The horizontal transfer of Pseudomonas aeruginosa PA14 ICE PAPI-1 is controlled by a transcriptional triad between TprA, NdpA2 and MvaT.

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. Genome sequences reveal that most P. aeruginosa strains contain a significant number of accessory genes gathered in genomic islands. Those genes are essential for P. aeruginosa to invade new ecological niches with high levels of antibiotic usage, like hospitals, or to survive during host infection by providing pathogenicity determinants. P. aeruginosa pathogenicity island 1 (PAPI-1), one of the largest genomic islands, encodes several putative virulence factors, including toxins, biofilm genes and antibiotic-resistance traits. The integrative and conjugative element (ICE) PAPI-1 is horizontally transferable by conjugation via a specialized GI-T4SS, but the mechanism regulating this transfer is currently unknown. Here, we show that this GI-T4SS conjugative machinery is directly induced by TprA, a regulator encoded within PAPI-1. Our data indicate that the nucleotide associated protein NdpA2 acts in synergy with TprA, removing a repressive mechanism exerted by MvaT. In addition, using a transcriptomic approach, we unravelled the regulon controlled by Ndpa2/TprA and showed that they act as major regulators on the genes belonging to PAPI-1. Moreover, TprA and NdpA2 trigger an atypical biofilm structure and enhance ICE PAPI-1 transfer.

The Pseudomonas aeruginosa lectin LecA triggers host cell signalling by glycosphingolipid-dependent phosphorylation of the adaptor protein CrkII.

The human pathogen Pseudomonas aeruginosa induces phosphorylation of the adaptor protein CrkII by activating the non-receptor tyrosine kinase Abl to promote its uptake into host cells. So far, specific factors of P. aeruginosa, which induce Abl/CrkII signalling, are entirely unknown. In this research, we employed human lung epithelial cells H1299, Chinese hamster ovary cells and P. aeruginosa wild type strain PAO1 to study the invasion process of P. aeruginosa into host cells by using microbiological, biochemical and cell biological approaches such as Western Blot, immunofluorescence microscopy and flow cytometry. Here, we demonstrate that the host glycosphingolipid globotriaosylceramide, also termed Gb3, represents a signalling receptor for the P. aeruginosa lectin LecA to induce CrkII phosphorylation at tyrosine 221. Alterations in Gb3 expression and LecA function correlate with CrkII phosphorylation. Interestingly, phosphorylation of CrkIIY221 occurs independently of Abl kinase. We further show that Src family kinases transduce the signal induced by LecA binding to Gb3, leading to CrkY221 phosphorylation. In summary, we identified LecA as a bacterial factor, which utilizes a so far unrecognized mechanism for phospho-CrkIIY221 induction by binding to the host glycosphingolipid receptor Gb3. The LecA/Gb3 interaction highlights the potential of glycolipids to mediate signalling processes across the plasma membrane and should be further elucidated to gain deeper insights into this non-canonical mechanism of activating host cell processes.

M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation.

Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation.

The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa.

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.

Functional Characterization of Pseudomonas Contact Dependent Growth Inhibition (CDI) Systems.

Contact-dependent inhibition (CDI) toxins, delivered into the cytoplasm of target bacterial cells, confer to host strain a significant competitive advantage. Upon cell contact, the toxic C-terminal region of surface-exposed CdiA protein (CdiA-CT) inhibits the growth of CDI- bacteria. CDI+ cells express a specific immunity protein, CdiI, which protects from autoinhibition by blocking the activity of cognate CdiA-CT. CdiA-CT are separated from the rest of the protein by conserved peptide motifs falling into two distinct classes, the "E. coli"- and "Burkholderia-type". CDI systems have been described in numerous species except in Pseudomonadaceae. In this study, we identified functional toxin/immunity genes linked to CDI systems in the Pseudomonas genus, which extend beyond the conventional CDI classes by the variability of the peptide motif that delimits the polymorphic CdiA-CT domain. Using P. aeruginosa PAO1 as a model, we identified the translational repressor RsmA as a negative regulator of CDI systems. Our data further suggest that under conditions of expression, P. aeruginosa CDI systems are implicated in adhesion and biofilm formation and provide an advantage in competition assays. All together our data imply that CDI systems could play an important role in niche adaptation of Pseudomonadaceae.

Pseudomonas aeruginosa Genome Evolution in Patients and under the Hospital Environment.

Pseudomonas aeruginosa is a Gram-negative environmental species and an opportunistic microorganism, establishing itself in vulnerable patients, such as those with cystic fibrosis (CF) or those hospitalized in intensive care units (ICU). It has become a major cause of nosocomial infections worldwide and a serious threat to Public Health because of overuse and misuse of antibiotics that have selected highly resistant strains against which very few therapeutic options exist. Herein is illustrated the intraclonal evolution of the genome of sequential isolates collected in a single CF patient from the early phase of pulmonary colonization to the fatal outcome. We also examined at the whole genome scale a pair of genotypically-related strains made of a drug susceptible, environmental isolate recovered from an ICU sink and of its multidrug resistant counterpart found to infect an ICU patient. Multiple genetic changes accumulated in the CF isolates over the disease time course including SNPs, deletion events and reduction of whole genome size. The strain isolated from the ICU patient displayed an increase in the genome size of 4.8% with major genetic rearrangements as compared to the initial environmental strain. The annotated genomes are given in free access in an interactive web application WallGene  designed to facilitate large-scale comparative analysis and thus allowing investigators to explore homologies and syntenies between P. aeruginosa strains, here PAO1 and the five clinical strains described.

Antiadhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalized patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multiresistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells, and destruction of alveolar tissues were performed. The antiadhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

Expeditive synthesis of trithiotriazine-cored glycoclusters and inhibition of Pseudomonas aeruginosa biofilm formation.

Readily accessible, low-valency glycoclusters based on a triazine core bearing D-galactose and L-fucose epitopes are able to inhibit biofilm formation by Pseudomonas aeruginosa. These multivalent ligands are simple to synthesize, are highly soluble, and can be either homofunctional or heterofunctional. The galactose-decorated cluster shows good affinity for Pseudomonas aeruginosa lectin lecA. They are convenient biological probes for investigating the roles of lecA and lecB in biofilm formation.

A lipid zipper triggers bacterial invasion.

Glycosphingolipids are important structural constituents of cellular membranes. They are involved in the formation of nanodomains ("lipid rafts"), which serve as important signaling platforms. Invasive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium--a key process in bacterial uptake. However, it is unknown whether glycosphingolipids directly take part in the membrane invagination process. Here, we demonstrate that a "lipid zipper," which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3, triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aeruginosa. In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was sufficient to achieve complete membrane engulfment of the bacterium. In addition, theoretical modeling elucidated that the adhesion energy of the LecA-Gb3 interaction is adequate to drive the engulfment process. In cellulo experiments demonstrated that inhibition of the LecA/Gb3 lipid zipper by either lecA knockout, Gb3 depletion, or application of soluble sugars that interfere with LecA binding to Gb3 significantly lowered P. aeruginosa uptake by host cells. Of note, membrane engulfment of P. aeruginosa occurred independently of actin polymerization, thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry. Our study sheds new light on the impact of glycosphingolipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes.

Characterization of a novel two-partner secretion system implicated in the virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen implicated in nosocomial infection and infecting people with compromised immune systems such as cystic fibrosis patients. Although multiple genes involved in P. aeruginosa pathogenesis have been characterized, the overall mechanism of virulence is not fully understood. In this study, we identified a functional two-partner secretion (TPS) system, composed of the PdtA exoprotein and its cognate pore-forming ß-barrel PdtB transporter, which is implicated in the virulence of P. aeruginosa. We found that the predicted PdtA exoprotein is related to the HMW-like adhesins subfamily TPS systems. We demonstrate here that limitation of inorganic phosphate (Pi) allows the production of PdtA protein. We show that PdtA is processed during its outer-membrane translocation, with an N-terminal domain released into the extracellular environment and a C-terminal domain associated with the outer membrane of the cell. We also obtained evidence that the transport of PdtA is strictly dependent on the production of PdtB, a result confirming that these proteins constitute a functional TPS system. Furthermore, using the Caenorhabditis elegans model of infection, we show that a pdtA mutant is less virulent than the wild-type strain.

Construction of Pseudomonas aeruginosa two-hybrid libraries for high-throughput assays.

In Pseudomonas aeruginosa, identification of new partners of a protein of interest could give precious clues to decipher a biological process in which this protein is involved. However, genes encoding for partners of a protein of interest are unknown and frequently scattered throughout the genome. We describe herein the construction and the use of pan-genomic bacterial two-hybrid libraries to identify new partners of a protein of interest encoded by P. aeruginosa.

Monitoring lectin interactions with carbohydrates.

Protein-carbohydrate interactions are often involved in the first step of infection and Pseudomonas aeruginosa produces several proteins that are able to bind specifically to glycan epitopes present on host epithelia. The experimental approaches for studying protein-carbohydrate interaction have been inspired, with some adaptations, from those commonly used for protein-protein or protein-ligand interactions. A range of methods are described herein for detecting lectin activity, screening for monosaccharide or oligosaccharide specificity, determining the affinity of binding together with thermodynamics and kinetics parameters, and producing crystal of lectin-carbohydrate complexes for further structural studies.

A gacS deletion in Pseudomonas aeruginosa cystic fibrosis isolate CHA shapes its virulence.

Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3' end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor.

Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage.

Within the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1∆lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1∆lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1∆lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier.

Phosphate starvation relayed by PhoB activates the expression of the Pseudomonas aeruginosa σvreI ECF factor and its target genes.

The cell-surface signalling (CSS) system represents an important regulatory mechanism by which Gram-negative bacteria respond to the environment. Gene regulation by CSS systems is particularly present and important in the opportunistic human pathogen Pseudomonas aeruginosa. In this bacterium, these mechanisms regulate mainly the uptake of iron, but also virulence functions. The latter is the case for the P. aeruginosa PUMA3 CSS system formed by the putative VreA receptor, the σ(VreI) extracytoplasmic function sigma factor and the VreR anti-sigma factor. A role for this system in P. aeruginosa virulence has been demonstrated previously. However, the conditions under which this system is expressed and activated have not been elucidated so far. In this work, we have identified and characterized the global regulatory cascade activating the expression of the PUMA3 system. We show that the PhoB transcriptional regulator, part of the PhoB-PhoR two-component signalling system, can sense a limitation of inorganic phosphate to turn on the expression of the vreA, vreI and vreR genes, which constitute an operon. Upon expression of these genes in this condition, σ(VreI) factor mediates transcription of most, but not all, of the previously identified σ(VreI)-regulated genes. Indeed, we found new σ(VreI)-targeted genes and we show that σ(VreI)-regulon genes are all located immediately downstream to the vreAIR gene cluster.

Identification of reciprocal adhesion genes in pathogenic and non-pathogenic Pseudomonas.

We used a combination of in silico and large-scale mutagenesis approaches to expand our current knowledge of the genetic determinants used by Pseudomonas putida KT2440 to attach to surfaces. We first identified in silico orthologues that have been annotated in Pseudomonas aeruginosa as potentially involved in attachment. In this search 67 paired-related genes of P. putida KT2440 and P. aeruginosa were identified as associated to adhesion. To test the potential role of the corresponding gene products in adhesion, 37 knockout mutants of KT2440, available in the Pseudomonas Reference Culture Collection, were analysed with regard to their ability to form biofilms in polystyrene microtitre plates; of these, six mutants were deficient in adhesion. Since mutants in all potential adhesion genes were not available, we generated a genome-wide collection of mutants made of 7684 independent mini-Tn5 insertions and tested them for the formation of biofilm on polystyrene microtitre plates. Eighteen clones that exhibited a reduction of at least twofold in biofilm biomass formation were considered candidate mutants in adhesion determinants. DNA sequencing of the insertion site identified five other new genes involved in adhesion. Phenotypic characterization of the mutants showed that 11 of the inactivated proteins were required for attachment to biotic surfaces too. This combined approach allowed us to identify new proteins with a role in P. putida adhesion, including the global regulator RpoN and GacS, PstS that corresponds to one of the paired-related genes for which a mutant was not available in the mutant collection, and a protein of unknown function (PP1633). The remaining mutants corresponded to functions known or predicted to participate in adhesion based on previous evidence, such as the large adhesion proteins LapA, LapF and flagellar proteins. In silico analysis showed this set of genes to be well conserved in all sequenced P. putida strains, and that at least eight reciprocal genes involved in attachment are shared by P. putida and P. aeruginosa.

Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB.

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.

Neglected but amazingly diverse type IVb pili.

This review provides an overview of current knowledge concerning type IVb pili in Gram-negative bacteria. The number of these pili identified is steadily increasing with genome sequencing and mining studies, but studies of these pili are somewhat uneven, because their expression is tightly regulated and the signals or regulators controlling expression need to be identified. However, as illustrated here, they have a number of interesting functional, assembly-related and regulatory features.

A bacterial two-hybrid genome fragment library for deciphering regulatory networks of the opportunistic pathogen Pseudomonas aeruginosa.

Bacterial gene regulation is controlled by complex regulatory cascades which integrate input environmental signals and adapt specific and adequate output cellular responses. These complex networks are far from being elucidated, in particular in Pseudomonas aeruginosa. In the present study, we developed bacterial two-hybrid genome fragment libraries of the P. aeruginosa PAO1 strain to identify potential partners involved in the HptB/HsbR/HsbA pathway. This powerful tool, validated by the interaction previously described between HsbR and HsbA proteins, allowed us to demonstrate that the HsbR response regulator dimerizes through its PP2C/ATPase C-terminal effector domain, an observation further confirmed by pull-down experiments. This will also allow us to identify further new partners in this cascade.