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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 Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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.

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.

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., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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., (© 2011 Blackwell Publishing Ltd.)

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., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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.

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.

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.

Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase-type plasminogen activator receptor (uPAR) participates in pericellular proteolysis and the adherence/migration of epithelial cells and leukocytes recruited during infection and shows functional regulation by various proteinases via limited endoproteolysis occurring within its three domains (D1 to D3). We thus examined the proteolytic activity of LasB on uPAR by using recombinant uPAR as well as uPAR-expressing, human monocytic, and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2 and at the carboxy terminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on amino-terminal protein microsequencing and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala(84)-Val(85) and Thr(86)-Tyr(87) (D1-D2) and Gln(279)-Tyr(280) (D3). Such a dual cleavage of uPAR led to the removal of amino-terminal D1, the generation of a truncated D2D3 species, and the shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for the alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa.

Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.

The type III secretion system (TTSS) of Pseudomonas aeruginosa was characterized genetically and phenotypically in 92 epidemiologically unrelated bacteremic strains. Four groups of strains (TTSS types) were defined according to the level of type III protein secretion and kinetics of cytotoxicity. Type 1 strains (n=26) were highly and rapidly cytotoxic and secreted ExoU, type 2 strains (n=48) exhibited slower cytotoxic rates and expressed ExoS but not ExoU, type 3 strains (n=14) were poorly cytotoxic, and type 4 strains (n=4) were not cytotoxic. Type 3 and 4 strains did not have detectable secretion phenotype; however, some type 4 strains were able to reach a level of cytotoxicity similar to that of type 1 and type 2 strains when complemented in trans by a functional exsA gene. A statistically significant association (P<.001) was found between TTSS types and detection of the mutually exclusive exoU and exoS genes. In addition, 24 of 25 serotype O:1, O:10, and O:11 strains contained exoU, whereas 54 of 55 serotype O:3, O:4, O:6, O:12, and O:16 strains contained exoS (P<.001). Our results demonstrate correlations among exoU or exoS genotype, TTSS phenotype, and O serotype in bacteremic P. aeruginosa isolates.

This study was designed to investigate the molecular mechanisms of Staphylococcus aureus adherence to human airway epithelium. Using a humanized bronchial xenograft model in the nude mouse and primary cultures of human airway epithelial cells (HAEC), we showed that S. aureus adhered mainly to undifferentiated HAEC whereas weak adherence (11- to 20-fold lower) to differentiated HAEC was observed (P < 0.01). A fibronectin (FN)-binding protein (FnBP)-deficient strain of S. aureus had a fivefold-lower adherence level to undifferentiated HAEC than did the parental strain (P < 0.005), suggesting that S. aureus FN-binding capacity is involved in the adherence to HAEC. We also showed that 97% of 32 S. aureus clinical strains, isolated from the airway secretions of cystic fibrosis patients (n = 18) and patients with nosocomial pneumonia (n = 14), possessed the two fnb genes. The strains from pneumonia patients had a significantly (P < 0.05) higher FN-binding capacity than did the strains from CF patients. This result was confirmed by the expression of FnBPs, investigated by Western ligand affinity blotting. Our results suggest a major role of FnBPs in the colonization of the airways by S. aureus and point to the importance of the adhesin regulatory pathways in the staphylococcal infectious process.

Pseudomonas aeruginosa is the most important bacterial pathogen associated with chronic airway infection, especially in cystic fibrosis. We addressed the question of whether the galactophilic internal lectin of P. aeruginosa (PA-I) could represent a virulence factor for the respiratory epithelium. PA-I lectin was localized in all the bacteria of P. aeruginosa ATCC 33347 as determined by immunofluorescence staining. We investigated the dose-dependent effect of P. aeruginosa PA-I lectin on the growth, the ciliary beating frequency, and the morphology of human respiratory cells in primary culture of nasal polyps collected from non-cystic fibrosis patients. PA-I lectin significantly (P < 0.01) inhibited the growth of respiratory cells at a concentration of > or = 10 micrograms/ml. The percentage of active ciliated cell surface of the cultures decreased significantly (P < 0.05) at a PA-I lectin concentration of 50 micrograms/ml. Exposed to a low concentration of PA-I lectin (10 micrograms/ml), respiratory epithelial cells showed intracytoplasmic vacuoles when examined by light and transmission electron microscopy. At a higher concentration of PA-I lectin (100 micrograms/ml), major cell damage and severe epithelial shedding occurred. These results demonstrate that the P. aeruginosa internal PA-I lectin has a dose-dependent cytotoxic effect on respiratory epithelial cells in vitro. The P. aeruginosa PA-I lectin may represent a virulence factor by contributing to the respiratory epithelial damage during P. aeruginosa respiratory infections.