Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.

The general secretory pathway (GSP) is a two-step process for the secretion of proteins by Gram-negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin-arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.

The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation.

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1-A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Colicin A hybrids: a genetic tool for selection of type II secretion-proficient Pseudomonas strains.

The gram-negative bacterium Pseudomonas aeruginosa secretes the majority of its extracellular proteins by the type II secretion mechanism, a two-step process initiated by translocation of signal peptide-bearing exoproteins across the inner membrane. The periplasmic forms are transferred across the outer membrane by a machinery consisting of 12 xcp gene products. Although the type II secretion machinery is conserved among gram-negative bacteria, interactions between the secreted proteins and the machinery are specific. The lack of a selectable phenotype has hampered the development of genetic strategies for studying type II secretion. We report a novel strategy to identify rare events, such as those that allow heterologous secretion or identification of extragenic suppressors correcting xcp defects. This is based on creating a host-vector system where the non-secretory phenotype is lethal. The original tool we designed is a hybrid protein containing elastase and the pore-forming domain of colicin A.

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system.

Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Two-component systems in Pseudomonas aeruginosa: why so many?

Screening the Pseudomonas aeruginosa genome has led to the identification of the highest number of putative genes encoding two-component regulatory systems of all bacterial genomes sequenced to date (64 and 63 encoding response regulators and histidine kinases, respectively). Sixteen atypical kinases, among them 11 devoid of an Hpt domain, and three independent Hpt modules were retrieved. These data suggest that P. aeruginosa possesses complex control strategies with which to respond to environmental challenges.

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2.

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.

Alteration of the lipopolysaccharide structure affects the functioning of the Xcp secretory system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes a wide range of hydrolytic enzymes into the external medium by the Xcp secretion machinery. To better understand the role played by envelope constituents in the functioning of this type II secretory system, we have studied the influence of lipopolysaccharide (LPS) on the secretion of two extracellular enzymes, the elastase LasB and the lipase LipA. Strains with defective LPS decreased production of LasB and altered the secretion processes of both LasB and LipA without any apparent effect on the composition of the Xcp machinery. The PAO1algC strain, defective in the outer core of LPS, was leaky, as shown by the extracellular release of the periplasmic beta-lactamase. Generation of an xcpR mutation in this mutant led only to a partial accumulation of LasB within the cells, indicating that in strain PAO1algC with a functional xcpR gene, LasB was released in the extracellular medium partly by leakage and partly by secretion. The pool of LasB released into the medium by leakage was not recovered in an active form, while extracellular LasB was active when secreted via the secretory machinery. Further analysis revealed that the presence of a functional Xcp machinery is strictly required for the activation process of LasB. Our results provide evidence that the Xcp system is not fully functional when the LPS structure of P. aeruginosa is altered.

Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells.

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.

Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa.

The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P. aeruginosa secretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.

Molecular organization of the xcp gene cluster in Pseudomonas putida: absence of an xcpX (gspK) homologue.

A DNA fragment containing xcp (gsp) gene homologues, required for extracellular protein secretion by the general secretory pathway (GSP) in various Gram-negative bacteria, was cloned from Pseudomonas putida (Pp) strain WCS358 and sequenced. The results presented here and those previously reported (de Groot, A., Krijger, J.-J., Filloux, A., Tommassen, J., 1996. Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358 Mol. Gen. Genet. 250, 491-504) complete the sequence of the xcp gene cluster of Pp. Unlike that of Pseudomonas aeruginosa (Pa), the xcp gene cluster of Pp contains a gspN homologue. More surprisingly, in contrast to all known gsp gene clusters, the xcpX (gspK) homologue is not found. In addition, genes flanking the xcp cluster of Pp are not related to those flanking the xcp genes of Pa. Overall, the xcp gene products of Pp are as much related to those of Pa as to gsp gene products of enterobacterial species, suggesting that the xcp clusters of Pp and Pa have evolved separately.

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone.

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.

The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family).

The xcp gene products in Pseudomonas aeruginosa are required for the secretion of proteins across the outer membrane. Four of the Xcp proteins, XcpT, U, V and W, present sequence homology to the subunits of type IV pili at their N-termini, and they were therefore designated pseudopilins. In this study, we characterized the xcpX gene product, a bitopic cytoplasmic membrane protein. Remarkably, amino acid sequence comparisons also suggested that the XcpX protein resembles the pilins and pseudopilins at the N-terminus. We show that XcpX could be processed by the prepilin peptidase, PilD/XcpA, and that the highly conserved glycine residue preceding the hydrophobic segment could not be mutated without loss of the XcpX function. We, therefore, classified XcpX (GspK) as the fifth pseudopilin of the system.

Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa.

The virulence of the opportunistic pathogen Pseudomonas aeruginosa is largely dependent upon the extracellular production of a number of secreted proteins with toxic or degradative activities. The synthesis of several exoenzymes is controlled in a cell-density-dependent manner by two interlinked quorum-sensing systems. Their secretion across the outer membrane occurs through the Xcp translocation machinery. The xcp locus located at 40 min on the chromosome consists of two divergently transcribed operons, namely xcpPQ and xcpR to xcpZ. In this study, transcriptional fusions were constructed between the xcpP and xcpR genes and the lacZ reporter. Transcriptional activation of the xcpP and xcpR genes in P. aeruginosa is growth-phase dependent and the lasR-lasI autoinduction system is required for this control. In the heterologous host Escherichia coli, the lasR gene product, together with its cognate autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), activates both the xcpP-lacZ and the xcpR-lacZ gene fusion. The second P. aeruginosa quorum-sensing modulon rhIR-rhII (vsmR-vsmI) is also involved in the control of the xcp genes. Expression of the lacZ fusions is strongly reduced in PANO67, a pleiotropic mutant defective in the production of N-acyl-homoserine lactones responsible for the activation of RhIR. Furthermore, introduction of the lasR mutation in PANO67 results in additional diminution of xcpR transcription, indicating that the two systems can regulate their target genes independently. These data demonstrate that expression of the xcp secretion system depends on a complex regulatory network involving cell-cell signalling which controls production and secretion of virulence-associated factors.

The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase.

The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhIR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhIR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhIR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhIR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhIR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.

A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS.

In Pseudomonas aeruginosa, the production of many virulence factors and secondary metabolites is regulated in concert with cell density through quorum sensing. Two quorum-sensing regulons have been identified in which the LuxR homologues LasR and RhlR are activated by N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) respectively. The lasR and rhlR genes are linked to the luxl homologues lasl and rhll, which are responsible for synthesis of OdDHL and BHL, respectively. As lasRl and rhlRl are both involved in regulating synthesis of exoenzymes such as elastase, we sought to determine the nature of their interrelationship. By using lacZ transcriptional fusions in both homologous (P. aeruginosa) and heterologous (Escherichia coli) genetic backgrounds we provide evidence that (i) lasR is expressed constitutively throughout the growth cycle, (ii) rhlR expression is regulated by LasR/OdDHL, and (iii) that RhlR/BHL regulates rhll. We also show that expression of the stationary-phase sigma factor gene rpoS is abolished in a P. aeruginosa lasR mutant and in the pleiotropic BHL-negative mutant PANO67. Furthermore, our data reveal that kin E. coli, an rpoS-lacZ fusion is regulated directly by RhlR/BHL. Taken together, these results indicate that P. aeruginosa employs a multilayered hierarchical quorum-sensing cascade involving RhlR/BHL and LasR/OdDHL, interlinked via RpoS, to integrate the regulation of virulence determinants and secondary metabolites with adaptation and survival in the stationary phase.

Protein secretion by heterologous bacterial ABC-transporters: the C-terminus secretion signal of the secreted protein confers high recognition specificity.

Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) Is released by its own specific secretion machinery which is an ABC-transporter. Despite sequence similarities between components of ABC-transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC-transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C-terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated alpha-haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the translocator which confers specificity to the secretion process. Secretion was size-dependent. However inclusion of glycine-rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20 kDa larger than the normal maximal size.

Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa.

Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa. Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features. Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane. To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization. We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.

Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the auto-inducer molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase-negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of lasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re-established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum-sensing modulons interactively regulate gene expression in P. aeruginosa.