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The virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen whose adaptability, ubiquitousness, and pathogenicity are closely related. Both cell-associated and extracellular products of P. aeruginosa contribute to its virulence. Surface structures, including pili and the polysaccharide capsule or glycocalyx, appear to mediate the initial attachment of P. aeruginosa to its prospective host, thus permitting colonization. Extracellular enzymes such as alkaline protease, elastase, phospholipase C, and exotoxin A degrade infected tissues and promote bacterial invasion. When dissemination occurs, systemic disease results, often with fatal consequences. Although extracellular enzymes of P. aeruginosa figure prominently in local disease processes, exotoxin A and endotoxin are primarily responsible for systemic disease. The most protective antibodies presently known are directed toward the nontoxic portions of P. aeruginosa lipopolysaccharides that serve no known virulence function per se. However, there is preliminary evidence that the protective activity of these opsonic antibodies may be augmented by toxin-neutralizing antibodies directed toward the lipid A moiety of endotoxin and exotoxin A.

Antibodies, Bacterial↗

The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections. The cell-density-dependent signaling mechanisms known as quorum sensing play a role in several of these infections including corneal, lung and burn wound infections. In addition, the quorum-sensing systems contribute to the ability of P. aeruginosa to form biofilms on medically important devices. The quorum-sensing systems accomplish their effect by controlling the production of different virulence factors and by manipulating the host immune response.

Animals↗

Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO. It was found that cyanide is produced in P. aeruginosa at similar levels irrespective of its complement of CIO, indicating that the CIO is not an obligatory electron sink for cyanide synthesis. However, MICs for cyanide and growth in its presence demonstrated that the CIO provides P. aeruginosa with protection against the effects of exogenous cyanide. Nevertheless, the presence of cyanide did not affect the viability of cio mutant strains compared to the wild-type during prolonged incubation in stationary phase. The detection of the fermentation end products acetate and succinate in stationary-phase culture supernatants suggests that P. aeruginosa, irrespective of its CIO complement, may in part rely upon fermentation for energy generation in stationary phase. Furthermore, the decrease in cyanide levels during incubation in sealed flasks suggested that active breakdown of HCN by the culture was taking place. To investigate the possibility that the CIO may play a role in pathogenicity, wild-type and cio mutant strains were tested in the paralytic killing model of Caenorhabditis elegans, a model in which cyanide is the principal toxic agent leading to nematode death. The CIO mutant had delayed killing kinetics, demonstrating that the CIO is required for full pathogenicity of P. aeruginosa in this animal model.

Acetic Acid↗

Silver sulfadiazine: effect on the ultrastructure of Pseudomonas aeruginosa.

Pseudomonas aeruginosa exposed to silver sulfadiazine (AgSu) were examined in an electron microscope. The treated cells were distorted in shape, and structures (blebs) protruded from the cell surface. These "blebs" appeared to arise from the cell wall. A strain of P. aeruginosa resistant to AgSu did not display these changes. Upon exposure of P. aeruginosa to silver nitrate, none of these changes was seen; rather, such cells are characterized by large, central aggregations of nuclear material. The results are consistent with previous findings which suggested that AgSu acted at the cell surface.

Microscopy, Electron↗

Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses at least two multiple drug efflux systems which are defined by the outer membrane proteins OprM and OprJ. We have found that mutants overexpressing OprM were two- and eightfold more resistant than their wild-type parent to sulfamethoxazole (SMX) and trimethoprim (TMP), respectively. For OprJ-overproducing strains, MICs of TMP increased fourfold but those of SMX were unchanged. Strains overexpressing OprM, but not those overexpressing OprJ, became hypersusceptible to TMP and SMX when oprM was inactivated. The wild-type antibiotic profile could be restored in an oprM mutant by transcomplementation with the cloned oprM gene. These results demonstrate that the mexABoprM multidrug efflux system is mainly responsible for the intrinsic resistance of P. aeruginosa to TMP and SMX.

Anti-Bacterial Agents↗

Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.

Amino Acid Sequence↗

Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-homoserine lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.

Bacterial Proteins↗

The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients. The molecular basis of the host-P. aeruginosa interaction and the effect of specific P. aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown. We examine interactions between P. aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P. aeruginosa human isolate PA14. Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-kappa B-like transcription factors, allow increased P. aeruginosa infectivity within fly tissues. In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an I kappa B-like factor that inhibits the Toll signaling, reduce infectivity. Our finding that Dorsal activity is required to restrict P. aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity. Additionally, our results provide the basis for future studies into interactions between P. aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans.

Animals↗

Vfr controls quorum sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa controls several genes in a cell density-dependent manner through a phenomenon termed quorum sensing. The transcriptional activator protein of the las quorum-sensing system is encoded for by the lasR gene, which is at the top of a quorum-sensing hierarchy. The activation of LasR as a transcriptional activator induces the expression of multiple genes that code for factors important for virulence, and rhlR, which encodes the transcriptional activator protein of the P. aeruginosa rhl quorum-sensing system. Elucidating the method of lasR regulation is crucial to understanding P. aeruginosa quorum sensing. In this report, we present studies on the transcriptional control of lasR. We identified two distinct transcriptional start sites for lasR that were located 201 bp (transcript T1) and 231 bp (transcript T2) upstream from the lasR start of translation. With the use of transcriptional lasRp-lacZ fusions, we showed that in P. aeruginosa, lasR expression is cell density dependent. This gene was expressed at a basal level until it was induced during the second half of log-phase growth, with expression becoming maximal during stationary-phase growth. We also showed that lasR expression was regulated through the cyclic AMP receptor protein (CRP)-binding consensus sequence in its promoter region. Our results from P. aeruginosa mutant studies and gel retardation assays indicated that this regulation was mediated by Vfr, a homolog of the Escherichia coli CRP.

Bacterial Proteins↗

The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors.

Amino Acid Sequence↗

Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common pathogen in mechanically ventilated patients and produces a wide array of virulence factors. Bismuth-thiols (BTs) are active in vitro against all bacterial lung pathogens, including P. aeruginosa. The objective of these studies was to examine the biochemical and morphologic effects of sublethal BT concentrations on P. aeruginosa and to evaluate virulence in cell culture. Bismuth-dimercaprol, at a fraction of the minimal inhibitory concentration, reduced alginate expression by 67% in P. aeruginosa, whereas subinhibitory bismuth-ethanedithiol (BisEDT) reduced alginate by 92% in P. syringae. BisEDT effects on lipopolysaccharide content and type III secreted cytoxins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subinhibitory BisEDT reduced cell-associated lipopolysaccharide, and inhibited processing of the secreted cytotoxic protein ExoU. BisEDT-induced outer membrane blebbing and aggregation of cytoplasmic material was noted in electron microscopy. Virulence of P. aeruginosa was assessed by adherence to epithelial cells and sensitivity to serum killing. BisEDT inhibited adherence of P. aeruginosa to 16HBE14o- cells by 28% and to a collagen matrix by 53%. BisEDT-treated bacteria were also 100-fold more sensitive to serum bactericidal activity. In summary, low BT concentrations affect P. aeruginosa in a variety of ways, the combination of which may help prevent or resolve respiratory tract infection.

Bacterial Adhesion↗

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.

Animals↗

[Laboratory and clinical study on combined effects of fosfomycin plus sulbactam/cefoperazone for mixed infections of MRSA and Pseudomonas aeruginosa].

Pseudomonas aeruginosa has been a major bacterial partner when MRSA (Methicillin-resistant Staphylococcus aureus) is isolated clinically. Hence, we studied the in vitro combined effect of fosfomycin (FOM) plus sulbactam/cefoperazone (SBT/CPZ) against mixed infections of MRSA and P. aeruginosa. The combined effect of FOM plus SBT/CPZ was observed through an FIC index using the checkerboard method, log reduction of colony counts on the time killing curve, and morphological changes observed using scanning electron micrography. Under serum concentration at 3 hours after administration in consider of pharmacokinetics, the combination effect was also evaluated. Furthermore, we evaluated a time-lag combination therapy in which FOM is administered 60 minutes before SBT/CPZ administration for mixed infections. Synergistic effects of FOM plus SBT/CPZ were clearly observed against mixed infections of MRSA and P. aeruginosa. Log reduction of colony counts on the time killing curve seemed to be an appropriate indicator in the evaluation of synergistic effects.

Aged↗

The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa.

Pseudomonas aeruginosa NM48, a non-mucoid derivative of an alginate-producing strain isolated from a cystic fibrosis patient, was grown in batch culture with glycerol, glucose or succinate as carbon source, and in continuous culture (D 0.05 h-1) under glycerol or glucose limitation. Glycerol uptake, glycerol kinase and glycerol-3-phosphate dehydrogenase were induced by glycerol, but not by glucose or succinate. Linear uptake of [14C]glycerol by washed cells (Km < or = 2 microM) was inhibited by unlabelled glycerol and glyceraldehyde, but not by cyanide or the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and was accompanied by substantial intracellular accumulation of glycerol-3-phosphate and/or dihydroxyacetone phosphate but not glycerol. Prolonged growth under glycerol limitation led to substantial increases in the activities and/or concentrations of the enzymes catalysing glycerol uptake and metabolism, together with a 48,000 M(r) outer-membrane protein which was also over-expressed following prolonged growth under glucose limitation. The N-terminal amino acid sequence (AEAFSPN-) and electrophoretic properties of this protein were the same as those of the previously characterized glucose porin (OprB) from P. aeruginosa, indicating that this porin is active with both glucose and glycerol. It is concluded that during growth under glycerol limitation, glycerol is transported into P. aeruginosa NM48 via OprB and a high-affinity, binding-protein-independent facilitated-diffusion system.

Adaptation, Biological↗

Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

Bacterial Proteins↗

Catabolism of L-arginine by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is known to break down arginine by the arginine deiminase pathway. An additional pathway has now been found whereby arginine is converted to putrescine with agmatine and N-carbamoylputrescine as intermediates. The following enzyme activities belonging to this pathway were detected in crude extracts: arginine decarboxylase (EC 4.1.1.19), which catalyses the release of CO2 from arginine to give agmatine; agmatine deiminase (EC 3.5.3.12), which degrades agmatine to N-carbamoylputrescine; and N-carbamoylputrescine amidinohydrolase (EC 3.5.3.-), which then removes the ureido group of carbamoylputrescine. In crude extracts, arginine decarboxylase activity was stimulated by pyridoxal phosphate, Mg2+ and by the products of the catabolic pathway, putrescine and spermidine. Growth of P. aeruginosa on arginine as the sole carbon and nitrogen source markedly increased the activity of arginine decarboxylase. Agmatine and N-carbamoylputrescine induced the synthesis of agmatine deiminase and N-carbamoylputrescine hydrolase. Addition of succinate or citrate to medium containing arginine or agmatine led to repression of the enzymes involved in the arginine decarboxylase pathway. Moreover, the repression of agmatine deiminase and N-carbamoylputrescine hydrolase was further increased when P. aeruginosa was grown in media with agmatine plus glutamine or agmatine plus succinate and ammonia. This suggests that the expression of the agmatine pathway may be regulated by carbon catabolite repression as well as nitrogen catabolite repression.

Agmatine↗

Siderophore activity of pyoverdin for Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces an extracellular compound with yellowish green fluorescence, called pyoverdin, which functions as a siderophore. The production of pyoverdin, formerly called fluorescein, is concomitant with the production of another siderophore, pyochelin. Pyoverdin is produced by P. aeruginosa in several forms, some of which were separated on gel filtration columns and on reverse-phase, high-pressure liquid chromatography columns. An active form of iron-free pyoverdin was purified to homogeneity. The elution of pyoverdin from the columns was monitored for absorbance, fluorescence, and siderophore activities. These activities, iron binding, and the stimulation of bacterial iron transport indicated that pyoverdin can function as a siderophore for P. aeruginosa. The siderophore function of pyoverdin may be related to the pathogenicity of this bacterium because pyoverdin stimulated growth not only in iron-deficient culture medium, but also in defined medium containing transferrin and in human serum or plasma.

Culture Media↗

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.

Bacterial Outer Membrane Proteins↗