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 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.

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.

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.

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.

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.

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.

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.

The 'P-usher', a novel protein transporter involved in fimbrial assembly and TpsA secretion.

We identified a new bacterial transporter, the Pseudomonas aeruginosa CupB3 protein, which is an outer membrane usher involved in pili assembly. In CupB3, the usher domain has fused during evolution with a POTRA (polypeptide-transport-associated)-like domain found in TpsB transporters of two-partner secretion systems. In TpsBs, the POTRA captures the TpsA passenger, which is then transported across the outer membrane through the TpsB beta-barrel. We named CupB3 a 'P-usher' for POTRA-like domain-containing usher. We showed that CupB3 assembles CupB1 fimbrial subunits into pili and secretes CupB5, a TpsA-like protein. The CupB3 usher domain has the function of a TpsB beta-barrel in CupB5 translocation. We revealed that the POTRA-like domain is neither essential for CupB1 fimbriae assembly nor for cell surface exposition of CupB5, but is crucial to coordinate bona fide transport of CupB1 and CupB5 through the usher domain. The P-usher defines a novel transport pathway involving a molecular machine made with old spare parts.

Inside the complex regulation of Pseudomonas aeruginosa chaperone usher systems.

Pseudomonas aeruginosa assembles several cell surface-associated organelles, including those of the chaperone usher (CU) pathway. Five different CU loci have been identified and characterized in various strains of P.aeruginosa. However, their potential functional redundancy, particularly in biofilm formation, is supported by the control of their expression by a complex and specific regulatory network. Here, we review recent findings relating to this network. The control exerted by this network involves transcriptional repressors and activators, a phase-variable mechanism, a second intracellular messenger (c-di-GMP) and chemosensory and two-component systems.

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.

Development of a genetic tool for activating chromosomal expression of cryptic or tightly regulated loci in Pseudomonas aeruginosa.

A method for replacing endogenous promoter by a constitutive promoter in Pseudomonas aeruginosa is described. Plasmid pKNG101, a broadly used shuttle suicide vector in P. aeruginosa, was improved to allow chromosomal introduction of a Plac promoter in front of any kind of gene especially those with unknown function. Using this strategy alleviates the need for cloning difficulties encountered in this bacteria and antibiotic marker selection.

The thermolysin-like metalloproteinase and virulence factor LasB from pathogenic Pseudomonas aeruginosa induces anoikis of human vascular cells.

Disruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the haematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasB-deficient pseudomonal strain PAO1lasBΔ had limited impact on human vascular cell adherence and viability, secretomes from the LasB-expressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.

The Pseudomonas aeruginosa opportunistic pathogen and human infections.

Pseudomonas aeruginosa, a Gram-negative environmental species and an opportunistic microorganism, establishes itself in vulnerable patients, such as those with cystic fibrosis or hospitalized in intensive care units. It has become a major cause of nosocomial infections worldwide (about 10% of all such infections in most European Union hospitals) and a serious threat to Public Health. The overuse and misuse of antibiotics have also led to the selection of resistant strains against which very few therapeutic options exist. How an environmental species can cause human infections remains a key question that still needs elucidation despite the incredibly high progress that has been made in the P. aeruginosa biology over the past decades. The workshop belonging to Current trends in Biomedicine series, which was held under the sponsorship of the Universidad International de Andalucia between the 8th and the 10th November 2010 brought in the most recent advances in the environmental life of P. aeruginosa, the human P. aeruginosa infections, the new animal models to study Pseudomonas infections, the new genetic aspects including metabolomics, genomics and bioinformatics and the community lifestyle named biofilm that accounts for P. aeruginosa persistence in humans. This workshop organized by Soeren Molin (Danemark), Juan-Luis Ramos (Spain) and Sophie de Bentzmann (France) gathered 46 researchers coming from 11 European and American countries in a small format and was hosted in the 'Sede Antonio Machado' in Baeza. It was organized in seven sessions covering animal models for P. aeruginosa pathogenesis, resistance to drugs, regulatory potency including small RNA, two component systems, extracytoplasmic function sigma factors and trancriptional regulators, new therapies emerging from dissection of molecular mechanisms, and evolutionary mechanisms of P. aeruginosa strains in patients.

The PprA-PprB two-component system activates CupE, the first non-archetypal Pseudomonas aeruginosa chaperone-usher pathway system assembling fimbriae.

The opportunistic pathogen Pseudomonas aeruginosa has redundant molecular systems that contribute to its pathogenicity. Those assembling fimbrial structures promote complex organized community lifestyle. We characterized a new 5.8 kb genetic locus, cupE, that includes the conserved usher- and chaperone-encoding genes. This locus, widely conserved in different bacterial species, contains four additional genes encoding non-archetypal fimbrial subunits. We first evidenced that the cupE gene cluster was specifically expressed in biofilm conditions and was responsible for fibre assembly containing at least CupE1 protein, at the bacterial cell surface. These fimbriae not only played a significant role in the early stages (microcolony and macrocolony formation) but also in shaping 3D mushrooms during P. aeruginosa biofilm development. Using wide-genome transposon mutagenesis, we identified the PprAB two-component system (TCS) as a regulator of cupE expression, and further demonstrated the involvement of the PprAB TCS in direct CupE fimbrial assembly activation. Thus, this TCS represents a new regulatory element controlling the transition between planktonic and community lifestyles in P. aeruginosa.

Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.

Activation of human pro-urokinase by unrelated proteases secreted by Pseudomonas aeruginosa.

Pathogenic bacteria, including Pseudomonas aeruginosa, interact with and engage the host plasminogen (Plg) activation system, which encompasses the urokinase (uPA)-type Plg activator, and is involved in extracellular proteolysis, including matrilysis and fibrinolysis. We hypothesized that secreted bacterial proteases might contribute to the activation of this major extracellular proteolytic system, thereby participating in bacterial dissemination. We report that LasB, a thermolysin-like metalloprotease secreted by Ps. aeruginosa, converts the human uPA zymogen into its active form (kcat=4.9 s-1, Km=8.9 microM). Accordingly, whereas the extracellular secretome from the LasB-expressing pseudomonal strain PAO1 efficiently activates pro-uPA, the secretome from the isogenic LasB-deficient strain PDO240 is markedly less potent in pro-uPA activation. Still, both secretomes induce some metalloprotease-independent activation of the human zymogen. The latter involves a serine protease, which we identified via both recombinant protein expression in Escherichia coli and purification from pseudomonal cultures as protease IV (PIV; kcat=0.73 s-1, Km=6.2 microM). In contrast, neither secretomes nor the pure proteases activate Plg. Along with this, LasB converts Plg into mini-Plg and angiostatin, whereas, as reported previously, it processes the uPA receptor, inactivates the plasminogen activator inhibitor 1, and activates pro-matrix metalloproteinase 2. PIV does not target these factors at all. To conclude, LasB and PIV, although belonging to different protease families and displaying quite different substrate specificities, both activate the urokinase-type precursor of the Plg activation cascade. Direct pro-uPA activation, as also reported for other bacterial proteases, might be a frequent phenomenon that contributes to bacterial virulence.

Organization and PprB-dependent control of the Pseudomonas aeruginosa tad Locus, involved in Flp pilus biology.

Bacterial attachment to the substratum involves several cell surface organelles, including various types of pili. The Pseudomonas aeruginosa Tad machine assembles type IVb pili, which are required for adhesion to abiotic surfaces and to eukaryotic cells. Type IVb pili consist of a major subunit, the Flp pilin, processed by the FppA prepilin peptidase. In this study, we investigated the regulatory mechanism of the tad locus. We showed that the flp gene is expressed late in the stationary growth phase in aerobic conditions. We also showed that the tad locus was composed of five independent transcriptional units. We used transcriptional fusions to show that tad gene expression was positively controlled by the PprB response regulator. We subsequently showed that PprB bound to the promoter regions, directly controlling the expression of these genes. We then evaluated the contribution of two genes, tadF and rcpC, to type IVb pilus assembly. The deletion of these two genes had no effect on Flp production, pilus assembly, or Flp-mediated adhesion to abiotic surfaces in our conditions. However, our results suggest that the putative RcpC protein modifies the Flp pilin, thereby promoting Flp-dependent adhesion to eukaryotic cells.

Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands.

Pseudomonas aeruginosa is a frequently encountered pathogen that is involved in acute and chronic lung infections. Lectin-mediated bacterium-cell recognition and adhesion are critical steps in initiating P. aeruginosa pathogenesis. This study was designed to evaluate the contributions of LecA and LecB to the pathogenesis of P. aeruginosa-mediated acute lung injury. Using an in vitro model with A549 cells and an experimental in vivo murine model of acute lung injury, we compared the parental strain to lecA and lecB mutants. The effects of both LecA- and Lec B-specific lectin-inhibiting carbohydrates (alpha-methyl-galactoside and alpha-methyl-fucoside, respectively) were evaluated. In vitro, the parental strain was associated with increased cytotoxicity and adhesion on A549 cells compared to the lecA and lecB mutants. In vivo, the P. aeruginosa-induced increase in alveolar barrier permeability was reduced with both mutants. The bacterial burden and dissemination were decreased for both mutants compared with the parental strain. Coadministration of specific lectin inhibitors markedly reduced lung injury and mortality. Our results demonstrate that there is a relationship between lectins and the pathogenicity of P. aeruginosa. Inhibition of the lectins by specific carbohydrates may provide new therapeutic perspectives.