The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.     

The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates

APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation-reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is -280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe-4S] center by either APS or the competitive inhibitor 5'-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe-4S] cluster.     

Effects of cell surface damage on surface properties and adhesion of Pseudomonas aeruginosa

Bacterial cell surfaces play a crucial role in their adhesion to surfaces. In the present study, physico-chemical cell surface properties of Pseudomonas aeruginosa, isolated from a case of contact lens associated keratitis, are determined for mid-exponential and early stationary phase cells and for cells after exposure to a lens care solution or after mechanical damage by sonication. Exposure to a lens care solution and mechanical cell surface damage reduced the cell surface hydrophobicity and water contact angles decreased from 129 degrees to 96 degrees and 83 degrees, respectively. Zeta potentials in saline (-9 mV) were hardly affected after mechanical damage, but tri-modal zeta potential distributions, with subpopulation zeta potentials at -11, -28 and -41 mV, were observed after exposure of bacteria to a lens care solution. X-ray photoelectron spectroscopy indicated changes in the amounts of oxygen-, nitrogen- and phosphorus-rich cell surface components. Mid-exponential phase cells had more nitrogen-rich cell surface components than early stationary phase cells, but water contact angles and zeta potentials were not very different. In addition, mid-exponential phase cells adhered better than early stationary phase cells to hydrophobic and hydrophilic substrata in a parallel plate flow chamber. The capacity of P. aeruginosa to adhere was decreased after inflicting cell surface damage. Exposure to a lens care solution yielded a larger reduction in adhesion capacity than sonication, likely because sonication left most of the cells in a viable state, in contrast to exposure to a lens care solution. It is argued that for clinically relevant experiments, it may be preferable to work with surface damaged cells rather than with gently harvested organisms.     

Rhamnolipid-induced shedding of flagellin from Pseudomonas aeruginosa provokes hBD-2 and IL-8 response in human keratinocytes

Several 'pathogen-associated molecular pattern' (PAMP) of the opportunistic pathogen Pseudomonas aeruginosa activate the innate immune system in epithelial cells. Particularly the production of antimicrobial peptides such as the human beta-defensin-2 (hBD-2) and proinflammatory cytokines as the interleukin (IL)-8 is boosted. In the present study culture supernatants of static grown P. aeruginosa were found to be potent hBD-2 and IL-8 inducers, indicating a soluble or shedded PAMP, comparable to that of heat-killed bacterial supernatants. In subsequent analyses this PAMP was identified as flagellin, the major structural protein of the flagella. Flagellin is known to be an immunostimulatory potent factor, but the mechanisms by which P. aeruginosa is able to remove flagellin from the flagella remain unknown. Here we provide evidence for the presence of a factor responsible for release of flagellin from the flagella. Purification of this factor and subsequent mass spectrometry analyses identified rhamnolipids as responsible agents. Our findings indicate that maybe upon adhesion to surfaces P. aeruginosa alters the outer membrane composition in a rhamnolipid-depending manner, thereby shedding flagellin from the flagella. In turn epithelial cells recognize flagellin and cause the synthesis of antimicrobial peptides as well as recruitment of inflammatory cells by induction of proinflammatory cytokines.     

The effect of lipopolysaccharide composition on the ultrastructure of Pseudomonas aeruginosa.

The surface structure of Pseudomonas aeruginosa PACl and PAClR and of lipopolysaccharide-defective mutants derived from them was studied by negative-staining and thin-section electron microscopy and compared with that of a rough mutant with wild-type lipopolysaccharide. The rough mutant and the parent strains had fairly smooth outer layers. Negatively stained preparations of all the mutants lacking polymerized O-antigenic sidechains, including a semi-rough mutant, showed numerous blebs on the surface. In thin sections of these mutants occasional extrusions from the surface were seen. They appeared to consist of material extruded from the outer membrane, but there was no evidence to suggest they were complete unit membranes. Polymerized O-antigenic side-chains in the lipopolysaccharide appear to be required to produce the wild-type appearance of the outer membrane in P. aeruginosa.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa

The effect of rhamnolipid-biosurfactant produced by Pseudomonas sp. PS-17 on cell surface structures of Pseudomonas aeruginosa NBIMCC 1390 was studied. The results demonstrated that the rhamnolipid at concentrations below and above CMC provoked a multi-component response of the bacterial cells without affecting their growth and viability. Above CMC, the rhamnolipid caused reduction of total cellular LPS content of 22%, which can be associated with an increase in cell hydrophobicity to 31% adherence. The rhamnolipid-biosurfactant at concentration below CMC did not affect the LPS component of the bacterial outer membrane but caused changes in OMP composition of P. aeruginosa. Examination of the OMP profiles revealed that the amount of major proteins (Opr F, Opr D, Opr J and Opr M) markedly decreased. To our knowledge this is the first report on the rhamnolipid-biosurfactant interactions with bacterial cells showing changes in outer membrane proteins of P. aeruginosa. In both concentrations, the biosurfactant caused changes in cell surface morphology. The results indicate that the rhamnolipid-biosurfactant from Pseudomonas sp. PS-17 has a potential application in the relatively new field of biomedicine.     

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded β-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.     

Substrate interaction with recombinant amidase from Pseudomonas aeruginosa during biocatalysis

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate–amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates.     

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.     

Interaction between hyaluronate and the cell surface of Pseudomonas aeruginosa

Abstract Treatment of Pseudomonas aeruginosa cells with the non-metabolizable polysaccharide hyaluronate led to a strong increase in extracellular lipase activity. Alteration of the cell surface either by treatment with the chelator EDTA or by selecting for phage-resistant mutants significantly altered the bacterial response to hyaluronate. Binding of ¹⁴C-labeled hyaluronate to the bacteria was shown to depend on polysaccharide concentration and on cell number. Cell-free exolipase interacted with chemically cross-linked hyaluronate. The results suggested an interaction between hyaluronate and the cell surface of P. aeruginosa as a prerequisite for the polysaccharide to be effective.     

Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Crystallization and some properties of leukocidin from Pseudomonas aeruginosa

A leukocidin was isolated and purified from autolysates of Pseudomonas aeruginosa strain 158 by a combination of procedures such as column chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100, and zone electrophoresis on pevikon. The purified preparation showed a single band in each experiment using electrophoreses in the presence or absence of sodium dodecyl sulfate (SDS), and the agar-gel Ouchterlony immunodiffusion test. The purified pseudomonal leukocidin was crystallized by salting out with saturated ammonium sulfate at pH 7.0 in a needle-leaf like form. The molecular weight of the leukocidin was estimated to be 42,500 by SDS-polyacrylamide gel electrophoresis, 40,000 by gel filtration, and 44,700 (3.3 S20,W) by sucrose density gradient centrifugation. The isoelectric point of the leukocidin was estimated to be at pH 6.3 by isoelectrofocusing. Morphological studies of a leukocidin-treated leukocyte showed that the formation of vacuoles of cytosolic granules and the swelling of the lobulated nuclei occurred prior to leukocyte enlargement. In a slide adhesion test with rabbit polymorphonuclear leukocytes (1 X 10(6], the minimum cytotoxic dose for the destruction of all leukocytes was 13-20 ng of the crystallized toxin. Rabbit lymphocytes were one-thirtieth as sensitive as rabbit leukocytes. Leukocidin did not act on rabbit erythrocytes or on platelets.     

Monolayer film behavior of lipopolysaccharide from Pseudomonas aeruginosa at the air-water interface

Lipopolysaccharide (LPS) is an essential biomacromolecule making up approximately 50% of the outer membrane of gram-negative bacteria. LPS chemistry facilitates cellular barrier and permeability functions and mediates interactions between the cell and its environment. To better understand the local interactions within LPS membranes, the monolayer film behavior of LPS extracted from Pseudomonas aeruginosa, an opportunistic pathogen of medical importance, was investigated by Langmuir film balance. LPS formed stable monolayers at the air-water interface and the measured lateral stresses and modulus (rigidity) of the LPS film in the compressed monolayer region were found to be appreciable. Scaling theories for two-dimensional (2D) polymer chain conformations were used to describe the pi-A profile, in particular, the high lateral stress region suggested that the polysaccharide segments reside at the 2D air-water interface. Although the addition of monovalent and divalent salts caused LPS molecules to adopt a compact conformation at the air-water interface, they did not appear to have any influence on the modulus (rigidity) of the LPS monolayer film under biologically relevant stressed conditions. With increasing divalent salt (CaCl2) content in the subphase, however, there is a progressive reduction of the LPS monolayer's collapse pressure, signifying that, at high concentrations, divalent salts weaken the ability of the membrane to withstand elevated stress. Finally, based on the measured viscoelastic response of the LPS films, we hypothesize that this property of LPS-rich outer membranes of bacteria permits the deformation of the membrane and may consequently protect bacteria from catastrophic structural failure when under mechanical-stress.     

Solution structure and properties of AlgH from Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the algH gene regulates the cellular concentrations of a number of enzymes and the production of several virulence factors, and is suggested to serve a global regulatory function. The precise mechanism by which the algH gene product, the AlgH protein, functions is unknown. The same is true for AlgH family members from other bacteria. In order to lay the groundwork for understanding the physical underpinnings of AlgH function, we examined the structure and physical properties of AlgH in solution. Under reducing conditions, results of NMR, electrophoretic mobility, and sedimentation equilibrium experiments indicate AlgH is predominantly monomeric and monodisperse in solution. Under nonreducing conditions intra and intermolecular disulfide bonds form, the latter promoting AlgH oligomerization. The high‐resolution solution structure of AlgH reveals alpha/beta‐sandwich architecture fashioned from ten beta strands and seven alpha helices. Comparison with available structures of orthologues indicates conservation of overall structural topology. The region of the protein most strongly conserved structurally also shows the highest amino acid sequence conservation and, as revealed by hydrogen‐deuterium exchange studies, is also the most stable. In this region, evolutionary trace analysis identifies two clusters of amino acid residues with the highest evolutionary importance relative to all other AlgH residues. These frame a partially solvent exposed shallow hydrophobic cleft, perhaps identifying a site for intermolecular interactions. The results establish a physical foundation for understanding the structure and function of AlgH and AlgH family proteins and should be of general importance for further investigations of these and related proteins. Proteins 2015; 83:1137–1150. © 2015 Wiley Periodicals, Inc.