Vol. 30, No. 2INFECTION AND IMMUNITY, Nov. 1980, p. 4024080019-9567/80/1 1-0402/07.$02.00/0

Slime Glycolipoproteins and the Pathogenicity of VariousStrains of Pseudomonas aeruginosa in Experimental Infection

G. DIMITRACOPOULOS'2 AND P. F. BARTELL'*Department of Microbiology, College ofMedicine and Dentistry of Neu, Jersey-Neu Jersey Medical School,Newark, New, Jersey 07103,' and Department of Microbiology, Faculty ofMedicine, Uniquersity ofAthens,

Athens, Greece2

Several strains of Pseudomonas aeruginosa were differentiated on the basis ofthe surface properties of the cells. Fisher immunotype, phage type, polysaccharidedepolymerase type, and indirect hemagglutination reactions were used for thispurpose. Each strain was then studied with respect to events known to occurduring the experimental infection of mice with P. aeruginosa. The virulence ofthe viable cells varied significantly, although all strains were virulent. Glycolipo-proteins were isolated from the slime of each strain, and they appeared similarchemically when they were analyzed for gross composition. The toxicity of theisolated glycolipoproteins varied insignificantly, except for that of one strain.Viable cells of each strain and their respective glycolipoproteins caused leuko-penia, which occurs in the course of the lethal infection. The antisera to theglycolipoproteins protected mice in every case against infection by the homolo-gous strains. In some cases, various degrees of cross-protection were observed.

Although the lethal events that follow exper-imental Pseudomonas aeruginosa infection ofmice are far from completely clear, some of thefactors that influence the course of events havebeen identified. The cumulative evidence indi-cates that the slime layer of the viable cellcontributes significantly to the pathogenesis andthat specifically it is the glycolipoprotein (GLP)that is the active component of the slime layer.The experimental infection of mice with strainBI has provided an especially informative line ofinvestigation in this regard. The isolated slimelayer of strain BI produces lethal effects wheninjected into mice, including a sequence ofevents comparable with those produced by theviable cell (5). The significance of this findingwas enhanced when it was demonstrated thatthe viable cell synthesizes slime in the infectedmouse and that the course of the lethal eventsis a function of the de novo synthesis and dis-persion of slime by means of the peripheralcirculatory system (9).

Furthermore, a GLP of reproducible physico-chemical characteristics can be isolated fromthe slime of strain BI (19). The GLP injected inmice produces precisely the pathogenic effectsof the viable cell, including leukopenia and in-hibition of phagocytosis (19). In addition, GLPacts as a specific antigen in rabbits, stimulatingthe appearance of an anti-GLP serum capable ofprotecting mice against the leukopenia, the in-hibition of phagocytosis, and the eventual le-thality caused by viable cells of the homologousstrain (21). The distinction of slime GLP from

endotoxin has been demonstrated (19). Re-cently, this distinction was corroborated indi-rectly by the work of Pier et al. (16). Using apolysaccharide depolymerase with a demon-strated substrate specificity in the GLP of strainBI (3, 6), they showed that lipopolysaccharidefrom their strain of P. aeruginosa is resistant tothe depolymerase, whereas the antigenicity of ahigh-molecular-weight polysaccharide from theslime of the same strain is destroyed by thedepolymerase.With these relationships well established, it

became possible to trace some of the steps in theinteraction of viable P. aeruginosa cells with thehost during the course of lethal infection. Thus,it has been demonstrated that GLP reducescirculating polymorphonuclear leukocytes, spe-cifically the neutrophils, thus creating a neutro-penic condition in the animal (14). Further evi-dence indicates that the neutropenia resultsfrom a selective attachment of GLP to neutro-phils, with the liver subsequently clearing thecoupled neutrophil from the circulation.Among other remaining problems is that of

determining how widely this model is applicableas an explanation of the lethal events in experi-mental P. aeruginosa infection. The presentreport deals with aspects of this problem. Sev-eral strains of P. aeruginosa were distinguishedwith respect to variables related to the surfacestructure: Fisher immunotype, phage-receptorspecificity, polysaccharide-depolymerase sub-strate specificity, and antigenic individuality.The biological activity of GLP from each strain

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SLIME GLYCOLIPOPROTEINS OF P. AERUGINOSA 403

was then studied and compared with the modelstrain BI system. The lethal events in every caseparalleled those of the model. Furthermore, theGLP provided the basis for passive protection inevery case against infection by the homologousviable cell.

MATERIALS AND METHODSOrganisms. P. aeruginosa strains BI and EI were

originally isolated from human clinical specimens andhave been described previously (4, 6). Strains 1547,1549, 1555, and 1556 were supplied by Joseph A. Bass(University of Texas, San Antonio). The Fisher im-munotypes of strains BI and EI were determined byViola Mae Young (National Cancer Institute, Balti-more, Md.) and those of the other strains, by Dr. Bass.

Bacteriophages and bacteriophage depo-lymerases. The temperate Pseudomonas phages andtheir associated depolymerases were isolated from ly-sogenic cells and purified by methods already de-scribed (3, 4). Bacteriophage typing was performed bythe method of Adams (1). Large quantities of depo-lymerases were obtained from soft-agar overlays ofphage-infected cultures. Harvested cultures weretreated with 10% chloroform and clarified by centrif-ugation. High-speed centrifugation was used to sedi-ment the phage particles. Depolymerases were precip-itated from the supernatant fluids with ammoniumsulfate at a concentration of 45% saturation. The sed-imented precipitates were dissolved in 0.1 M sodiumphosphate buffer (pH 7.5) at approximately 4% of theinitial volume. After dialysis in the cold until thedialyzing buffer was negative for sulfate, the prepara-tions were stored in 1.0-ml amounts at -20'C.GLP. GLP fractions were obtained from the extra-

cellular slime layers of the strains as indicated andwere purified by methods described previously (5, 19).Slime was extracted in 0.15 M NaCl from 18-h bacte-rial cultures grown on sheets of cellophane overlayingTrypticase soy agar (BBL Microbiology Systems,Cockeysville, Md.). The extract was precipitated withethanol, clarified by centrifugation, and dialyzedagainst distilled water. The dialysate was centrifugedat 105,000 x g for 3 h, and the supernatant fluid, whichcontained the GLP fraction, was lyophilized. The GLPwas then filtered through gels and subjected to anion-exchange chromatography, in which it was eluted at aKCl molarity of 0.3 to 0.4. Purity and homogeneitywere demonstrated by chromatography, sedimenta-tion pattern, and immunodiffusion (19). LyophilizedGLP was stored in vacuo at 40C. Chemical analyseswere determined on independently prepared lots bystandard methods. Hexoses were determined by theanthrone reaction (22); hexosamines, by the methodof Belcher et al. (7); protein, by the method of Lowryet al. (13); and lipid, by the gravimetric procedure ofSalton (17).Animals. White male rabbits weighing 3 to 4 kg

were housed individually. White male Swiss miceweighing 18 to 20 g were housed 10 per cage. Allanimals were supplied with water and Purina chow adlibitum.Anti-GLP sera. On day 0, rabbits received subcu-

taneous injections in four separate sites for a total of

2 mg of GLP in equal volumes of 0.01 M phosphate-buffered saline (PBS; pH 7.2) and Freund incompleteadjuvant. Groups of three rabbits were bled 18 to 20days after immunization, and the pooled sera werestored at -20°C. Indirect hemagglutination titers re-mained undiminished for more than 1 year. These seraproduced a single band when allowed to react withcrude cellular extracts in gel (Special Noble agar, DifcoLaboratories, Detroit, Mich.) immunodiffusion tests.Animal challenge. Bacterial cells were prepared

as described previously (9). Briefly, 18-h cultures werewashed from Trypticase soy agar slants, sedimented,washed once, and suspended finally in 0.01 M PBS(pH 7.2) at a turbidity equivalent to 1 x 10'( to 5 x10"' viable cells per ml. Colony counts on Trypticasesoy agar were made on each experimental suspensionto determine the numbers of viable bacteria. Themethod of Miller and Tainter (15), employing probitvalues, was used to determine the mouse 50% lethaldose (LDro) for viable cells and GLPs. Groups of 10white male Swiss mice (weighing approximately 20 geach) were injected intraperitoneally with 0.5 ml ofeach of five twofold dilutions of the bacteria or GLP.Animals were observed daily for up to 7 days. Inpassive experiments mice were passively immunizedwith 0.5 ml of rabbit anti-GLP serum injected intra-peritoneally 3 h before bacterial challenge with ap-proximately five LD-,'s. Protective capacity is ex-pressed as the percent survival 7 days after bacterialchallenge. All experiments were repeated three to fivetimes.

Leukocyte counts. Leukocyte counts were madeon peripheral blood collected retroorbitally as re-ported previously (19). The results are presented asaverages for groups of five mice.

Indirect hemagglutination inhibition. Hemag-glutinating activity of the sera was determined indi-rectly as described previously (9). In brief, formalinizedsheep erythrocytes (Difco Laboratories) were sensi-tized with GLP (200 ag/ml) in PBS at room tempera-ture for 30 min. After several washes in PBS, thesensitized erythrocytes were resuspended in PBS to aconcentration of 5% (packed vol/vol) and were addedin 0.05-ml amounts to serial dilutions (0.5 ml) of theappropriate serum preparation. Titers are expressedas the reciprocal of the highest dilution producing apositive hemagglutination pattern after 2 h at roomtemperature. The inhibitory activity of the GLP prep-arations was determined by indirect hemagglutinationinhibition. The GLP sample (10 ug in 0.1 ml) wasadded to 0.5-ml serial dilutions of the appropriate anti-GLP sera. After incubation at room temperature for60 min, 0.05 ml of sensitized sheep erythrocytes wasadded to each tube, and the indirect hemagglutinationinhibition titer was determined after a 2-h incubationperiod at room temperature.

RESULTSSurface characteristics. Although several

systems are available for serotyping strains ofP. aeruginosa, we chose the immunotypeschema of Fisher et al. (10) for our initial selec-tion of strains for study for two reasons. Sinceone of our primary aims was to investigate the

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404 DIMITRACOPOULOS AND BARTELL

protective antigens of a group of P. aeruginosa,we found in the Fisher system a basis for assur-

ing a selection of diverse protective capacities.Secondly, 94% of the bacterial cultures studiedby Fisher were typable by the schema. There-fore, it seemed reasonable that a selection ofstrains based on that schema would also repre-sent a fair sample of the range of P. aeruginosastrains isolated from clinical sources. In addition,to ensure further the variety of surface charac-teristics in our study group, we reduced dupli-cation of serotypes through use of other criteriaof surface diversity. Thus, by means of a groupofphages isolated in this laboratory from clinicalsources, we were able to distinguish a group ofstrains that differed additionally through phagetype (Table 1). Another means of differentiationwas available through the use of phage 2 and 8depolymerases. The specific substrates for thesedepolymerases have been shown to exist in theslime layers of a very restricted number of P.aeruginosa strains (4). Strains El, 1547, and1556 have the same phage type even thoughthey are distinguishable on the basis of immu-notype. For these three strains, phage 8 depo-lymerase allowed a further differentiationthrough the presence of substrate in strain El.What is more, this property distinguishes strainEL from all other strains in this experimentalgroup.

Strains 1555 and 1556 are, respectively, Fisherimmunotypes 3 and 7. These immunotypes are

reported to evoke cross-reacting antibody (10).Hence, the differing phage types of these strainsprovide additional assurance of the surface dif-ferences indicated by immunotype. The phagetypes of strains BI, 1549, and 1555 were clearlydifferent, and strain 1554 was nontypable. Thus,each strain was distinguishable on the basis ofat least one surface property, and all but onewere distinguishable on the basis of more thanone property.The purified GLPs from each strain were an-

TABLE 1. Surface characteristics of various strainsof P. aeruginosa

PhageFisher Phage receptor depo-

Strain immu- lymer-

notype

2 8 23 29 34 67 75 84 2 8

RI 1 + .+ - + -

El 6 + + - - +

1547 4 + + - - -

1549 5 + + + + + - -

1554 2

1555 3 .-- + -

1556 7 L + I + I

alyzed chemically and were found to containhexose, hexosamine, lipid, and protein (data notshown). However, these analyses were not usefulfor strain differentiation because no qualitativedifferences were observed from strain to strain.Certain quantitative differences were obtained,but they did not enhance significantly the dis-tinctions already made by other methods.Antigenic relatedness of slime GLPs. Pre-

vious studies of the role of the slime layer inexperimental infections have suggested that an-tigenic diversity exists among GLPs extractedfrom slime (8). The antigenic diversity can bedemonstrated even though the GLPs evoke sim-ilar patterns of leukopenia and toxicity. Becausethe strains used in the present study were se-lected on the basis of surface diversity, it wasexpected that they would also exhibit a corre-sponding antigenic diversity. Such were the re-sults when rabbit antisera raised against purifiedGLPs were tested against homologous and het-erologous slime inhibitors in an indirect hemag-glutination inhibition system (Table 2). As ex-pected, homologous GLP by far showed thestrongest inhibition and showed inhibition inevery instance. Heterologous GLP inhibitors,even at their most effective (for example, theGLP from strain BI against anti-strain 1549 GLPserum), were 32-fold less active than the homol-ogous strains. Note particularly that strains 1547and 1556, which showed some evidence of sur-face relatedness by the criteria which we usedfor the initial selection of the present strains(Table 1), are readily distinguishable antigeni-cally.Virulence of strains. The capacity of each

TABLE 2. Antigenic relatedness of slime GLPsobtained from various strains of P. aeruginosa asdetermined by indirect hemagglutination inhibition

(HAI)Antise- Titer Indirect HAI indexb with indicated GLPrum with- inhibitorraised out

to in-GLP hibi- BI El 1547 1549 1554 1555 1556from: tore

BI 256 128 4 2 2 1 2 4EI 256 1 128 1 1 1 2 41547 256 2 4 128 4 1 4 41549 256 4 4 2 128 1 4 41554 128 1 1 1 1 128 1 11555 128 2 2 2 2 1 128 41556 256 1 4 1 1 1 1 256" Reciprocal of the indirect hemagglutination titer

without inhibitor." The index was calculated by dividing the reciprocal

of the indirect hemagglutination titer by the reciprocalof the indirect HAI titer. Both titers were determinedas described in the text.

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SLIME GLYCOLIPOPROTEINS OF P. AERUGINOSA 405

strain to produce a lethal infection was assessedby the intraperitioneal injection of viable cellsinto mice (Fig. 1). Variations in virulence ofmore than a log were found between somestrains. Of the seven strains tested, strain BIappeared to be the least virulent, requiring an

average of 6.8 x 108 viable cells to kill 50% of themice injected. Most virulent was strain 1556, forwhich the average LDro was significantly lowerat 3.3 x 107 viable cells than that of strain BI (P< 0.01). The average LDro's for the other fivestrains were distributed between these extremes.Strains El and 1549 fell somewhat below strainBI, and together they formed the least virulentgroup. Strains 1554 and 1555 were close to strain1556 as the most virulent group. Strain 1547occupied an intermediate position with regardto these groups of relatively high and low viru-lence.Toxicity of slime GLP. Earlier reports from

this laboratory have demonstrated that GLPextracted from the exopolysaccharide of theslime from strains BI and El can act as a viru-lence factor in the experimental mouse infection

10br

-

-j

0

z 8coanEo0

W7BI El 1547 1549 1554 1555 1556

STRAIN DESIGNATION

FIG. 1. Mouse LDr,( of viable cells of variousstrains of P. aeruginosa. Each point represents thenumber of viable cells per LDr,o as calculated from asingle experiment; the horizontal bar represents thearithmetic mean of the five values.

(9, 19). Accordingly, GLP was extracted fromeach of the representative strains under studyhere. All the extracted GLPs showed toxic ef-fects when they were injected intraperitoneallyinto mice (Fig. 2). With the exception of strain1547, all the average LD-,o values were groupedbetween 23.0 and 31.4 Ag per g of mouse, withstrains 1556 and 1554 occupying the lower andupper boundaries, respectively. None of the var-iations in this group were significant. However,the average LDi, for strain 1547 was estimatedat 46.0 Ig per g of mouse, which indicated sig-nificantly less toxicity (P < 0.01) than for any

other strain tested.Leukopenia. Leukopenia characteristically

occurs in the peripheral circulation of the mouseimmediately after intraperitoneal injection witha lethal dose of viable cells of strain BI, and itspersistence constitutes an event of decisive im-portance for the survival of the infected animal(19). Therefore, it was necessary to determinewhether leukopenia was also characteristic oflethal infection with the present strains. Theresults (Fig. 3) indicate that all seven strainsproduced a similar degree of leukopenia, follow-ing a similar time course, as a result of the samelethal dose of5 LDro ofviable cells. Furthermore,most of the mice exhibiting leukopenia diedduring the 24-h period after injection, and theremaining 10 to 20% died by the end of the next24-h period. Thus, none of the strains differed inthe capacity ofviable cells to produce leukopeniaduring the course of a lethal infection.

60

50-

40

A, 30enmo

20

10

BI EI 1547 1549 1554

STRAIN DESIGNATION

1555 1556

FIG. 2. Mouse LD5,, for the slime GLPs from var-

ious strains of P. aeruginosa. Each point representsthe LDrA as calculated from a single experiment; thehorizontal bar represents the arithmetic mean of thefive values.

I

- Is~~~~~~

* 0

0

I 1 1 1

S~~~~~~~~

-0

LU

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406 DIMITRACOPOULOS AND BARTELL

8.

0

EE

CL0~z

ui

0IC-

-i

o

CONTROL

So

Ecr.ILJI3-

C-)

CoCT ISTRAIN ui

CONTROL)PBS

STRAIN

0 1 3 5 0 1 3 5HOURS HOURS

FIG. 3. Leukopenia in the peripheral circulation FIG. 4. Leukopenia in the peripheral circulationof mice after intraperitoneal injection of viable cells ofmice after intraperitoneal injection ofpurified GLP(5 LD5, ofP. aeruginosa strains. (50 fig per g of mouse) from various strains of P.

aeruginosa.Similarly, purified GLP from strain BI in ap-

propriate doses provokes the onset of a leuko-penia resembling that produced by the viablecell (19). Accordingly, purified GLP from eachstrain was investigated for its capacity to reducecirculating leukocytes. The results indicate thateach strain had this capacity (Fig. 4). However,no significant differences could be detected fromstrain to strain.Passive protection. In the context of the

present study, one of the most important prop-erties of the extracted GLP is its demonstratedcapacity to elicit the production of antibody thatprotects mice against experimental challenge byviable cells of the strain from which the GLPwas derived (19). We were greatly interested indetermining whether the present strains pos-sessed a similar protective potential. Accord-ingly, mice were challenged with 5 LDro of viablecells after passive immunization with antisera topurified GLPs. Specific rabbit anti-GLP serawere able to protect 70 to 90% of the micechallenged with the homologous strain (Table3). Although there was cross-protection betweenstrains, each antiserum protected most effec-tively against the homologous strain. However,cross-protection of various degrees was ob-served, and in some cases it reached considerablelevels: anti-strain EI-GLP serum protected

TABLE 3. Immunogenic relatedness of slime GLPsobtained from various strains of P. aeruginosa, as

determined by mouse passive protectionChal- Survival (%) of mice passively immunized withlenge rabbit anti-GLP sera

strain (5LD,,) BI EI 1547 1549 1554 1555 1556 NRS"

BI 90 0 30 40 20 10 0 0EI 0 80 0 10 0 50 50 01547 10 0 90 40 0 30 0 01549 30 30 30 70 0 10 20 01554 20 0 10 40 90 10 10 01555 0 60 0 0 0 80 70 01556 0 70 0 10 0 40 80 0

" Normal rabbit serum.

against viable cells of strains 1555 (60%) and1556 (70%), anti-strain 1555-GLP serum pro-tected against strain EI cells (50%), and anti-strain 1556-GLP serum protected against cells ofstrains 1555 (70%) and EI (50%). There were alsoslight indications ofminor cross-protective activ-ity of other antisera against heterologous strains.

DISCUSSIONThe extracellular slime layer of P. aeruginosa

is a characteristic of the species. For this reason

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SLIME GLYCOLIPOPROTEINS OF P. AERUGINOSA 407

there has been for many years a special interestin its possible role as a virulence factor in thepathogenesis of P. aeruginosa infection (2, 12,18). With the application ofpurification methodsto the extraction of slime, it has been possible todemonstrate the homogeneity of purified slimeand identify its major chemical components (19)and to demonstrate that the GLP of strain BIacts as a mediator of virulence and as a protec-tive antigen (21). Considerable interest now cen-ters on the question of whether GLP influencesthe course of infection in strains other than BI.In the present study we addressed ourselves tothis question.A group of P. aeruginosa strains was selected

for comparison with the model system on thebasis of immunotype and other methods thatdepend on surface properties. With the distinc-tiveness of the strains assured, we characterizedsome of the effects of injection of mice with theviable cells and the extracted GLP ofeach strain.The LD50 values for viable cells ranged over afull log, whereas the LD5o values for the GLPsvaried only insignificantly from one to the other,with the exception of strain 1547. Leaving asidestrain 1547 for the moment, the reader may havenoted the occasional lack of correlation betweenthe lethality of the viable cell and that of theGLP. The toxicity of the GLPs from strains BI,El, and 1549, for example, is essentially the sameas for the other strains; however, the lethality ofthe viable cells was less than that of the otherviable cells. We previously observed an occa-sional lack of correlation between the lethalityof a viable cell and the toxicity of the homolo-gous GLP (8). No experimentally supported ex-planation yet exists, and several possible eventscould cause the phenomenon. In general, therelatively reduced lethality of a viable cell incomparison with other strains for which theGLPs possess the same level of toxicity may beexplained by variations in the rate of GLP pro-duction. Thus, the less lethal cell may simplyproduce less GLP.For strain 1547, however, another explanation

must be sought, for its GLP was significantlyless toxic. Furthermore, its reduced toxicity didnot correlate with the lethality of the viablecells, which exhibited an intermediate value be-tween high and low extremes. Possibly the GLPof strain 1547 differs structurally to a sufficientdegree to affect its pathological capacity, at leastquantitatively, whereas the rate of GLP produc-tion is sufficiently high so that the lethality ofthe viable cell appears similar to that of otherstrains.Leukopenia has been identified as a crucial

event in the lethal experimental infection of themouse with viable cells (19). Furthermore, its

occurrence as one of the events preceding deathhas been rationalized in a hypothetical modelthat accounts for both the leukopenia and theaccumulation of GLP in the liver of fatally af-fected animals after inoculation with viable cells(14). Accordingly, the occurrence of leukopeniawas investigated and demonstrated after infec-tion with viable cells of each strain and afterinjection of the GLP isolated from each strain.

Antisera to the GLPs provided a considerabledegree of protection against infection with someheterologous strains, particularly anti-strain1555 and anti-strain 1556 sera against strain Elcells. Tempting as it is to suggest that a commonantigen may have been made active here, wereserve judgment on this point until the activefragments of the GLP can be identified moreprecisely. For strain BI, this laboratory has re-ported a series of experiments that suggest thatthe carbohydrate moiety of the GLP molecule isresponsible for antigenicity and inhibition ofphagocytosis and the lipid moiety is responsiblefor leukopenic and lethal effects (20).

Clearly, in some, perhaps many, strains of P.aeruginosa, the slime layer contributes heavily,perhaps decisively, to the pathogenesis of Pseu-domonas infection. Although the strains inves-tigated here vary definitively with respect toseveral surface variables, the purified GLP fromeach strain caused leukopenia and eventualdeath. This supports the view that the GLPpossesses identical or similar pathogenic prop-erties regardless of the strain of origin. Quitepossibly, the pathogenic mechanism involves ad-herence ofGLP to the neutrophilic polymorpho-nuclear leukocytes and sequestration of theGLP-neutrophil complex in the liver with theconsequence of reduced nonspecific host defen-ses, as previous studies from this laboratory haveindicated for strain BI (14).

It has been suggested that other factors mayalso play a role in the lethal effects of infectionwith viable P. aeruginosa cells (11). Exotoxin Aand proteases have been frequently advancedrecently as the likely sources of Pseudomonasvirulence. Significantly, in the present study,mice passively immunized with anti-GLP serumraised against purified homogeneous GLP werefully protected against a lethal challenge withan untreated suspension ofviable organisms thatwould have contained labile toxic substancessuch as exotoxin A and proteolytic enzymes ifthese substances had been synthesized. More-over, in the present study, as well as in ourprevious studies, our methods of preparation ofGLP systematically exclude the presence of anyother toxic substance reported to date (14).Therefore, the results of this study support ourprevious conclusion that the GLP is an impor-

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408 DIMITRACOPOULOS AND BARTELL

tant pathogenic product of the P. aeruginosacell.

ACKNOWLEDGMENTSWe thank Thomas Orr for a critical reading of the manu-

script and editorial assistance. We thank Sara Lathi for hertechnical contribution.

This investigation was supported by Public Health Servicegrant AI-08504 from the National Institute of Allergy andInfectious Diseases.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Interscience Pub-lishers, Inc., New York.

2. Alms, T. H., and J. A. Bass. 1967. Immunization againstPseudomonas aeruginosa. II. Induction of protectionby an alcohol-precipitated fraction from the slime layer.J. Infect. Dis. 117:249-256.

3. Bartell, P. F., G. K. H. Lam, and T. E. Orr. 1968.Purification and properties of polysaccharide depolym-erase associated with phage-infected Pseudomonasaeruginosa. J. Biol. Chem. 243:2077-2080.

4. Bartell, P. F., and T. E. Orr. 1969. Distinct slime poly-saccharide depolymerases of bacteriophage-infectedPseudomonas aeruginosa: evidence of close associationwith the structured bacteriophage particle. J. Virol. 4:580-584.

5. Bartell, P. F., T. E. Orr, and B. Chudio. 1970. Purifi-cation and chemical composition of the protective slimeantigen of Pseudomonas aeruginosa. Infect. Immun. 2:543-548.

6. Bartell, P. F., T. E. Orr, and G. K. H. Lam. 1966.Polyvsaccharide depolymerase associated with bacterio-phage infection. J. Bacteriol. 92:56-62.

7. Belcher, R., A. J. Nutten, and C. M. Sambrook. 1954.The determination of glucosamine. Analyst (London)79:201-208.

8. Dimitracopoulos, G., and P. F. Bartell. 1979. Phage-related surface modifications of Pseudomonas aerugi-nosa: effects on the biological activity of viable cells.Infect. Immun. 23:87-93.

9. Dimitracopoulos, G., J. W. Sensakovic, and P. F.Bartell. 1974. Slime of Pseudomonas aeruginosa: in

vivo production. Infect. Immun. 10:152-156.10. Fisher, M. W., H. B. Devlin, and F. J. Gnabasik. 1969.

New immunotype schema for Pseudomonas aerugi-nosa based on protective antigens. J. Bacteriol. 98:835-836.

11. Liu, P. V. 1974. Extracellular toxins of Pseudomonasaeruginosa. J. Infect. Dis. Supply. 130:594-99.

12. Liu, P., Y. Abe, and J. Bates. 1961. The role of variousfractions of Pseudomonas aeruginosa in its pathogen-esis. J. Infect. Dis. 108:218-228.

13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

14. Lynn, M., J. W. Sensakovic, and P. F. Bartell. 1977.In vivo distribution of Pseudomonas aeruginosa slimeglycolipoprotein: association with leukocytes. Infect.Immun. 15:109-114.

15. Miller, L. C., and M. L. Taintor. 1944. Estimation of theE.D.50 and its error by means of logarithmic-probitgraph paper. Proc. Soc. Exp. Biol. Med. 57:261-264.

16. Pier, G. B., H. F. Sidberry, S. Zolyomi, and J. Sadoff.1978. Isolation and characterization of a high-molecu-lar-weight polysaccharide from the slime of Pseudom-onas aeruginosa. Infect. Immun. 22:908-918.

17. Salton, M. R. J. 1953. Studies of the bacterial cell wall.IV. The composition of the cell walls of some gram-positive and gram-negative bacteria. Biochim. Biophys.Acta 10:512-523.

18. Schwartzmann, S., and J. Boring. 1971. Antiphago-cytic effect of slime from a mucoid strain of Pseudom-onas aeruginosa. Infect. Immun. 3:762-767.

19. Sensakovic, J. W., and P. F. Bartell. 1974. The slimeof Pseudomonas aeruginosa: biological characteriza-tion and possible role in experimental infection. J. In-fect. Dis. 129:101-109.

20. Sensakovic, J. W., and P. F. Bartell. 1975. Biologicalactivity of fragments derived from the extracellularslime glycolipoprotein of Pseudomonas aeruginosa. In-fect. Immun. 12:808-812.

21. Sensakovic, J. W., and P. F. Bartell. 1977. Glycolipo-protein from Pseudomonas aeruginosa as a protectiveantigen against P. aeruginosa in mice. Infect. Immun.18:304-309.

22. Spiro, R. G. 1966. Analysis of sugars found in glycopro-teins. Methods Enzymol. 8:3-26.

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