Vol. 56, No. 11INFECTION AND IMMUNITY, Nov. 1988, p. 2788-27930019-9567/88/112788-06$02.00/0Copyright © 1988, American Society for Microbiology

Alginase Treatment of Mucoid Pseudomonas aeruginosa EnhancesPhagocytosis by Human Monocyte-Derived Macrophages

FERESHTEH EFTEKHAR"2 AND DAVID P. SPEERTl 2,3*

Departments of Paediatrics' and Microbiology,3 University of British Columbia, Vancouver, British Columbia V6T I W5,and Division of Infectious Diseases, British Columbia's Children's Hospital, Vancouver,

British Columbia V5Z 4H4,2 Canada

Received 29 June 1988/Accepted 10 August 1988

Mucoid Pseudomonas aeruginosa colonizes and infects the respiratory tract of most older patients with cysticfibrosis. These bacteria resist both opsonin-dependent and -independent phagocytosis by human polymorpho-nuclear leukocytes and monocyte-derived macrophages. Resistance to phagocytosis is thought to be mediatedin part by the mucoid exopolysaccharide associated with the bacterial surface. The purpose of this study wasto determine whether degradation of the mucoid exopolysaccharide by alginase enhances bacterial suscepti-bility to nonopsonic phagocytosis by macrophages. Eight phagocytosis-resistant mucoid P. aeruginosa isolatesfrom patients with cystic fibrosis were studied. The bacteria were treated with a preparation of alginase fromBacillus circulans, and phagocytosis by macrophages was measured by a visual inspection assay. Alginasedegradation of mucoid exopolysaccharide was measured by the periodate-thiobarbituric acid assay and byindirect immunofluorescence with a mouse monoclonal antibody to the mucoid exopolysaccharide. Alginasedegraded the mucoid exopolysaccharide of all eight mucoid strains tested. Phagocytosis was enhanced in fiveof the eight strains. Alginase-enhanced phagocytosis was magnesium dependent and heat labile. Alginase maybe a useful tool for studying the biological properties of P. aeruginosa mucoid exopolysaccharide.

Mucoid strains of Pseudomonas aeruginosa frequentlycolonize the respiratory tract of older patients with cysticfibrosis (CF). The resulting chronic infection is the majorcontributing factor to morbidity and mortality in these pa-tients (19). Mucoid P. aeruginosa resists both nonopsonicphagocytosis by human monocyte-derived macrophages(MO) and by neutrophils (3) and opsonic phagocytosis byneutrophils in the absence of specific antibody (1). Themucoid exopolysaccharide (MEP) of mucoid P. aeruginosaenhances adherence of this organism to tracheal epithelialcells (21), and MEP from some mucoid strains binds to bothhuman buccal and tracheal epithelial cells (6). All efforts toeradicate mucoid P. aeruginosa from infected lungs of CFpatients have been unsuccessful. In this paper we proposethe use of the enzyme alginase as a tool to study the role ofbacterial alginate in resistance of mucoid P. aeruginosa tononopsonic phagocytosis by M+.

Alginases or alginate-degrading enzymes are produced bya number of environmental organisms, including marinebacteria, algae, and marine mollusks (5, 7, 13, 27, 28). Othergram-negative bacteria such as Azotobacter vinelandii (12)(and a bacteriophage specific for A. vinelandii [4]), Klebsi-ella aerogenes (2), Pseudomonas maltophilia and Pseudo-monas putida (28, 29), and P. aeruginosa (8, 15) have alsobeen reported to produce alginases. Hansen et al. (10, 11)isolated an extracellular alginase from strains of Bacilluscirculans. Not all of the reported alginases have beenextensively studied, but those which have been character-ized seem to be capable of degrading both bacterial and/oralgal alginates and partially hydrolyzed polyuronic acidsubstrates (4, 5, 7, 8, 10, 15, 28, 29). Alginates are polyuronicacid complexes composed of 1,4-linked D-mannuronic andL-guluronic acids. Bacterial alginates are distinguished fromalgal polymers by possession of 0-acetyl groups, mainly ontheir mannuronate blocks (9, 16, 22, 23). A well-character-

* Corresponding author.

ized bacterial alginate is the MEP elaborated in large quan-tities by mucoid strains of P. aeruginosa which colonize thelungs of older CF patients. The purpose of this study was todetermine whether an alginase from B. circulans coulddegrade MEP from the surface of mucoid P. aeruginosa andrender the bacteria susceptible to nonopsonic phagocytosisby M+.

MATERIALS AND METHODSBacterial strains and growth media. B. circulans ATCC

15518 was obtained from the American Type Culture Col-lection, Rockville, Md. Clinical isolates of mucoid P. aeru-ginosa were obtained from cultures of sputum or throatsamples of patients with CF treated at the Cystic FibrosisAssessment Clinic, British Columbia's Children's Hospital.The Pseudomonas strains used in phagocytosis experimentswere C1712M, C1628M, C1900M, C2141M, C2101M, andC2208M (all polytypeable with the International AntigenicTyping System [Difco Laboratories, Detroit, Mich.]),LM3M (type 3), and C2146M (type 1,10). All strains wereresistant to phagocytosis by M+. MEP was prepared fromstrains C46M, PlM (type 9,10), C96M (nontypeable), andC1712M. All bacterial cultures were stored at -70°C inTrypticase soy broth (BBL Microbiology Systems, Cockeys-ville, Md.) with 8% dimethylsulfoxide and were subculturedon Trypticase soy agar (BBL) plates prior to each experi-ment. Batch cultures used to produce alginase were grown inmedium containing 0.5% yeast extract (Difco), 0.5% sodiumalginate (Sigma Chemical Co., St. Louis, Mo.), and 2 mMMgCl2.

Alginase preparation. B. circulans was grown in alginate-yeast extract medium at 26°C with vigorous aeration for 3 to4 days. The culture was centrifuged (8,000 x g) for 20 min at4°C, and the supernatant was concentrated 10-fold with aMini-Tan microfiltration system (Millipore Corp., Bedford,Mass.) with a membrane filter exclusion size of 10,000daltons. Ammonium sulfate was added to the concentrated

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supernatant to 70% saturation. The precipitate was collectedafter centrifugation at 20,000 x g for 30 min at 4°C, sus-pended in 10 mM Tris hydrochloride (pH 7.4) (Tris), anddialyzed overnight against Tris. This crude alginase prepa-ration was then frozen at -20°C. A partially purified alginasewas prepared by passing the crude alginase extract througha DEAE-Sephacel column (2.5 by 20 cm), washing thecolumn with Tris, and eluting the enzyme with NaCl (0.05 to0.5 M). Alginase, eluted at 0.1 M NaCl, was detected byperiodate-thiobarbituric acid (TBA) assay of the collectedfractions (see below). The enzyme was then dialyzed againstdistilled water overnight and lyophilized. The alginase prep-aration was devoid of bacterial contamination as assessed bymicroscopic inspection and absence of viable counts onTrypticase soy agar plates. Crude alginase was tested forprotease activity by using Hide Azure powder (Sigma).Dilutions of crude alginase and the protease control (typeXIV; Sigma) were made in 3.0 ml of phosphate-bufferedsaline (PBS) (pH 7.4). Tubes were incubated in a 37°C waterbath for 15 min, and 20 mg of Hide Azure powder was addedto each tube. The test tubes were then incubated at 37°C for30 to 60 min and were centrifuged to remove the particulatedye. Protease activity was determined by measuring theoptical density of the released dye at 595 nm.

Determination of alginase activity. The yield of unsaturatedmaterial from alginase action on alginate substrates wasdetermined by the TBA procedure of Weissbach and Hur-witz (30). Crude or partially purified alginase was added tosodium alginate at a concentration of 2.5 mg/ml in a volumeof 500 p.l. Samples (50 ,ul) were taken at 10-min intervals forup to 60 min and were tested by the TBA assay. One unit ofenzyme activity was defined as the amount of enzymerequired to liberate an equivalent of 1 nmol of beta-formylpyruvate per min per ml; 0.01 ,umol of beta-formylpy-ruvate produces an increase in optical density of 0.29 at 549nm (20). Alginase activity was also assayed by measuring theincrease of optical density at 235 nm, which assesses forma-tion of unsaturated uronic acids of the intermediate oligosac-charides (20). The specific activity of the enzyme is ex-

pressed as units of enzyme activity per milligram of protein.Protein concentration was determined by the method ofLowry et al. (17).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresiswas carried out by the procedure of Laemmli and Favre (14).The crude alginase extract and material eluted from DEAE-Sephacel were electrophoresed, using a 4% stacking gel anda 12% separating gel. Molecular weight standards were fromAmersham Corp., Arlington Heights, Ill., and protein gelswere stained with Coomassie brilliant blue G-250 (EastmanKodak Co., Rochester, N.Y.).

Preparation of bacterial MEP. MEP from mucoid P. aeru-

ginosa was prepared by a modification of the procedure ofEvans and Linker (9), as described previously (24).

Alginase treatment of mucoid P. aeruginosa strains. MucoidP. aeruginosa isolates from CF patients were grown on

Trypticase soy agar plates directly from frozen cultures(37°C for 24 h). The bacteria were swabbed from the plates,suspended in Tris, washed twice, and adjusted spectropho-tometrically to 109 bacteria per ml. The bacterial suspensionwas incubated with alginase (125 U/ml; specific activity,7.7), heat-inactivated (60°C for 2 h) alginase, or Tris at 37°Cwith rotation. Depending on the experiment, the total vol-ume varied between 300 and 500 ,ul. In some experiments,enzyme treatment of bacteria and other control treatmentswere carried out in the presence of various amounts of

Mg2 . Samples were removed at various time intervals andcentrifuged. The supernatants were tested by the TBA assayfor enzyme activity, and the bacteria were suspended inRPMI 1640 at a concentration of 109/ml.

Indirect immunofluorescence of mucoid P. aeruginosa be-fore and after alginase treatment. Immunofluorescence ofmucoid P. aeruginosa was determined as described byMutharia and Hancock (18). Briefly, P. aeruginosa suspen-sions treated with alginase, heat-inactivated alginase, or Triswere smeared on glass slides, air dried, and heat fixed. Amouse monoclonal antibody to MEP (a gift from G. Pier,Harvard University, Boston, Mass.) was diluted (1:100) inPBS (pH 7.4) with 1% fetal calf serum (PBS/FCS). Thesmears were incubated with the monoclonal antibody or PBSat room temperature for 30 min and washed three times withPBS/FCS. Fluoresceinated anti-mouse immunoglobulin G(Organon Teknika, Malvern, Pa.) was added to each smearat a dilution of 1:20, and the slides were incubated at roomtemperature for another 30 min. The slides were washed fourtimes with PBS/FCS, air dried, and examined with a Zeissepifluorescence microscope. The intensity of fluorescencewas graded as 0 to 4+.

Preparation of M+. M4~were prepared from human mono-nuclear cells as previously described (26). Cultured mo-nocytes in Teflon beakers were used after 4 to 7 days of invitro maturation.

Phagocytosis. Phagocytosis of unopsonized P. aeruginosawas performed as described previously (25) with M4 adher-ent to glass coverslips. Suspensions of 107 to 108 mucoidbacteria treated with alginase, heat-treated alginase, or Tris(0.1 ml) were added to each well of a 24-well tissue cultureplate (Becton Dickinson Labware, Oxnard, Calif.) contain-ing a glass coverslip on which M4~had been plated in a totalvolume of 1 ml. The plates were incubated at 37°C in 5% CO2with gentle rocking for 1 h, and phagocytosis was thenassessed visually by light microscopy. In some experiments,alginase was directly added to the phagocytic mixture in thetissue culture plate wells and bacteria were not preincubatedwith the enzyme. A total of 100 M( were examined for eachtest condition, and the number of bacteria per M4 wasrecorded.

Statistical analysis. Differences between conditions (bac-teria treated with alginase, heat-inactivated alginase, or Tris)were analyzed by analysis of variance to allow for day-to-day variation. All hypotheses were based on the standarderror estimates obtained from the analysis of variance. Thesignificance of differences between conditions is reported.

RESULTSIsolation and partial purification of alginase. The crude

alginase extract was prepared from the supernatant of 72-hbatch cultures of B. circulans by 70% ammonium sulfateprecipitation (Fig. 1, lane 1). The protease content of thecrude alginase as measured by the Hide Azure assay was0.86 ,ug (protease equivalents [Sigma])/mg of protein. Thecrude alginase was further purified by subsequent ion-exchange chromatography on a DEAE-Sephacel column;the alginase was eluted with 0.1 M NaCl. The purifiedenzyme yielded a major protein band of about 50,000 dal-tons. Two minor bands were also observed, one with amoleclar mass of 200,000 daltons which proved to be sodiumalginate when the gel was stained with Alcian blue and asecond with an apparent molecular mass of 90,000 daltons(Fig. 1, lane 2).

Digestion of MEP by alginase. MEP was extracted fromfour mucoid strains of P. aeruginosa (C96M, C46M,

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2 3- 200 k

- 92.5 k

---69k

46 k

-30 k

_a - 21.5 k

-14.3 k

FIG. 1. Sodium dodecyl sulfate-polyacrylamide (12%) gel analy-sis of crude alginase (lane 1) and DEAE-Sephacel-purified alginase(lane 2) from B. circulans. Alginase preparations were mixed withthe sample buffer (1% sodium dodecyl sulfate, 0.125 M Tris, 25%glycerol, 3% mercaptoethanol) and heated at 100°C for 5 min beforeelectrophoresis. Lane 3, molecular weight markers (molecularweights shown in thousands).

C1712M, and P1M). The crude alginase extract (30 U/ml)from B. circulans degraded commercial sodium alginate aswell as each of the MEP preparations as measured by theTBA assay (Fig. 2). All MEPs were used in their native formwithout deacetylation. The addition of Mg2' did not enhanceenzyme degradation of the tested substrates. Increase ofoptical density at 235 nm was rapid for both crude andpartially purified alginase when they were incubated withsodium alginate.

Effect of the duration of alginase treatment of mucoid P.aeruginosa on susceptibility to phagocytosis by M4+. Thedegree of phagocytosis of a mucoid P. aeruginosa strain(C1712M) by M4 is shown in Table 1. This strain was chosento standardize the experimental conditions throughout thestudy. The bacteria were treated with alginase in the pres-ence of 10 mM Mg2+ for different time intervals prior tophagocytosis by M+. Alginase treatment of strain C1712Mfor 90 to 120 min was optimum for enhancing phagocytosis

2-

0 Z

TIME (min)FIG. 2. Degradation of commercial sodium alginate (SA) and

MEP from four mucoid strains of P. aeruginosa by alginase from B.circulans. Each line represents MEP from a strain of P. aeruginosa.The optical density at 549 nm (O.D.549) represents the degree ofenzyme activity as determined by the TBA assay.

TABLE 1. Effect of duration of alginase treatment of mucoidP. aeruginosa C1712M on susceptibility to phagocytosis by MO

Alginase treatment PaoySiaRelease of(min) Phagocytosisa MEPl

0 2.59 ± 0.26' 0.0030 5.11 ± 0.49 0.6960 5.50 ± 0.49 0.6990 8.67 ± 0.76 1.09120 8.74 ± 0.82 2.09180 9.82 ± 0.81 0.69

a Bacteria were treated with alginase (125 U/ml). Samples were removed atintervals and used for phagocytosis experiments. See the text for details.

b Release of MEP from P. aeruginosa by alginase was measured by theTBA assay. The values represent nanomoles of beta-formylpyruvate released.See the text for details.

' Mean number of bacteria ingested per macrophage + standard error. Dataare from one representative experiment.

by M+. Longer incubation periods did not further enhancethe uptake significantly but may have resulted in bacterialgrowth or degradation of alginase. Therefore, mucoid strainswere incubated with alginase for 2 h in all subsequentexperiments. MEP degradation as measured by the TBAassay (Table 1) was also greatest after 2 h.

Effect of Mg2+ on alginase activity. The effect of Mg2+concentration on alginase activity as assessed by phagocy-tosis of C1712M by M4 is shown in Table 2. A Mg2+concentration of 2 mM resulted in the greatest bacterialuptake with 7-day-old M+. In general, the 4-day-old M4 hadlower uptake compared with that by older M+, and theoptimum Mg2+ concentration was 2 to 5 mM. A Mg2+concentration of 2 mM was therefore used for alginasetreatment of all eight mucoid strains.

Effect of alginase treatment on different mucoid P. aerugi-nosa strains. Eight mucoid strains of P. aeruginosa whichwere resistant to phagocytosis by M4 were used (Table 3).Each strain was studied on multiple days with M4X fromdifferent donors. The mean values and standard errors werecalculated and compared for different conditions, usinganalysis of variance to adjust for day-to-day variation.Phagocytosis of strains C1712M, C1628M, C1900M, LM3M,and C2141M was enhanced after pretreatment of bacteriawith alginase compared with pretreatment with Tris (P <0.0001 for each of the five strains). Heat-inactivated alginaseenhanced bacterial uptake compared with that by Tris con-trols, but the increase was significantly less than that ob-tained with native alginase (P < 0.0001). Strains C2101M,C2146M, and C2208M were not phagocytosed by M( underany experimental condition and were classified as "non-

TABLE 2. Effect of Mg2+ concentration on alginase-mediatedenhancement of phagocytosis by M<[4

Phagocytosis of mucoid P. aeruginosa C1712M"

Mg2Mconcn 4-day-old MN 7-day-old MX

Alginase Tris Alginase Tris

0 3.0 ± 0.51b 0.69 ± 0.12 8.2 ± 0.73 1.9 ± 0.201 4.3 ± 0.54 0.76 ± 0.11 19.2 ± 1.00 2.4 ± 0.252 4.6 ± 0.61 1.2 ± 0.15 21.2 ± 1.32 1.3 ± 0.165 5.5 ± 0.71 0.97 ± 0.16 16.9 ± 1.50 2.5 ± 0.2510 4.7 ± 1.08 0.79 ± 0.13 8.7 ± 0.82 3.8 ± 0.33

"Bacteria were treated with alginase (125 U/ml) or Tris for 2 h at 37°C priorto phagocytosis by Mo. See the text for details.

b Mean number of bacteria ingested per macrophage ± standard error.

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TABLE 3. Effect of alginase on phagocytosis of mucoid P. aeruginosa by M4

Phagocytosis of bacteria treated witha:

Strain Alginase Tris Heated alginase No. ofexpts

Mean no. of % M4X Mean no. of % M4 Mean no. of % M4bacteria/M4 with bacteria bacterialM4 with bacteria bacteria/MX with bacteria

C1712M 6.60*b 71.2 1.63 39.0 3.82* 55.5 4C1628M 7.34* 87.5 1.38 45.5 3.68* 67.4 8C1900M 8.31* 87.2 1.09 32.5 4.54* 61.5 3LM3M 3.08* 74.0 0.86 34.3 1.89* 53.3 3C2141M 2.92* 57.0 0.37 16.5 0.89* 27.5 4C2101M 0.26 6.5 0.16 4.0 0.15 6.5 4C2146M 0.27 9.7 0.03 1.7 0.04 2.5 4C2208M 0.04 3.0 0.04 3.5 0.13 6.0 2

a Bacteria were treated with alginase (125 U/ml), heat-inactivated alginase (125 U/ml), or Tris at 37°C for 2 h in the presence of 2 mM Mg2" prior to phagocytosisby M4 as described in the text.

b Mean number of bacteria ingested per M4. *, Value is significantly greater than that for the Tris control for the same strain (P < 0.0001). See the text formethods of statistical analysis.

responders." The percentage of M4 which phagocytosedmucoid bacteria in each experiment is also shown for allconditions (Table 3). Alginase treatment of C1712M,C1628M, C1900M, LM3M, and C2141M rendered theseorganisms more susceptible to phagocytosis by MW. Asignificantly larger population of M4 phagocytosed alginase-treated organisms than those treated with heat-inactivatedalginase or Tris.A comparison between phagocytosis of strain C1900M

after preincubation of the bacteria with alginase, heat-treated alginase, or Tris and phagocytosis when the enzymewas added directly to the phagocytic mixture is shown inTable 4. Only the native enzyme enhanced bacterial uptakeby M+~, and heat-inactivated alginase had no effect onphagocytosis. Both crude and partially purified alginaseenhanced phagocytosis of strain C1900M to the same extent(Table 4).The results of the TBA assay performed on the superna-

tants of bacteria treated with alginase, heat-inactivated algi-nase, or Tris buffer are shown in Table 5. The presence ofTBA-positive material, measured by an increase of opticaldensity at 549 nm, was observed only when the organisms

TABLE 4. Comparison of M4 uptake of strain C1900Mpreincubated with alginase and phagocytosis in the presence of

alginase in the phagocytic mixture

Mean no.of bacteria ± SE

Treatment Alginase- Alginase addedpretreated to phagocytic algiasctobacteria' mixture'

Tris 1.11 ± 0.20 1.81 ± 0.24Crude alginase 13.92 ± 1.10 7.43 ± 0.70 7.8Heated crude alginase 11.37 + 2.53 1.82 ± 0.26Pure alginased 12.44 ± 1.04 6.94 ± 0.64 97.0Heated pure alginase 7.27 00.70 1.75 ± 0.25

a Bacteria were treated with alginase (125 U of crude alginase per ml or 15.7U of pure alginase per ml), heated alginase, or Tris at 37°C for 2 h in thepresence of 2 mM Mg2+ prior to phagocytosis by M4F as described in the text.Values are the averages of three experiments.

b Alginase (12.5 U of crude alginase per ml or 1.57 U of pure alginase perml), heated alginase, or Tris was added to the phagocytic mixture. Thenumber of bacteria per M4 was determined after 1 h at 37°C. Values are theaverages of three experiments.

Specific activity is expressed as units of alginase per milligram of protein.d Partially purified alginase was eluted from a DEAE-Sephacel column (see

the text for details).

were treated with native alginase. The enzyme appeared todegrade MEP from all eight organisms regardless of theirsubsequent susceptibility to phagocytosis by M+. The extentof release of TBA-positive material was not correlated withthe susceptibility of the organisms to phagocytosis. Forexample, strain C1712M, which was very well phagocytosedby M4) after alginase treatment, had the lowest release ofTBA-positive material of all strains tested.

Immunofluorescence of alginase4treated bacteria. The effectof alginase on three mucoid P. aeruginosa strains wasmeasured by indirect immunofluorescence, using a monoclo-nal antibody to MEP (Table 6). All three strains showedintense imnmunofluorescence with a minimally fluorescentbackground under control conditions. After alginase treat-ment, fluorescence of the bacteria and the background(MEP) was substantially decreased in both strains C1900Mand LM3M. Alginase treatmnent enhanced phagocytosis ofboth of these strains. Fluorescence was only minimallydecreased after alginase treatment of strain C2146M; phago-cytosis of this strain was not enhanced by alginase. Heat-treated alginase had little or no effect on the immunofluores-cence of any of the three strains.

DISCUSSIONAlginate-degrading enzymes from different sources have

been shown to digest the MEP of mucoid P. aeruginosa (8,

TABLE 5. Effect of alginase on release of MEP from mucoidP. aeruginosa strains, as measured by the TBA method

Release of MEPa No. ofStrain

Alginase Tris Heated alginase expts

C1712M 1.32 ± 0.42 0.00 ± 0.00 0.00 ± 0.00 4C1628M 7.82 ± 1.15 0.02 ± 0.01 0.04 ± 0.02 6C1900M 9.44 ± 2.89 0.00 ± 0.00 0.23 + 0.16 7LM3M 3.43 + 0.29 0.01 ± 0.01 0.06 ± 0.06 3C2141M 2.90 ± 0.54 0.08 t 0.03 0.61 ± 0.16 4C2101M 6.65 ± 1.93 0.00 ± 0.00 0.02 ± 0.02 4C2146M 9.74 ± 1.59 0.00 ± 0.00 0.30 ± 0.30 4C2208M 11.50 ± 1.80 0.00 + 0.00 0.22 + 0.22 2

a Release of MEP from P. aeruginosa by alginase was measured by theTBA assay. The values represent nanomoles (mean ± standard error) ofbeta-formylpyruvic acid liberated (measured as optical density at 549 nm).Release of 0.01 micromoles of beta-formylpyruvic acid results in an increaseof optical density at 549 nm of 0.29. Bacteria were treated with alginase (125U/ml), heat-inactivated alginase (125 U/ml), or Tris at 37°C in the presence of2 mM Mg2+ prior to phagocytosis by M<4. See the text for details.

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TABLE 6. Immunofluorescence of alginase-treatedmucoid P. aeruginosa

Bacteria treated with":Strain

Tris Alginase Heated alginase

C1900M 4+ 1+ 3+LM3M 3+ 1+ 2-3+C2146M 4+ 3+ 3-4+

a Bacteria were incubated with a monoclonal antibody to MEP and thenwith fluoresceinated anti-immunoglobulin G. See the text for details. Thebacteria were treated with alginase (125 U/ml), heat-inactivated alginase (125U/ml), or Tris for 2 h at 37°C prior to immunofluorescent staining. Theintensity of fluorescence was graded from 1 to 4+.

10, 15, 28). MEP in its native, acetylated form is lesssusceptible to the action of alginase than when it is deacety-lated (15). Nevertheless, both forms have been shown to bedegraded to various degrees by mannuronic acid-specificalginases (8, 15).

In the current investigation, we showed that an alginasefrom B. circulans was capable of degrading MEP from fourmucoid strains of P. aeruginosa from CF patients. TheseMEPs were in their native form and were therefore acetyla-ted. Alginase treatment of mucoid P. aeruginosa cells re-sulted in degradation of the surface MEP of all eight teststrains and enhancement of phagocytosis by Mi for five.The enzyme removed the MEP from the bacterial cellsurface as measured by a chemical assay (TBA) and immu-nofluorescence staining of the MEP before and after alginasetreatment. The specificity of alginase for digestion of MEPfrom mucoid P. aeruginosa was examined by using threenonmucoid isolates from the patient from whom strainC1900M was isolated. Neither alginase nor heat-treatedalginase enhanced phagocytosis of any of these nonmucoidbacteria compared with phagocytosis of Tris controls, andthere was no TBA-positive material released (unpublisheddata).Although alginase degraded MEP from the surface of all

eight P. aeruginosa strains, phagocytosis was enhanced foronly five. This may have been due to differences amongstrains in the ratio of mannuronic acid to guluronic acid and/or the degree of acetylation of the different polysaccharides.Mucoid P. aeruginosa strains have been shown to vary inthe ratio of the two uronic acid constituents; since 0-acetylgroups are associated only with mannuronic acid residues,the degree of acetylation also varies from strain to strain (22,23). MEPs with a higher percentage of mannuronate residueswould likely be more richly acetylated and more resistant toalginase degradation.The quantity of MEP on the surface of bacteria varies

among strains, and large amounts of MEP could be moredifficult to degrade with alginase. This may explain whythree strains resisted phagocytosis even though their MEPswere degraded. Strains C2101M and C2146M producedcopious amounts of polysaccharide, as judged by the viscos-ity of the supernatant after each wash and the difficulty ofsuspending the bacterial pellets, compared with the othermucoid organisms studied. Both of these strains resistedphagocytosis both before and after alginase treatment. Wedid not quantitate the amount of MEP or the degree ofacetylation of MEP in the organisms tested. Chemical anal-ysis of the MEPs from these strains may provide a betterunderstanding of their relative susceptibilities to enzymaticdigestion.

Heat-inactivated crude or partially purified alginase wasincapable of degrading the bacterial surface MEP but en-

hanced phagocytosis by M4 when bacteria were preincu-bated with the enzyme for 2 h prior to phagocytosis exper-iments. On the other hand, when heated alginase was addeddirectly to the phagocytic mixture, the results were similar tothose for the Tris controls. These results suggest that theenhancement of mucoid P. aeruginosa phagocytosis aftertreatment with heated alginase is nonspecific. The 2-h incu-bation of bacteria and alginase perhaps allows these organ-isms to be coated by the denatured proteins or secretedbacterial products, which may make them susceptible tononspecific uptake by MP. In fact, preincubation of bacteriawith other proteins such as bovine serum albumin alsoenhances bacterial uptake by M4X to some degree (unpub-lished data). The heat-inactivated alginase might simply havealtered the hydrophobic and/or electrostatic properties of thebacterial surface, thereby enhancing susceptibility to nonop-sonic phagocytosis (25). Nevertheless, the native alginasewas superior to the heat-inactivated alginase in enhancingphagocytosis for each of the five strains (P < 0.0001).The alginase used in this study resembles the mannuroni-

dase from B. circulans JBH2 characterized by Hansen et al.(10). We have cloned the alginase gene in Escherichia coli,but gene expression is poor (F. Eftekhar, W. Paranchych,and D. P. Speert, Abstr. Annu. Meet. Am. Soc. Microbiol.1987, B258, p. 67). Other alginases with different specificities(guluronidases versus mannuronidases) may prove to have awider range of activity against mucoid P. aeruginosa. Weare also screening CF isolates of P. aeruginosa for alginaseproduction. It has been found that alginase production isgreater in mucoid than in nonmucoid strains of P. aerugi-nosa (8, 15). In situ production of alginase by mucoid P.aeruginosa could modulate the severity of CF lung infec-tions by decreasing the mass of MEP in respiratory secre-tions. Alginase-producing bacteria may therefore prove to beless virulent by being more susceptible to phagocytosis andkilling by pulmonary phagocytes.

ACKNOWLEDGMENTS

Lisa Thorson provided excellent technical assistance. The gift ofmonoclonal antibodies from Gerald Pier is greatly appreciated. Wealso thank Martin Puterman for statistical analysis of the data.

This work was supported by grants to D.P.S. from the CanadianCystic Fibrosis Foundation, the Medical Research Council of Can-ada, and the British Columbia Medical Services Foundation. D.P.S.is a Scholar of the Canadian Cystic Fibrosis Foundation.

LITERATURE CITED1. Baltimore, R. S., and M. Mitchell. 1980. Immunologic investi-

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