. Here, we used DAP-seq to map the genome-wide binding sites of all 55 DNA-binding two-component systems (TCSs) response regulators (RRs) across the three major P. aeruginosa lineages. The resulting networks encompass about 40% of all genes in each strain and contain numerous new regulatory interactions across most major physiological processes. Strikingly, about half of the detected targets are specific to only one or two strains, revealing a previously unknown large functional diversity of TFs within a single species. Three main mechanisms were found to drive this diversity, including differences in accessory genome content, as exemplified by the strain-specific plasmid in IHMA87 outlier strain which harbors numerous binding sites of conserved chromosomally-encoded RRs. Additionally, most RRs display potential auto-regulation or RR-RR cross-regulation, bringing to light the vast complexity of this network. Overall, we provide the first complete delineation of the TCSs regulatory network in P. aeruginosa that will represent an important resource for future studies on this pathogen.

Species-specific recruitment of transcription factors dictates toxin expression.

Tight and coordinate regulation of virulence determinants is essential for bacterial biology and involves dynamic shaping of transcriptional regulatory networks during evolution. The horizontally transferred two-partner secretion system ExlB-ExlA is instrumental in the virulence of different Pseudomonas species, ranging from soil- and plant-dwelling biocontrol agents to the major human pathogen Pseudomonas aeruginosa. Here, we identify a Cro/CI-like repressor, named ErfA, which together with Vfr, a CRP-like activator, controls exlBA expression in P. aeruginosa. The characterization of ErfA regulon across P. aeruginosa subfamilies revealed a second conserved target, the ergAB operon, with functions unrelated to virulence. To gain insights into this functional dichotomy, we defined the pan-regulon of ErfA in several Pseudomonas species and found ergAB as the sole conserved target of ErfA. The analysis of 446 exlBA promoter sequences from all exlBA+ genomes revealed a wide variety of regulatory sequences, as ErfA- and Vfr-binding sites were found to have evolved specifically in P. aeruginosa and nearly each species carries different regulatory sequences for this operon. We propose that the emergence of different regulatory cis-elements in the promoters of horizontally transferred genes is an example of plasticity of regulatory networks evolving to provide an adapted response in each individual niche.

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa.

Many proteobacteria, such as Escherichia coli, contain two main types of quinones: benzoquinones, represented by ubiquinone (UQ) and naphthoquinones, such as menaquinone (MK), and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been discovered recently in E. coli Its physiological relevance is not yet understood, because MK and DMK are also present in E. coli Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV, and ubiU genes, respectively, are essential for UQ9 biosynthesis and, thus, for denitrification in P. aeruginosa These three genes here are called ubiTPa , ubiVPa , and ubiUPa We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for the pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a target for antibiotics development to manage P. aeruginosa infections.

cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers.

The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [Keq] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS) and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death.

Pathogenic bacteria secrete protein toxins that provoke apoptosis or necrosis of eukaryotic cells. Here, we developed a live-imaging method, based on incorporation of a DNA-intercalating dye into membrane-damaged host cells, to study the kinetics of primary bone marrow-derived macrophages (BMDMs) mortality induced by opportunistic pathogen Pseudomonas aeruginosa expressing either Type III Secretion System (T3SS) toxins or the pore-forming toxin, Exolysin (ExlA). We found that ExlA promotes the activation of Caspase-1 and maturation of interleukin-1ß. BMDMs deficient for Caspase-1 and Caspase-11 were resistant to ExlA-induced death. Furthermore, by using KO BMDMs, we determined that the upstream NLRP3/ASC complex leads to the Caspase-1 activation. We also demonstrated that Pseudomonas putida and Pseudomonas protegens and the Drosophila pathogen Pseudomonas entomophila, which naturally express ExlA-like toxins, are cytotoxic toward macrophages and provoke the same type of pro-inflammatory death as does ExlA+ P. aeruginosa. These results demonstrate that ExlA-like toxins of two-partner secretion systems from diverse Pseudomonas species activate the NLRP3 inflammasome and provoke inflammatory pyroptotic death of macrophages.

Phenotype and toxicity of the recently discovered exlA-positive Pseudomonas aeruginosa strains collected worldwide.

We recently identified a hypervirulent strain of Pseudomonas aeruginosa, differing significantly from the classical strains in that it lacks the type 3 secretion system (T3SS), a major determinant of P. aeruginosa virulence. This new strain secretes a novel toxin, called ExlA, which induces plasma membrane rupture in host cells. For this study, we collected 18 other exlA-positive T3SS-negative strains, analyzed their main virulence factors and tested their toxicity in various models. Phylogenetic analysis revealed two groups. The strains were isolated on five continents from patients with various pathologies or in the environment. Their proteolytic activity and their motion abilities were highly different, as well as their capacity to infect epithelial, endothelial, fibroblastic and immune cells, which correlated directly with ExlA secretion levels. In contrast, their toxicity towards human erythrocytes was limited. Some strains were hypervirulent in a mouse pneumonia model and others on chicory leaves. We conclude that (i) exlA-positive strains can colonize different habitats and may induce various infection types, (ii) the strains secreting significant amounts of ExlA are cytotoxic for most cell types but are poorly hemolytic, (iii) toxicity in planta does not correlate with ExlA secretion.

ExsB is required for correct assembly of the Pseudomonas aeruginosa type III secretion apparatus in the bacterial membrane and full virulence in vivo.

Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.

Structural basis of cytotoxicity mediated by the type III secretion toxin ExoU from Pseudomonas aeruginosa.

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB.

BACKGROUND: Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. RESULTS: The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. CONCLUSIONS: The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA.

An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions.

Genomic erosion and horizontal gene transfer shape functional differences of the ExlA toxin in Pseudomonas spp.

Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA Pch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.

PtrA is a periplasmic protein involved in Cu tolerance in Pseudomonas aeruginosa.

In this work, we demonstrate that PtrA (U. H. Ha et al., Mol. Microbiol. 54:307-320, 2004) is a periplasmic protein, specifically synthesized in the presence of copper, that is involved in the tolerance of Pseudomonas aeruginosa to copper. Our biochemical and genetic analyses challenge its role in transcriptional inhibition of the type III secretion system.

Transcription Inhibitors with XRE DNA-Binding and Cupin Signal-Sensing Domains Drive Metabolic Diversification in Pseudomonas.

Transcription factors (TFs) are instrumental in the bacterial response to new environmental conditions. They can act as direct signal sensors and subsequently induce changes in gene expression leading to physiological adaptation. Here, by combining transcriptome sequencing (RNA-seq) and cistrome determination (DAP-seq), we studied a family of eight TFs in Pseudomonas aeruginosa This family, encompassing TFs with XRE-like DNA-binding and cupin signal-sensing domains, includes the metabolic regulators ErfA, PsdR, and PauR and five so-far-unstudied TFs. The genome-wide delineation of their regulons identified 39 regulatory interactions with genes mostly involved in metabolism. We found that the XRE-cupin TFs are inhibitors of their neighboring genes, forming local, functional units encoding proteins with functions in condition-specific metabolic pathways. Growth phenotypes of isogenic mutants highlighted new roles for PauR and PA0535 in polyamines and arginine metabolism. The phylogenetic analysis of this family of regulators across the bacterial kingdom revealed a wide diversity of such metabolic regulatory modules and identified species with potentially higher metabolic versatility. Numerous genes encoding uncharacterized XRE-cupin TFs were found near metabolism-related genes, illustrating the need of further systematic characterization of transcriptional regulatory networks in order to better understand the mechanisms of bacterial adaptation to new environments.IMPORTANCE Bacteria of the Pseudomonas genus, including the major human pathogen Pseudomonas aeruginosa, are known for their complex regulatory networks and high number of transcription factors, which contribute to their impressive adaptive ability. However, even in the most studied species, most of the regulators are still uncharacterized. With the recent advances in high-throughput sequencing methods, it is now possible to fill this knowledge gap and help the understanding of how bacteria adapt and thrive in new environments. By leveraging these methods, we provide an example of a comprehensive analysis of an entire family of transcription factors and bring new insights into metabolic and regulatory adaptation in the Pseudomonas genus.

ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

Electrochemical detection of redox molecules secreted by Pseudomonas aeruginosa - Part 1: Electrochemical signatures of different strains.

During infections, fast identification of the microorganisms is critical to improve patient treatment and to better manage antibiotics use. Electrochemistry exhibits several advantages for rapid diagnostic: it enables easy, cheap and in situ analysis of redox molecules in most liquids. In this work, several culture supernatants of different Pseudomonas aeruginosa strains (including PAO1 and its isogenic mutants PAO1ΔpqsA, PA14, PAK and CHA) were analyzed by square wave voltammetry on glassy carbon electrode during the bacterial growth. The obtained voltamograms shown complex traces exhibiting numerous redox peaks with potential repartitions and current amplitudes depending on the studied bacterium and/or growth time. Among them, some peaks were clearly associated to the well-known redox toxin Pyocyanin (PYO) and the autoinducer Pseudomonas Quinolone Signal (PQS). Other peaks were observed that are not yet attributed to known secreted species. Each complex electrochemical response (number of peaks, peak potential and amplitude) can be interpreted as a fingerprint or "ID-card" of the studied strain that may be implemented for fast bacteria strain identification.