Components of intestinal epithelial hypoxia activate the virulence circuitry of Pseudomonas

We have previously shown that a lethal virulence trait in Pseudomonas aeruginosa, the PA-I lectin, is expressed by bacteria within the intestinal lumen of surgically stressed mice. The aim of this study was to determine whether intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression. A fusion construct was generated to express green fluorescent protein downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P. aeruginosa. Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1-0.3% O2) for 1 h, with or without a recovery period of normoxia (21% O2) for 2 h, and then inoculated with P. aeruginosa containing the PA-I reporter construct. Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 h; P < 0.001). Similar activation of PA-I was also induced by cell-free apical, but not basal, media from hypoxic Caco-2 monolayers. PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia. We conclude that the virulence circuitry of P. aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.     

Recognition of intestinal epithelial HIF-1alpha activation by Pseudomonas aeruginosa

Human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and reoxygenation release soluble factors into the apical medium that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier-dysregulating protein PA-I lectin/adhesin. In this study, we defined the role of hypoxia-inducible factor (HIF)-1alpha in this response. We tested the ability of medium from Caco-2 cells with forced expression of HIF-1alpha to increase PA-I expression in P. aeruginosa and found that medium from Caco-2 cells overexpressing HIF-1alpha increased PA-I expression compared with medium from control cells (P < 0.001, ANOVA). To identify the components responsible for this response, medium was fractionated by molecular weight and subjected to mass spectroscopy, which identified adenosine as the possible mediator. Both adenosine and its immediate downstream metabolite inosine induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the medium of Caco-2 cells exposed to hypoxia or overexpressing HIF-1alpha, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned medium enhanced the extracellular accumulation of inosine. Together, these results provide evidence that P. aeruginosa can recognize and respond to extracellular end products of intestinal hypoxia that are released after activation of HIF-1alpha. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.     

Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin

The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.     

Type II restriction endonucleases from Bacillus sphaericus

Abstract The galactophilic lectin of the bacterium Pseudomonas aeruginosa (PA-I) was used for mitogenic stimulation of peripheral bloodlymphocytes from cancer-bearing patients and healthy subjects. This lectin, which preferentially stimulates sialidase-treated lymphocytes, was shown to be useful in the detection of an impairment in the mitogenic response of the patients' lymphocytes. Its efficiency was at least as that of the Phaseolus vulgaris lectin (PHA), which is widely used for the diagnosis and prognosis of deficient immunocompetence states.     

Analysis of the amino acid sequence of the Pseudomonas aeruginosa galactophilic PA-I lectin.

Based on the NH2-terminal 30-amino acid sequence of Pseudomonas aeruginosa galactophilic PA-I lectin, two degenerate primer oligonucleotides were synthesized and used in polymerase chain reaction with the bacterial chromosomal DNA as a template. A predominant DNA fragment of the appropriate size was radiolabeled and used as a probe for screening a P. aeruginosa genomic lambda gt11 library. One positive clone carrying an insert of about 630 base pairs encompassing the entire PA-I lectin gene was isolated and found to contain a 369-base pair open reading frame between an initiation codon (19 base pairs downstream from the insertion site, subsequent to a Shine-Dalgarno sequence) and two consecutive stop codons, followed by an oligo (seven) A sequence, in a partial dyad symmetry. The deduced amino acid sequence shows excellent agreement with the quantitative amino acid analysis and a perfect match with the NH2-terminal amino acid sequence of the purified lectin. It reveals that the PA-I lectin subunit contains 121 amino acids (M(r) 12,754; pI 4.94) with a predominant central hydrophilic core between two hydrophobic domains. Secondary structure algorithms predict that it is rich in beta sheets and contains several highly antigenic epitopes, but no signal peptide. In the carboxyl region a potential glycosylation site (Asn-Asn-Ser) was identified. Comparative analyses of this lectin sequence with those of lectins from other sources, reported in the protein and gene data banks, did not reveal any extensive homology.     

Effects of growth phase and boiling of enteropathogenic Escherichia coli strains on their interaction with Pseudomonas aeruginosa lectins

Escherichia coli strains from' serotypes O86, 0128 and O111 varied in their reactivity with Pseudomonas aeruginose lectins (PA-I with D-galactose specificity and PA-II which binds L-fucose, D-mannose, L-galactose and D-fructose). Generally, cells of O86 strains were agglutinated by PA-I, but not by PA-II, and those of O128 serotype were agglutinated by PA-II, and not by PA-I. Adsorption tests showed that cells of E. coli O86 strains adsorb PA-I to a greater extent than PA-II, while most E. coli O128 strains adsorbed higher amounts of PA-II. Cells of E. coli O111B4 which were not agglutinated by either Pseudomonas lectin could still adsorb both. Boiling of O86 and O128 cells frequently enhanced their agglutinability as well as their lectin adsorption capacity. The agglutinability enhancement was somewhat more prominent in boiled stationary phase cells than in log phase cells probably due to late synthesis of the O antigen components concomitantly with the heat-sensitive components (K antigens) which masked them. PA-I agglutinating activity was inhibited by the lipopolysaccharide (LPS) extracted from E. coli O86 cells, while PA-II was inhibited by the LPS extracted from E. coli O128 cells. These findings indicate that the receptors to the Pseudomonas lectins probably reside in the terminal part of the O-specific-polysaccharide of the LPSs of these bacteria.     

On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D-galactose and thiogalactose.

The D-galactose-binding lectin (PA-I) from the bacterium Pseudomonas aeruginosa, isolated by affinity chromatography on Sepharose, was examined for its relative affinities for simple sugars and their derivatives using equilibrium dialysis and hemagglutination inhibition tests. The lectin, which was found to bind 0.68 mol of D-galactose per subunit of 12.8 kDa, exhibited an association constant (Ka) of 3.4 x 10(4) M-1 for D-galactose and higher affinities for hydrophobic and thio derivatives of D-galactose (with highest affinity for the hydrophobic thio derivatives). alpha-Methyl-galactoside was a stronger inhibitor than the beta-methyl derivative and alpha-lactose was a weak inhibitor but the hydrophobic phenylated derivatives of the beta-configuration of D-galactose were more potent inhibitors than the respective alpha-galactosides.     

The intracellular localization of Pseudomonas aeruginosa lectins

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).     

First report of an arabinose-specific fungal lectin

To date, arabinose-binding lectins have been reported only from the human opportunistic pathogen Pseudomonas aeruginosa, the plant aggressive pathogen Ralstonia solanacearum, and the sponge Pellina semitubulosa. An arabinose-binding lectin with mitogenic activity toward splenocytes and a high specific hemagglutinating activity was isolated in the present study from a wild discomycete mushroom, Peziza sylvestris. The maximal mitogenic activity was induced by a lectin concentration of 8 microM. The lectin was a single-chained protein with a molecular mass of 20 kDa. Its N-terminal sequence showed only slight resemblance to other mushroom lectins. It was adsorbed on both diethylaminoethyl-cellulose and carboxymethyl-cellulose. Unlike previously reported mushroom lectins, the hemagglutinating activity of the lectin was inhibited by arabinose, but not by a large variety of other carbohydrates. The lectin activity was adversely affected in the presence of 0.05 M NaOH or 0.025 M HCl, and when the ambient temperature was elevated above 35 degrees C.     

Blocking of Pseudomonas aeruginosa lectins by human milk glycans

The opportunistic human pathogen Pseudomonas aeruginosa produces a D-galactophilic (PA-IL) lectin and another lectin (PA-IIL) that binds L-fucose > D-arabinose > D-mannose in close association with its host-attacking factors. These lectins contribute to the virulence of P. aeruginosa by their involvement in the production, adhesion, and pathogenic effects of its biofilm on host cells. Therefore, they are considered targets for anti-Pseudomonas therapy. The present study compares their blocking by human milk samples with that of the plant lectin Con A. It demonstrates that human milk inhibits the hemagglutinating activities of the three lectins, with PA-IIL much more strongly inhibited than PA-IL or Con A. Using these lectins, Western blots of the milk samples accord with the hemagglutination inhibition data and disclose the distribution of the human milk glycoproteins that inhibit each lectin. The data of this paper reveal the high efficiency of human milk components in blocking the P. aeruginosa lectins and the usefulness of these lectins for detecting milk glycoprotein saccharides, which may protect the infant against infections.     

Binding of Pseudomonas aeruginosa Lectin to Rhizobium sp.

Binding sites for the mannosephilic lectin of Pseudomonas aeruginosa and for concanavalin A were found on the nitrogen-fixing symbiont Rhizobium sp. which infects Phaseolus lathyroides , and on the root cells of its host. The interaction between these lectins and the Rhizobium or the root cells was demonstrated by adsorption tests and by mannose-specific peroxidase-binding to the lectin-coated bacteria. Treatment of the bacteria with formaldehyde, phenol, ethanol or boiling did not alter their ability to adsorb the lectins. The growth-rate of the Rhizobium was unaffected when the Pseudomonas lectin was added to the culture medium. Addition of these lectins to the Rhizobium cells used for seedling infection led to an increased bacterial adsorption to seedling roots and to enlargement of nodule size, but did not increase nodulation.     

Glycosylation is required for outer membrane localization of the lectin LecB in Pseudomonas aeruginosa

The fucose-/mannose-specific lectin LecB from Pseudomonas aeruginosa is transported to the outer membrane; however, the mechanism used is not known so far. Here, we report that LecB is present in the periplasm of P. aeruginosa in two variants of different sizes. Both were functional and could be purified by their affinity to mannose. The difference in size was shown by a specific enzyme assay to be a result of N glycosylation, and inactivation of the glycosylation sites was shown by site-directed mutagenesis. Furthermore, we demonstrate that this glycosylation is required for the transport of LecB.     

Use of fluorescent lectin probes for analysis of footprints from Pseudomonas aeruginosa MDC on hydrophilic and hydrophobic glass substrata

Microbial footprints of Pseudomonas aeruginosa MDC attached for 1 h to clean or silanized glass were analyzed with fluorescently labeled lectin probes. Footprint composition varied, depending on cell physiology and substratum surface chemistry. This suggests that substratum physicochemistry affected the structure of cell surfaces of adsorbed organisms.     

Lectin-based affinity tag for one-step protein purification

The production of pure protein is indispensable for many applications in life sciences, however protein purification protocols are difficult to establish, and the experimental procedures are usually tedious and time-consuming. Therefore, a number of tags were developed to which proteins of interest can be fused and subsequently purified by affinity chromatography. We report here on a novel lectin-based affinity tag using the D-mannose-specific lectin LecB from Pseudomonas aeruginosa. A fusion protein was constructed consisting of yellow fluorescent protein and LecB separated by an enterokinase cleavage site. This protein was overexpressed in Escherichia coli Tuner (DE3), and the cell extract was loaded onto a column containing a mannose agarose matrix. Electrophoretically pure fusion protein at a yield of 24 mg/L culture was eluted with a D-mannose containing buffer The determination of equilibrium adsorption isotherms revealed an association constant of the lectin to the mannose agarose matrix of Ka = 3.26 x 10(5)/M. Enterokinase treatment of the purified fusion protein resulted in the complete removal of the LecB-tag. In conclusion, our results indicate that the lectin LecB of P. aeruginosa can be used as a tag for the high-yield one-step purification of recombinant proteins.     

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.     

A new mitogenic D-galactosephilic lectin isolated from seeds of the coral-tree Erythrina corallodendron. Comparison with Glycine max (soybean) and Pseudomonas aeruginosa lectins

The lectin of Erythrina corallodendron (Caesalpiniaceae) seeds was purified by heating, ammonium sulfate fractionation, and affinity chromatography on acid-treated Sepharose. The purified lectin is similar to the soybean lectin in being a glycoprotein of molecular weight around 110 000 - 120 000 and having D-galactosephilic activity. This lectin, like the soybean and Pseudomonas aeruginosa lectins, binds to D-galactosamine, N-acetyl-D-galactosamine, alpha- and beta-galactosides as well as to D-galactose. Like these lectins it absorbs onto either untreated or enzyme (papain or neuraminidase) treated human red blood cells, but exhibits a considerable mitogenic activity towards human lymphocytes (predominantly T cells) only after their treatment with neuraminidase. This mitogenic stimulation of lymphocytes is inhibited by D-galactose and its derivatives. Despite the great similarity between them, the E. corallodendron, soybean, and Pseudomonas lectins differ in regard to the intensity of their agglutinating activity towards erythrocytes obtained from different animals and human donors of diverse ABO blood groups. This phenomenon may be attributed to the difference in the affinities of the three lectins to the various D-galactose derivatives and to their molecular properties.     

铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

Lytic manifestations in the bacterial strains of Pseudomonas aeruginosa (bacteriophages bacteriocines, autoplaques).

Ps. aeruginosa strains--a frequement resuet of an irresponsible antibiotic therapy--represent a common agent of nosocomial infektions. At the same time, gravity of Pseudomonas diseases is also increasing. Lysogeny, bacteriocinogeny and frequent occurrence of autoplaques are the lytic manifestations of Pseudomonas aeruginosa strains which play a great role in the complexity of solving diagnostic, epidemiological end therapeutical problems connected with infections induced by these microbes. A survey is presented of the importance and utilization of the lytic properties of bacterial strains of Pseudomonas aeruginosa during the differentiation, epidemiological typing and further expansion of therapeutical possibilities in infections caused by Pseudomonas aeruginosa bacteria.