Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

Microbial Pathogenesis 1991 ; 10 : 173-182

Extracellular lipase of Pseudomonas aeruginosa : biochemical characterization and effect on human neutrophil and monocyte function in vitro Karl-E . Jaeger,' Arsalan Kharazmi 2 * and Niels Hoiby 2 'Lehrstuhl fur Biologie der Mikroorganismen, Ruhr-Universitat, D-4630 Bochum, Germany and 2 Statens Seruminstitut, Department of Clinical Microbiology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark (Received September 21, 1990; accepted in revised form November 12, 1990)

Jaeger, K .-E . (Lehrstuhl fur Biologie der Mikroorganismen, Ruhr-Universitat, D-4630 Bochum, Germany), A . Kharazmi and N . Hoiby . Extracellular lipase of Pseudomonas aeruginosa : biochemical characterization and effect on human neutrophil and monocyte function in vitro . Microbial Pathogenesis 1991 ; 10 : 173-182 . Lipase was isolated from P. aeruginosa by ultrafiltration of sterile-filtered culture supernatant . Gel filtration on Sepharose 4B yielded a broad peak corresponding to a molecular mass range of 100 to 1000 kDa . Electron microscopy of a negatively stained lipase preparation after Sepharose 4B chromatography revealed spherical particles with diameters ranging from 5 to 20 nm . Biochemical characterization and SDS polyacrylamide gel electrophoresis suggested that these particles consisted of protein and carbohydrate including lipopolysaccharide with the major enzyme activity being lipase . Various concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes . The chemotaxis and chemiluminescence of these cells were then determined . It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect on neutrophils . The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100°C . Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused by P. aeruginosa . Key words: Pseudomonas aeruginosa ; lipase ; neutrophil ; monocyte ; chemotaxis; chemiluminescence .

Introduction Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both chronic and acute infections . A prominent example is the chronic lung infection in cystic fibrosis (CF) which is regarded as the main cause of mortality in CF patients .' A large number of extracellular enzymes produced by P. aeruginosa seem to contribute to its virulence . Many of them, including exotoxin A, exoenzyme S, at least two proteases, and a phospholipase C, have been characterized biochemically and genetically, and their role as virulence factors has been determined .' Most clinical isolates 'Author to whom correspondence should be addressed at : Statens Seruminstitut, Department Microbiology, Rigshospitalet 7806, Tagensvej 20, DK-2200, Copenhagen N, Denmark . 0882-4010/91/030173+1 O X03 .00/0

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0.8

900

r

0.6 0.4 II 0.2 52 5.4 5.6 5.8 log Mr 0

600

Q

0) C N 0 0

J

300

J1 20 40 Fraction number

I

60

Fig . 1 . Gel filtration chromatography on Sepharose 4B of extracellular lipase from P . aeruginosa . Lipase activity ( •-•) was determined with p-nitrophenolpalmitate as substrate and protein concentration ( ) by measuring the absorbance at 280 nm . Insert : estimation of molecular mass of lipase by plotting the partition coefficients (Ka ,) of four marker proteins against the log of their molecular mass . Marker proteins were thyroglobulin (669 kDa), apoferritin (443 kDa), ii-amylase (200 kDa), and alcohol dehydrogenase (150 kDa) . Arrows indicate the Ka , values for lipase peaks I and 11 .

of P . aeruginosa including mucoid strains also produce an extracellular lipase (triacylglycerol acylhydrolase, EC 3 .1 .1 .3) 3-5 which has been purified to electrophoretic homogeneity and shown to be a 29 kDa protein with an isoelectric point of 5 .8 . 6 In the course of purification we observed that lipase existed in culture supernatants as high molecular mass particles which in this study will be referred to as lipase-LPS micelles . We further characterized these micelles assuming that they represent the native enzyme, i .e . the form originally excreted by P. aeruginosa cells . Since it has been shown that the inflammatory cells in the lungs seem to be important in the pathogenesis of lung infections caused by this bacterium,' we studied the effect of the native lipase on the function of human phagocytic cells .

Results Gel filtration of lipase

Cell-free culture supernatant of P. aeruginosa was concentrated by ultrafiltration and fractionated on a Sepharose 4B column . Figure 1 shows that lipase was eluted as a broad peak with two maxima of enzyme activity corresponding to estimated molecular masses of 840 kDa for peak I and 280 kDa for peak II . Comparison of several parallel experiments revealed slight variations in the elution positions corresponding to a range of 130-1300 kDa . It is interesting to note that the lipase activity peak did not correlate with the protein peak .

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P. aeruginosa lipase and phagocyte function

Table 1

Enzyme activities and biochemical composition of Sepharose 4B-pool

Enzymes Lipase Phospholipase C Alkaline phosphotase Protease

Activity (nKat) 1852 0 .16 nd' nd

Constituents Dry mass Protein Carbohydrate Lipopolysaccharide

Mass (Fig) 450 64 300 63

Values are given per millilitre of pool . "nd : not detectable .

Fig . 2 . Electron micrograph of negatively stained (uranyl acetate) lipase preparations after chromatography on Sepharose 4B . Circled structures (1-7) show approximately spherical particles with diameters ranging from 5 to 20 nm . Bar = 100 nm .

Biochemical composition After Sepharose 4B chromatography lipase-containing fractions were pooled and concentrated about 20-fold by ultrafiltration . Table 1 shows the biochemical composition of the pool . Lipase was the major enzyme activity present whereas only trace amounts of phospholipase C activity could be detected . The overall composition showed that only about 15% of the total pool was protein as already suggested by the elution profile of the protein peak in Fig . 1 and the major component was carbohydrate including lipopolysaccharide (LPS) . Electron microscopy The Sepharose 4B pool contained spherical particles with diameters ranging from 5 to 20 nm as shown in Fig . 2 . By assuming an average density of 1 .3 g cm -3 for spherical particles consisting primarily of protein and LPS we estimated a molecular mass range of 50-250 kDa roughly corresponding to the range we had determined by gel filtration chromatography .

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B

C

29 kDa-~

Fig . 3 . Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of Sepharose 4B pool . Gels were stained with silver as described by Oakley et al." (lane A) and Tsai and Frasch 24 (lanes B and C) . Lane A, Sepharose 4B-pool ; lane B, Sepharose 4B-pool digested with proteinase K prior to electrophoresis ; lane C, LPS purified from P. aeruginosa.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis Analysis of components of the Sepharose 4B pool on silver stained SDS gels showed a complex banding pattern with lipase appearing as a major band at 29 kDa (Fig . 3, lane A) . Digestion of the sample with proteinase K $ resulted in the typical ladder structure of LPS molecules with different length 0-antigens' as shown by comparison with the banding pattern of LPS purified from the same strain (Fig . 3, lanes B and C) . Cell viability Viability of neutrophils and monocytes as determined by trypan blue and nigrosin dye exclusion after 120 min incubation with up to 141 nKat/ml of lipase was greater than 95% . Chemotaxis The results on the effect of lipase on neutrophil and monocyte chemotaxis are shown in Table 2 . Lipase at concentrations higher than 35 nKat/ml inhibited neutrophil chemotaxis to F-Met-Leu-Phe by 25% and at 282 nKat/ml by about 50% . Preincubation of monocytes with 141 and 282 nKat/ml lipase inhibited the chemotaxis of these cells towards F-Met-Leu-Phe completely . At 70 nKat/ml there was about 20% inhibition .

P . aeruginosa lipase and phagocyte function

Table 2

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Effect of P . aeruginosa lipase on neutrophil and monocyte chemotaxis . Neutrophil

Monocyte

Lipase concs (n Kat/ml)

Non-heated

Heated

Non-heated

Heated

282 141 70 35

52+29 67+31 78+25 73+5

110+17 138+61 122+41

0+0 0+0 78+15 105+25

89 104 80

a

Not done . The cells were preincubated with various concentrations of lipase for 30 min and the neutrophil response to F-Met-Leu-Phe (10" 5 M) and monocyte response to ZAS was determined . Results are given as percentages of control cells response preincubated with buffer (mean ±SD of three experiments) . The response of control neutrophils to F-Met-Leu-Phe was 85±48 cells/field and that of monocytes to ZAS was 69± 18 cells/field .

Table 3 Effect of P. aeruginosa lipase on monocyte chemiluminescence Lipase concentration (nKat/ml) 141 70 35 18 9 2

Preincubation time 30 min <1 99+6 110+11 -

60 min

120 min

0 <1 49+2

<1 <1 49+1 36+2 36+1 96+3

'Not done . The cells were preincubated with various concentrations of lipase and then stimulated with opsonized zymosan (10 mg/ml) . Results are given as percentages of control cells response preincubated with buffer . The response of control monocytes was 76x 103-138 x 10 3 cpm/106 cells .

Heat treatment of lipase at 100°C for 10 min abolished the inhibitory effect of the lipase . Lipase by itself did not exhibit any chemotactic activity .

Chemiluminescence Preincubation of neutrophils with lipase at concentrations of 282, 141, 70, and 35 nKat/ml resulted in 75, 103, 88 and 100% respectively, of the chemiluminescence response of control cells preincubated with buffer . The results of monocyte chemiluminescence response are shown in Table 3 . There was both a preincubation-timedependent and a dose-dependent inhibition of monocyte chemiluminescence response by lipase . Preincubation of monocytes for 30 min with 141 nKat/ml resulted in total inhibition of monocyte response . At 60 min preincubation, lower concentrations of lipase were inhibitory (70 nKat/ml resulted in complete inhibition) and the strongest inhibitory effect was observed when the cells were preincubated for 120 min with lipase . With 120 min preincubation, lipase at as low as 9 nKat/ml inhibited the monocyte chemiluminescence by 65% . Preincubation of monocytes with heat-treated lipase (100°C, 10 min) at concentrations of 70 and 35 nKat/ml for 120 min resulted in 161 and 98% of control cells response, respectively . Preincubation of cells with 10 jig/ml of purified LPS from the same strain of P. aeruginosa did not have any

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effect on chemiluminescence (83X10 3 cpm/10 6 cells as compared to control value of 87 X 10 3 cpm/10 6 cells) .

Discussion This study demonstrated that extracellular lipase is released by P. aeruginosa as biochemically heterogenous lipase-LPS micelles in a molecular mass range of approximately 100-1000 kDa having diameters between 5 and 20 nm . In contrast, the purified monomeric enzyme is a 29 kDa protein .' We have no reason to assume that the lipaseLPS micelles contain significant amounts of other P . aeruginosa enzymes . In the cases of exotoxin A, exoenzyme S, proteases, cytotoxin, and phospholipase C, there is no evidence so far for high molecular mass micelles or association with LPS . 2 ' 10 Furthermore, cytotoxin has been shown to be localized in the periplasm of P . aeruginosa cells ." The release of heterogenous enzyme-LPS complexes has been described for alpha-hemolysin of Escherichia co/i. 12 .13 The size of the lipase-LPS micelles is probably too small to account for true outer membrane (OM) vesicles consisting of an LPS-phospholipid bilayer with different proteins inserted into it . Instead we may be dealing with structures mainly composed of LPS, proteins, and additional carbohydrate material of unknown origin . This assumption is consistent with our results of biochemical (Table 1) and SDS-PAGE analyses (Fig . 3) as well as with the estimation of the size of these micelles (Figs 1 and 2) . The existence of OM particles consisting of one or more protein species and LPS residing at the outer face of the OM has been described for E. coli and P. aeruginosa. 14 The presence or absence of major OM proteins strongly influenced the total number of OM particles as shown with appropriate E. coli mutant strains ." We obtained comparable results with respect to the number of extracellular lipase-LPS micelles by testing different P. aeruginosa mutants lacking either OM proteins or part of their LPS (K .-E . Jaeger and R . E . W . Hancock, unpublished data) . It is not yet known whether LPS and lipase are released from P . aeruginosa cells separately or as complexes pre-existing at the OM . We favour the latter hypothesis because we have not observed monomeric 29 kDa lipase in culture supernatants, except after rigid solubilization steps using surplus amounts of detergent . The release of these lipase-LPS micelles may be due to a membrane fragmentation or blebbing process which may occur spontaneously when the cells enter the early stationary growth phase, or may be regulated by an unknown mechanism . A better understanding of this process will result from experiments with antibodies recently raised against an amino-terminal peptide of P. aeruginosa lipase (K .-E . Jaeger and R . E . W . Hancock, unpublished data) . The lipase-LPS micelles seem to represent the native enzyme as it should be present e .g . in a P. aeruginosa infected lung . We therefore used this preparation in comparison with purified P. aeruginosa LPS to study its effect on human phagocytic cells . The data on the effect of lipase on human neutrophil and monocyte functions demonstrated that lipase exhibited a more profound inhibitory effect on monocytes than on neutrophils . Lipase had no effect on neutrophil chemotaxis and a slight inhibitory effect on neutrophil chemiluminescence . On the other hand lipase at 141 nKat/ml inhibited the monocyte chemotaxis completely and at as low as 9 nKat/ml the monocyte chemiluminescence response was strongly inhibited . The inhibition of monocyte chemiluminescence was dependent on both the preincubation time and the concentration of the lipase . At longer preincubation periods lower concentrations of lipase were required to inhibit the monocyte chemiluminescence . The inhibitory effect of lipase and its enzymatic activity was abolished by heat treatment of the enzyme at

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100°C for 10 min . The inhibition of monocyte function was not due to any cytotoxicity effect of lipase on these cells . The mechanisms involved in chemotaxis and oxidative burst response pathways in these two cell types are similar ." It is not clear why lipase inhibited the monocyte function and not the neutrophil . One possibility could be that the affinity of binding of lipase to monocytes is higher than that to neutrophils . It is known that bacterial products interacting with membrane lipid components exert quite different effects on human neutrophils and monocytes . 16, " Thus inhibition of monocyte function might be associated with the interaction of lipase with the lipid bilayer of the monocyte membrane . It has been shown that other lipases such as lipoprotein lipase increase the uptake of cholesteryl ester in various cell types by a mechanism which does not involve hydrolysis of the ester bonds .' $ Presumably, the lipase enhances receptormediated uptake of the cholestryl ester . Further studies are needed to clarify the mechanisms involved in the differential effect of lipase on neutrophils and monocytes . Although the lipase preparation contained some LPS the inhibitory effect observed could not be due to LPS . We and others have shown that LPS primes the neutrophils and monocytes for enhanced oxidative burst response .' 9 .21' 6 It has been shown that most clinical isolates of P. aeruginosa produce lipase .' However, the role of lipase in the pathogenesis of P . aeruginosa infections is not known . Rollof et al." have shown that Staphylococcus aureus lipase at low concentrations enhanced the direct migration of human neutrophils and at higher concentrations inhibited the chemotaxis of these cells towards the chemotactic peptide F-Met-Leu-Phe . Furthermore, lipase inhibited the phagocytic killing of S . aureus in a dose dependent manner ." The inhibition by lipase of the function of monocytes/macrophages, the important cells of the host defence system, may contribute to the pathogenesis of infections caused by P . aeruginosa .

Materials and methods Isolation of culture supernatant . Pseudomonas aeruginosa strain PAC 1 R was grown in nutrient broth medium to early stationary growth phase and the culture supernatant was isolated as described previously .' The supernatant fluid was mixed with Hepes buffer (pH 7 .5) to give final concentrations of 10 mm Hepes, 50 mm NaCl, 0 .1 mm CaCl 2 , and 0 .02% (w/v) NaN 3 . The supernatant was sterilized by filtration through a Durapore membrane with 0 .45 Jim pore size (Millipore, Eschborn, Germany) . The filtrate was concentrated approximately 50-fold by ultrafiltration with a YM 30 membrane (Amicon, Witten, Germany) at 5°C under a pressure of 2 .5 bar N 2 . The concentrated supernatant was stored in sterile tubes at 4°C . Ge/ filtration chromatography . A 93 cm x2 .5 cm bed (bed volume V,=457 ml) of Sepharose 4B (Pharmacia-LKB, Freiburg, Germany) was used for gel filtration chromatography . Concentrated culture supernatant (7-10 ml) was loaded on top of the gel and eluted with Hepes buffer pH 7 .5 without NaN 3 . The column was run at a flow rate of 36 ml/h overnight at room temperature and about 100 fractions were collected (6 ml/fraction) . Protein was detected by recording the absorbance at 280 nm (A 280 ) of the column effluent with a UV monitor . The void volume (V0 =138 .6 ml) of the gel was determined by elution of 10 ml of a suspension of phage T7 (2x10" phage particles/ml) under identical conditions . Calibration was achieved by calculating partition coefficients (ka° ) of four proteins obtained from Sigma, Munich, Germany, according to the equation : ka,=(Ve-VO)/(V,-Vo) with Ve being the volume at which protein peaks eluted . Sodium dodecyl sulphate polyacrylamide gel electrophoresis . Polyacrylamide gel electrophoresis in the presence of SDS and pre-treatment of samples with proteinase K were performed essentially as described .' Gels were stained with silver either by the method of Oakley et al." or by the method of Tsai and Frasch . 24

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Isolation of LPS . Lipopolysaccharide was isolated from P. aeruginosa PAC 1 R as described by Darveau and Hancock ." Concentration of 2-keto-3-deoxyoctonate was determined by the method of Osborn 26 and LPS concentrations were estimated using a value of 2 .4% (w/w) KDO in smooth LIPS of P. aeruginosa PAC 1 R . 27 Electron microscopy. Samples of the Sepharose 4B-pool were negatively stained with a solution of 3% (w/v) uranyl acetate pH 4 .5 . Samples were observed with a Philips EM 301 electron microscope at 80 kV and primary magnifications ranging from x 10000 to x32400 . Chemical determinations . Protein was determined by the method of Bradford 28 with bovine serum albumin as the standard . Total carbohydrate was determined by the method of Dubois 29 with glucose as the standard . Dry mass of samples was calculated after drying appropriate amounts of the Sepharose 4B pool under vacuum . Enzyme assays . Lipase, phospholipase C, and alkaline phosphatase were assayed by determination of A 410 of the corresponding p-nitrophenol derivatives of palmitate, 30 phosphorylcholine, 37 and phosphate . 32 Protease was assayed by determination of A430 with azocasein as substrate ." All substrates were obtained from Serva, Heidelberg, Germany . Neutrophils and monocytes . Neutrophils and monocytes were isolated from the peripheral blood of healthy individuals by dextran sedimentation followed by sodium metrizoate Ficoll (Lymphpoprep, Nyegaard, Oslo, Norway) separation ." The purity of neutrophil preparations was greater than 98% . The monocytes consisted of 15-32% of the mononuclear cell suspensions as judged by morphology after Wright and the non-specific esterase staining . The cells were suspended in appropriate buffers and media . Chemotaxis . A modified Boyden chamber assay was used for neutrophils35 and for monocytes" as previously described . Briefly, the chemotaxis chambers with a cellulose filter with 3 lrm pore size (Millipore, Bedford, Massachusetts) for neutrophils and 5 µm pore size (Nuclepore, Pleasanton, Massachusetts) for monocytes were filled with 0 .5 ml of the chemotactic peptide f-Met-Leu-Phe (10 -8 M), or zymosan activated serum (ZAS) at a dilution of 1 :20 . The upper compartment of the chambers contained 0 .5 ml of either neutrophil (1 x 10 6 /ml), or monocyte suspension (0 .5x10 6 /ml) . The cells have been preincubated with various concentrations of lipase for 30 min at 37°C . The chambers were incubated at 37°C for 150 min for neutrophils and 90 min for monocytes . After incubation the filters were removed, fixed on glass slides, stained with hematoxylin and mounted . The cells that had migrated through the filter to the other side were counted by direct microscopy in five random fields for neutrophils and 10 fields for monocytes on each filter . Appropriate controls were included in each experiment . The assays were performed in triplicate . Chemiluminescence . A previously described luminol enhanced chemiluminescence assay was used ." The assay was performed in a total volume of 5 .5 ml at ambient temperature in glass scintillation vials . A Beckman L 8000 scintillation counter placed under air-conditioned thermostat-controlled, 21 ± 1 °C conditions was used in the out of coincidence mode . A given volume of either neutrophil or monocyte suspension was preincubated with an equal volume of various concentrations of lipase at 37°C for different time periods . After preincubation, the cells were stimulated with either F-Met-Leu-Phe or opsonized zymosan in glass vials . The reaction mixture contained 1 ml of 5 x 10 5 preincubated neutrophils or 2 .5 x 105 monocytes, 0 .5 8M ml of F-Met-Leu-Phe (10 -5 M) or 0 .4 ml of opsonized zymosan (10 mg/ml), 50 µl of 5 x 10 luminol (5-amino-2,3-dihydro-1,4 phthalazinedione), and 4 .1 ml of Krebs Ringer solution . Reagents and vials were dark adapted before use, and the experiments were performed in duplicates under red light . Sequential 0 .5 min counts were taken on each vial over a period of 90 min . Statistics. Differences of more than two standard deviations were considered significant .

We thank F . Mayer, Institut fur Mikrobiologie, Unversitat Gottingen, Germany, for his help with electron microscopy . Expert technical assistance of Corina Strunk, Anne Asanovski and Glen Williams is acknowledged

P. aeruginosa lipase and phagocyte function

and Forschung Nordrhein-Westfalen, Germany, Az . : IV A6-10801090 01 38-DK from the Commission of the European Community .

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References 1 . Hoiby N . Microbiology of lung infections in cystic fibrosis patients . Acta Paediatr Scand Suppl 1982, 301 : 33-54 . 2 . Nicas TI, Iglewski BH . Toxins and virulence factors of Psuedomonas aeruginosa. In : Sokatch JR ed . The biology of Pseudomonas. Orlando : Academic Press, 1986 : 195-213 . 3 . Janda JM, Bottone EJ . Pseudomonas aeruginosa enzyme profiling : predictor of potential invasiveness and use as an epidemiological tool . J Clin Microbiol 1981 ; 14 : 55-60 . 4 . Wingender J, Winkler UK . A novel biological function of alginate in Pseudomonas aeruginosa and its mucoid mutants : stimulation of exolipase . FEMS Microbiol Lett 1984 ; 21 : 63-9 . 5 . Wretlind B, Heden L, Sjoberg L, Wadstrom T . Production of enzymes and toxins by hospital strains of Pseudomonas aeruginosa in relation to serotype and phage-typing pattern . J Med Microbiol 1973, 6 : 91-100 . 6 . Stuer W, Jaeger K-E, Winkler UK . Purification of extracellular lipase from Pseudomonas aeruginosa . J Bacteriol 1986 ; 168 : 1070-4 . 7 . Kharazmi A . Interactions of Pseudomonas aeruginosa proteases with the cells of the immune system . Antibiot Chemother 1989 ; 42 : 42-9 . 8 . Hitchock PJ, Brown TM . Morphological heterogeneity among Salmonella Iipopolysaccharide chemotypes in silver-stained polyacrylamide gels . J Bacteriol 1983 ; 154 : 269-77 . 9 . Rivera M, Bryan LE, Hancock REW, McGroarty EJ . Heterogeneity of lipopolysaccharide from Pseudomonas aeruginosa : analysis of lipopolysaccharide chain length . J Bacteriol 1988 ; 170 : 512-21 . 10 . Hayashi T, Kamio Y, Terawaki Y . Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa . Microbial Pathogenesis 1989 ; 6 : 103-12 . 11 . Kluftinger JL, Lutz F, Hancock REW . Pseudomonas aeruginosa cytotoxin : periplasmic location and inhibition of macrophages . Infect Immun 1989 ; 57 : 882-6 . 12 . Bonach GA, Snyder IS . Chemical and immunological analysis of the complex structure of Escherichia coli alpha-hemolysin . J Bacteriol 1985 ; 164 : 1071-80 . 13 . Bohach GA, Snyder IS . Composition of affinity-purified alpha-hemolysin of Escherichia coli Infect Immun 1986 ; 53 : 435-7 . 14 . Lugtenberg B, Van Alphen L . Molecular architecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria . Biochim Biophys Acta 1983 ; 737 : 51-115 . 15 . Snyderman R . Regulation of leukocyte function . New York : Plenum Publishing 1984 : 247-81 . 16 . Kharazmi A, Bibi Z, Neilsen H, Hoiby N . Effect of Pseudomonas aeruginosa rhamnolipid on human neutrophil and monocyte function . Actal Pathol Microbiol Immunol Scand 1989 ; 97 : 1068-72 . 17 . Wilkinson PC . Inhibition of leukocyte locomotion and chemotaxis by lipid-specific bacterial toxins . Nature 1975 ; 255 : 485-7 . 18 . Chajek-Saul T, Friedman G, Halperin 0, Stein Y . Uptake of chylomicron ( 3 H)cholestryl linoleyl either by mesenchymal rat heart cell cultures . Biochim Biophys Acta 1981 ; 666 : 147-55 . 19 . Kharazmi A, Andersen LW, Baek L, Valerius NH, Laub M, Rasmussen JP . Endotoxemia and enhanced generation of oxygen radicals by neutrophils from patients undergoing cardiopulmonary bypass . J Thorac Cardiovasc Surg 1989 ; 98 : 381-5 . 20 . Kharazmi A, Fomsgaard A, Conrad RS, Galanos C, Hoiby N . Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide : effect on human neutrophil chemotaxis and oxidative burst . J Leukocyte Biol 1990; In press . 21 . Guthrie LA, McPhail LC, Henson PM, Johnston RB . Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide J . Exp Med 1984 ; 160 : 1656-71 . 22 . Rollof R, Braconier JH, Soderstrom C, Nilsson-Ehle P . Interference of Staphylococcus aureus lipase with human granulocyte function . Eur J Clin Microbiol Infect Dis 1988; 7 : 505--10 . 23 . Oakley BR, Kirsch DR, Morris NR . A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels . Anal Biochem 1980 ; 105 : 361--3 . 24 . Tsai C-M, Frasch CE . A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels . Anal Biochem 1982 ; 119 : 115-19 . 25 . Darveau RP, Hancock REW . Procedure for the isolation of bacterial lipopolysaccharide from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains . J Bacteriol 1983, 155 :831--8 26 . Osborn MJ . Studies on the Gram-negative cell wall . Evidence for the role of 2-keto-3-deoxyoctonate in the Iipopolysaccharide of Salmonella typhimurium . Proc Natl Acad USA 1963 ; 50 : 499-507 . 27 . Chester JR, Meadow PM . Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa . Eur J Biochem 1975 ; 58 : 273-82 . 28 . Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem 1976 ; 72 : 248--52 . 29 . Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F . Colorimetric method for determination of sugars and related substances . Anal Chem 1956 ; 28 : 350-6 .

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30 .

Winkler UK, Stuckmann M . Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens . J Bacteriol 1979 ; 138 : 663--70 . Kurioka S, Matsuda M . Phopholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme . Anal Biochem 1976 ; 75 : 281-9 . Garen A, Levinthal C . A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of Escherichia coli. Biochim Biophys Acta 1960 ; 38 : 470-83 . Prestidge L, Gage V, Spizizen J . Protease activities during the course of sporulation in Bacillus subtilis . J Bacteriol 1971 ; 107 : 815-23 . Boyum A . Isolation of mononuclear cells and granulocytes from human blood . Scand J Clin Lab Invest 1968;21 :77-89 . Kharazmi A, Valerius NH, Hoiby N . Effect of antimalarial drugs on human neutrophil chemotaxis in vitro . Acta Pathol Microbiol Immunol Scand (C) 1983 ; 91 : 293-8 . Nielsen H, Olesen Larsen H . Human monocyte chemotaxis in vitro : influence of in vitro variables in the filter assay . Acta Pathol Microbiol Immunol Scand (C) 1983 ; 91 : 109-15 . Kharazmi A, Hoiby N, Daring G, Valerius NH . Pseudomonas aeruginosa exoproteases inhibit human neutrophil chemiluminescence . Infect Immun 1984 ; 44 : 587-91 .

31 .

32 . 33 . 34 . 35 . 36 . 37 .