Vol. 23, No. 3INFECTION AND IMMUNITY, Mar. 1979, p. 863-8720019-9567/79/03-0863/10$02.00/0

Relationship Between Superoxide Dismutase and PathogenicMechanisms of Listeria monocytogenesD. F. WELCH,*t C. P. SWORD,4 S. BREHM, AND D. DUSANIC

Department ofLife Sciences, Indiana State University, Terre Haute, Indiana 47809

Received for publication 14 December 1978

Listeria monocytogenes was examined for superoxide dismutase (SOD) activity.Two catalase-negative strains possessed at least twofold greater SOD activitiesthan the catalase-positive L. monocytogenes strains examined. Growth conditionssuch as aeration and iron concentration influenced the specific activity of SODobtained from cells cultured in defined media. L. monocytogenes SOD from crudeextracts and after partial purification was analyzed by polyacrylamide gel electro-phoresis. Iron was associated with the single band ofSOD activity detected in thegels. SOD activity appeared to be primarily extracytoplasmic. Survival of orga-

nisms in a superoxide-generating medium was studied, with photoactivation ofriboflavin used as the source of free radical formation. Virulent, catalase-positiveL. monocytogenes strains were relatively resistant to killing in a pH 7 superoxide-containing medium. An intact-cell assay for SOD was developed, which used thesuperoxide-generating system and employed the superoxide-dependent oxidationof sulfite, added to the medium, and inhibition of this oxidation by SOD. MaximalSOD activities of intact cells were observed when 100 to 400 ,g (dry weight) ofviable Listeria cells per ml was added to the medium. A possible role for SOD inthe pathogenesis of listeric infection is discussed.

Facultative intracellular pathogens such asListeria monocytogenes must possess means ofovercoming the nonspecific immune responsesmediated by phagocytic cells and various hu-moral factors. The sequence in phagocytic kill-ing of bacteria includes the formation of a toxicsuperoxide (*02) free radical (5) which is latereliminated by the enzyme superoxide dismutase(SOD) (22). Indirect evidence also exists for arole of * 02 in leukocyte-mediated antimicrobialactivity. Baehner et al. (6) showed that thenitroblue tetrazolium reduction by leukocytes isdependent on *°02 generated by phagocytic cellmetabolism, and Curnutte et al. (11) observedthat neutrophils obtained from patients withchronic granulomatous disease produced low orundetectable quantities * °2SODs from Escherichia coli have been found

to contain manganese (16) or iron (31). Yost andFridovich (32) demonstrated that E. coli cellsgrown in iron-rich media are more resistant tokilling by phagocytes than E. coli grown in iron-deficient media. Increased resistance appearedto correlate with increased levels of iron-SODrather than with catalase or other effects of iron.Marked reduction ofLD50 values for mice occurs

t Present address: Department of Pathology, University ofUtah Medical Center, Salt Lake City, UT 84132.

t Present address: Graduate School, South Dakota StateUniversity, Brookings, SD 57007.

when iron compounds are injected at the timeof infection with L. monocytogenes (25). Viru-lence enhancement by iron has also been wellrecognized from experimental infections withseveral other bacteria (9). The present study wasundertaken to determine the nature of SOD inListeria with respect to iron and to assess thepotential of SOD in host-parasite interactions inlisteric infections.

MATERLALS AND METHODSOrganisms. The catalase-positive strains of L.

monocytogenes employed were A4413, 34-S, JHH, and9037-7. The catalase-negative L. monocytogenesstrains included 1370 and A2419. Strains A4413, JHH,and 9037-7 were obtained from the U.S. Army Biolog-ical Laboratories at Fort Detrick, Frederick, Md.Strain 34-S was an isolate of ovine origin (4). Strain1370 was obtained from the Center for Disease Con-trol, Atlanta, Ga., and strain A2419, from E. H. Kam-pelmacher, Utrecht, The Netherlands. The 50% aver-age lethal doses (LD50) for mice range from 103 to 104cells for strains A4413, 34S, and JHH and from 107 to10' cells for the catalase-negative strains; strain 9037-7 is avirulent at doses less than 109. Organisms weremaintained at -20'C on Difco tryptose agar slants.Stock slants grown at 370C for 18 to 24 h were frozenuntil used as inocula. Cultures to be grown with aera-tion were placed on a rotatory shaker (New BrunswickScientific Co., New Brunswick, N.J.) operated at 200rpm.

863

864 WELCH ET AL.

Culture media and reagents. All media and re-agents were prepared with double-distilled water. De-hydrated media (Difco Laboratories, Detroit, Mich.)were prepared according to the manufacturer's direc-tions. Defined Listeria medium (DLM) was preparedas described by Welshimer (29). It contained minimalsalts, glucose, amino acids, and vitamins, and wassupplemented where indicated with a membrane filter-sterilized (0.22 Mm) solution of purified ferric ammo-nium citrate (Mallinckrodt, St. Louis, Mo.) Selectivemedia chosen for enumeration of viable organismsincluded McBride Listeria agar.

Plate counts. Enumeration of the viable bacteriafrom survival studies was performed by the pour platemethod after serial dilution. Plates were incubated at370C for 24 to 48 h. Plates containing 30 to 300 colonieswere counted with a New Brunswick colony counter.

Determinations of protein and dry cell weight.Crude cell extract protein was measured by themethod of Lowry et al. (20), with bovine albumin(Miles Laboratories, Kankakee, Ill.) as a standard. Drycell weights were determined by collecting 1.0 ml ofwashed-cell suspensions on preweighed membrane fil-ters (0.22 jum; Millipore Corp., Bedford, Mass.). Thefilters were placed in a drying oven and maintained at60'C until a constant weight was established. Nodetectable changes in filter weights due to the heatexposure alone were observed.SOD assay. SOD assays were performed at am-

bient room temperature with minor modifications ofthe procedure described by McCord and Fridovich(22). Reaction mixtures contained 10-5 M ferricyto-chrome c (type III, from horse heart, Sigma ChemicalCo.), 5 x 10-5 M xanthine (Sigma), 0.05 M potassiumphosphate, i0' M ethylenediaminetetraacetic acid(EDTA), pH 7.8, and approximately 6 x 109 M xan-thine oxidase (Sigma, grade III from buttermilk). Suf-ficient xanthine oxidase was added to cause a rate ofcytochrome c reduction of 0.05 absorbance unit permin when measured at 418 nm. The rate of cytochromec reduction was recorded by use of a Gilford model2000 or a Gilford model 2400-S spectrophotometer.SOD inhibits the rate of cytochrome c reduction byxanthine oxidase, and 1 U of SOD activity is definedas the amount of enzyme causing 50% inhibition of therate of cytochrome c reduction.

Preparation of cell extracts. Cell extracts wereobtained by use of a French pressure cell (Aminco,Silver Spring, Md.). Cells were harvested in the latelogarithmic to early stationary phase of growth bycentrifugation at 10,400 X g for 15 min in a SorvallRC-2B refrigerated centrifuge. The cells were sus-pended and washed once. Washed cells were sus-pended in cold water (0 to 40C) to a density of 101) to101/ml and were disrupted at 40C by use of a cellpressure of 20,000 psi. Unbroken cells and debris weresedimented by centrifugation at 15,900 x g for 20 min.The supernatant fluid was removed and stored at 4°Cuntil used.

Partial purification of SOD. DLM or brain heartinfusion shake cultures (1 liter) of strain A4413, incu-bated for 18 to 24 h, were harvested, and cell extractswere prepared. The extract was diluted to approxi-mately 200 ml with distilled water, and 3 M KCl wasadded to a final concentration of 0.1 M. The solution

INFECT. IMMUN.

was placed in a water bath at 60°C for 5 min, cooledin ice, and clarified by centrifugation at 15,900 x g for20 min. The precipitate was discarded, and the super-natant solution was brought to 52% ammonium sulfatesaturation. The mixture was stirred for 30 min, andthe precipitate was removed by centrifugation (15,900x g, 20 min). The supernatant solution was brought to85% ammonium sulfate saturation, cooled, and stirredfor 1 h. The precipitate was collected by centrifugationas above and suspended in 5 mM potassium phos-phate, pH 7.8. The solution was dialyzed overnight at4°C against 20 volumes of the same buffer.

Polyacrylamide gel electrophoresis. Slab gelelectrophoresis was performed with a Hoefer appara-tus (Hoefer Scientific, San Francisco, Calif.). Gels wereprepared with 10% acrylamide (Eastman Kodak Co.)which was recrystallized prior to use (19). Methylene-bisacrylamide (recrystallized) was purchased fromSigma Chemical Co., and N,N,N',N'-tetramethylene-diamine (TEMED) and Uniblue A were products ofEastman Kodak Co. The buffer system employed wasthat of Davis (13). One milligram of protein was ap-plied to the entire gel. Electrophoresis was initiated at10 mA and conducted at 20 mA after entry of thebromophenol blue tracking dye into the lower gel.Electrophoresis was continued until the dye front ap-proached the bottom of the gel (approximately 4 h).Gel slabs were sliced with a razor blade, and each slicewas stained individually to make various comparisons.Staining for protein was performed by the method of.Datyner and Finnimore (12). SOD activity was visu-alized by use of a nitroblue tetrazolium negative stain-ing technique (7). After electrophoresis the gels wereimmersed in 0.16% nitroblue tetrazolium and incu-bated at 36°C for 15 min. The gels were transferred toa solution containing 0.028 M TEMED, 2.8 x 10-5 Mriboflavin, and 0.036 M potassium phosphate at pH7.8, and were incubated at 36°C for 10 min. The gelswere removed from the incubator and exposed to afluorescent light for 3 to 5 min. The gels becameuniformly blue as a result of the production of forma-zan except in areas containing SOD, which appearedas achromatic zones.

Iron-containing compounds were identified in gelsby employing a modification of the transferrin stainingtechnique of Ornstein (24a). The iron reagent 2,4,-dinitroso-1,3-naphthalenediol was a product of East-man Organic Chemicals (Rochester, N.Y.).

Spheroplast formation and cell fractionation.A modification of the method of Ghosh and Murray(14) was used to obtain spheroplasts of L. monocyto-genes 1370. Tryptose broth cultures (100 or 200 ml),incubated for 18 to 24 h, were harvested by centrifu-gation at 10,400 X g for 15 min at 4°C. Cells weresuspended in 10 ml of a solution containing 0.3 mg oflysozyme/ml (Sigma, grade I), 0.4 M sucrose, and 0.03M tris(hydroxymethyl)aminomethane buffer, pH 6.7.The optical density of a 1:10 dilution of the finalmixture was 0.3 as determined in a Bausch & LombSpectronic 20 colorimeter at 600 nm. After incubationfor 10 min at 37°C with stirring, MgCl2 (1 M solution)was added to a final concentration of 0.02 M. Incuba-tion without stirring was continued for another 60 min.Phase-contrast microscopy was employed to observethe progress of spheroplast formation and occurrence

SUPEROXIDE DISMUTASE OF LISTERIA 865

of cell lysis. The spheroplasts were sedimented bycentrifugation (10,400 x g, 15 min); the supernatantfluids were removed and designated as the periplasmicfraction. The pellet was suspended in 0.1 M NaCl andgently shaken for 10 to 15 min. The lysate, designatedthe cytoplasmic fraction, was recovered in the super-

natant fluids after centrifugation at 15,900 x g for 20min. The membrane fraction (pellet) was solubilizedin 0.1% Triton X-100. The pellet was suspended in theproper volume of Triton to yield approximately 2 mgof protein/ml and incubated at 370C for 3 min (aftercentrifugation at 10,400 x g, 10 min). The solubilizedmembrane fraction was chilled and maintained at 4°Cuntil SOD assays were performed.

Superoxide generating system and survivalstudies. Survival of organisms in the presence ofexogenously generated superoxide was determined byexposure of cells to photoactivated riboflavin. Reac-tants consisted of 10-' M riboflavin, 0.025 M methio-nine, 0.05 M potassium phosphate, and i0-' M EDTA,pH 7.0. Sodium carbonate buffer (0.05 M) was usedfor pH 10 studies; total volume was 4.0 ml. Freshlyharvested, washed cells (5 x 107 to 1 x 108) were addedto each of two tubes containing the reaction mixtures.The control tube was wrapped in foil, and both were

incubated at 37°C at a distance of 25 cm from a bluelight source (Sylvania reflector-type lamp). The lightsource was maintained at 80 V by use of a variableautotransformer (Staco, Inc., Dayton, Ohio). Sampleswere removed for viable-cell counts just prior to Wiu-mination and at intervals during the period of incu-bation. The survival ratios were determined by com-

paring percentages of viable cells in the experimentaltube and control tube at the various time intervals. Noloss or significant increase of strain A4413 or 1370viability was observed in the control tubes at eitherpH 7 or pH 10.

Estimation of intact cell SOD activity. A modi-fication of the above pH 7 reaction mixture to include16 mM sodium sulfite allowed the measurement ofsuperoxide formation by observing the superoxide-de-pendent oxidation of sulfite. The mixture was allowedto equilibrate at 370C, and superoxide formation was

initiated by addition of 80 ,tl of riboflavin (4 x 10-5 M)to a final concentration of 8 x 10-7 M. This preparationwill be referred to throughout as the "riboflavin me-dium." Sulfite oxidation was followed with a model 55YSI oxygen monitor. The percentages of the totaloxygen availability, based on 100% at zero time, were

recorded in experimental tubes containing purifiedSOD, Listeria cell extract, or intact cells, and in con-

trol tubes containing heat-denatured enzyme or heat-killed cells. Decreased rates of oxygen consumption inexperimental tubes as compared to controls, due tocompetitive inhibition of sulfite oxidation by SOD,were used to calculate SOD activity in terms of thepercentage of inhibition of sulfite oxidation.

RESULTS

Survey of strains. Table 1 shows specificactivities of SOD in various strains of L. mono-

cytogenes. SOD activities of the virulent cata-lase-positive strains A4413, 34-S, and JHHranged approximately 10 to 15 U/mg greater

than that of strain 9037-7. Both catalase-nega-tive strains, 1370 and A2419, consistently dem-onstrated specific activities of 90 to 100 U/mg orgreater in crude cell extracts.Influence ofiron and oxygen tension. The

effect of aeration and various concentrations ofiron on the specific activity of SOD obtainedfrom strain A4413 grown in a defined medium ispresented in Table 2. Nearly a 50% increase inspecific activity was observed in SOD from cellsgrown in aerated cultures over that obtainedfrom cells grown in the same medium withoutshaking. Doubling the iron concentration in theshake cultures further increased the specific ac-tivity of SOD. The same strain grown anaero-bically on tryptose agar yielded a relatively lowspecific activity of SOD of only 14 U/mg ofprotein.Nitroblue tetrazolium assay for SOD ac-

tivity in crude cell extracts. SOD activity wasdetected in gels as one of the proteins with arapid electrophoretic mobility in crude cell ex-

TABLE 1. Superoxide dismutase activities of L.monocytogenes

Strain' Catalase Specific activity

A4413 Positive 44.8 ± 3.9 (6)34-S Positive 50.0 ± 0.8 (4)JHH Positive 51.0 ± 4.1 (3)9037-7 Positive 35.9 ± 1.9 (3)1370 Negative 119.1 ± 6.1 (3)A2419 Negative 91.0 ± 7.2 (4)

aExtracts were obtained from cells grown in brainheart infusion broth with shaking.

b Units of superoxide dismutase activity per milli-gram of protein. One unit of activity is the amount ofenzyme which causes 50% inhibition of the rate ofcytochrome c reduction. Mean values and standarderror terms were derived from the numbers of separateexperiments shown in parentheses.

TABLE 2. Strain A4413 superoxide dismutaseactivities from cells grown in defined Listeria

medium (DLM)Iron sup- Specific ac-DLM culture element' Ptivityb

Stationary 5 37Shake 5 53Shake 10 81Shake 100 87Shake (heated extract)c ....... 100 0

a Extracts were prepared from 18- to 24-h culturessupplemented with iron in the form of ferric ammo-nium citrate.

b Units per milligram of protein. Activity representsmean of at least two independent determinations.

c Heated extract obtained by exposure to boilingwater for 30 min.

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866 WELCH ET AL.

tracts of L. monocytogenes A4413 (Fig. 1). Theachromatic zone indicating SOD activity corre-

sponds to a band in the gel stained for protein.The achromatic zone at the bottom of the gelcorresponded to the tracking dye and is a resultof the ammonium persulfate used to polymerizethe gel. The other acromatic zone just above themajor SOD band may be an artifact or may bedue to a slightly modified form of the protein.Listeria appears to contain only one major formof SOD.

Partial purification ofListeria SOD. Heat-ing crude extracts to 60°C for 5 min in thepresence of 0.1M KCl followed by centrifugationresulted in a small decrease in the amount oftotal soluble protein and a slight increase in totalunits ofSOD activity (Table 3). Very little SODactivity (3%) was precipitated by the addition ofammonium sulfate to 52% saturation. Subse-quent addition of ammonium sulfate to 85%saturation precipitated 68% of the original SODactivity and resulted in a sevenfold purification.A significant loss ofSOD activity occurred whenthe initial ammonium sulfate saturation was in-creased to above 52%, as well as when the finalsaturation of ammonium sulfate was less than85%. Attempts to precipitate more protein byincreasing the time of precipitation or increasing

.

.

E w

A)BFIG. 1. Polyacrylamide gel electrophoresis of L.

monocytogenes A4413 crude cell extract. Strip B was

stained for protein and strip A was stained for SODactivity. Achromatic zone (arrow) indicated SODwhich corresponds with the lower band in strip A(arrow).

INFECT. IMMUN.

TABLE 3. Partial purification of superoxidedismutaseTotal

b Specific YieldStep protein Activity" activity iel%

(mg)

Crude extract 188 15,191 80 100KCL, 600C 175 15,208 87 1000-52% 83 473 6 352-85% 19 10,400 547 68

a Cells of strain A4413 were obtained from 18- to 24-h DLM shake cultures supplemented with 10 [Lg ofFe/ml. The crude extract, which was prepared bypassage of the cell suspension through a French pres-sure cell, was diluted to 200 ml, brought to 0.1 M KCl,and heated for 5 min at 60'C. After removal of dena-tured protein by centrifugation, the ammonium sulfatetreatments were carried out.

b Activity and specific activity refer to total unitsand units of SOD per milligram of protein, respec-tively.

the concentration of ammonium sulfate did notresult in an appreciable gain of total protein atthis step.

Identification of iron-containing pro-teins. The partially purified cell extracts fromstrain A4413 were applied to gels, subjected toelectrophoresis, and stained for protein, SOD,and iron (Fig. 2). In contrast to the gels of crudecell extracts (Fig. 1), only four major proteinstaining bands were observed. The most rapidlymigrating protein, SOD, appeared to be in great-est quantity, as indicated by the relative inten-sity of staining. The SOD activity stain likewiseshowed evidence of high enzyme concentrationby a large, rather diffuse achromatic zone. Ironin the SOD region was demonstrated by thestaining procedure for iron-containing proteinsin gels. Though not evident from Fig. 2, the iron-positive region stained blue-green. Catalase wasdetected as the third iron staining band from thetop by its reaction with hyrogen peroxide.Fractionation of cells and distribution of

SOD in Listeria. Spheroplasts were induced inL. monocytogenes 1370 by lysozyme treatment.Greater than 90% spheroplast formation wasobtained, as judged by phase-contrast micros-copy. Fractions collected were designated theperiplasm (the lysozyme incubation mixture),the cytoplasm (the spheroplast lysate), and thesolubilized membrane fraction. That the cellswere adequately fractionated by the proceduresemployed was demonstrated by polyacrylamidegel electrophoresis. Each fraction contained ma-jor protein staining bands which were not foundin common with either of the other fractions(data not shown).Table 4 shows the distribution ofSOD activity

associated with the fractions obtained from Lis-

SUPEROXIDE DISMUTASE OF LISTERIA 867

teria spheroplast preparations from cells grownunder various conditions. Total SOD activitywas approximately threefold greater when cellswere obtained from shake cultures than whencells were grown without shaking. The mem-brane fraction of L. monocytogenes grown intryptose or DLM broth with or without shakingpossessed the highest SOD specific activities.The distribution ofSOD activity varied with thegrowth conditions. The greatest percentage ofthe total SOD activity was associated with theperiplasm of cells from tryptose shake cultures.The cytoplasmic fraction contained the highest

-"-4.

A B CFIG. 2. Polyacrylamide gel electrophoresis ofpar-

tiallypurified cell extract (strain A4413). Gels stainedwith UniblueA (A), 2,4-dinitroso-1,3-naphthalenediolfor detection of iron (B), and nitroblue tetrazolium(C) were compared to determine the presence ofSODand iron-containingproteins. The presence ofa blue-green band in B corresponding to SOD bands in Aand C indicates association of iron with L. monocy-togenes SOD.

SOD activity when cells were grown in the samemedium without shaking. The distribution ofSOD activity was quite different when cells weregrown in DLM broth. In this medium, whencells were obtained from shake cultures, thehighest activity was associated with the mem-

brane fraction; however, the periplasmic fractioncontained the greatest percentage of SOD activ-ity when cells were grown in the same mediumwithout shaking.These data indicate that the distribution of

SOD in Listeria varies with growth conditionsbut is most often associated with the outer por-tion of the cell.Survival of organisms in a superoxide-

generating medium. Catalase-negative strain1370 and catalase-positive strain A4413 were

harvested from tryptose broth shake cultures,washed, and tested for resistance to killing byphotochemically generated superoxide in mediaat pH 7 and at pH 10. Strain 1370 lost viabilityat pH 7 and 10, but loss at the higher pHoccurred at a somewhat slower rate than at thelower pH (Fig. 3). Greater variation in survivalwas observed for strain A4413 at the two differ-ent pH values. Illumination in the presence ofsuperoxide or its derivatives apparently stimu-lated the growth of A4413 at pH 7 during theinitial hour of incubation. Greater loss of viabil-ity by A4413 as compared to 1370 at pH 10 maybe explained by the greater content of SOD instrain 1370 and thus its response to the higherconcentration of superoxide obtainable at pH 10.Superoxide-generating system and influ-

ence of SOD. The superoxide-dependent oxi-dation of sulfite in the presence of photoacti-vated riboflavin was followed by use of a Clark-type oxygen electrode. Figure 4 shows the rateof oxygen uptake determined directly from theoxygen monitor scale. The effect of the additionof 50 ng of purified bovine SOD per ml to theriboflavin medium is also shown. This amountof SOD reduced the rate of oxygen depletionfrom the medium by more than 50%. Thus,

TABLE 4. Distribution of superoxide dismutase activities in L. monocytogenes A4413

Units/mg of protein Units/fraction'Culture Total units

P C M P C M

TryptoseShake.. 68.5 45.0 94.0 362 (62) 122 (21) 98 (17) 582Still.. 6.0 24.0 48.0 22 (11) 96 (47) 87 (42) 205

DLMcShake... 5.5 14.5 95.5 23 (12) 32 (17) 133 (70) 189Still .. .. .. 5.0 9.5 57.0 23 (40) 14 (25) 20 (35) 57a P, Periplasm; C, cytoplasm; M, membrane.b Numbers in parentheses refer to percentage of the total units.c Supplemented with 10 tug of Fe/ml.

VOL. 23, 1979

868 WELCH ET AL.

0.8

-

0.4

* A4413 pH7o A4413 pHIO* 1370 pH7t 1370 pH10

0.5 1.5Time (hours)

FIG. 3. Survival of catalase-negative (strain 1370)and catalase-positive (strain A4413) L. monocyto-genes in a superoxide-generating medium. Washedcells from tryptose broth shake cultures were exposed,at a density of 5 X 107 to 1 x 108 cells/ml, to photo-chemically generated superoxide. Controls were in-cubated in the dark. Results are expressed as asurvival ratio, which is the ratio of the percentage ofbacterial viability at the time intervals in the exper-imental tube to the percentage of viability in thecontrol. Points represent the mean of two or threeseparate experiments in whichplate counts were donein duplicate.

inhibition of the rate of sulfite oxidation corre-lated with SOD activity, and a more directmethod of plotting this activity is in terms ofpercentage of inhibiiton. Figure 5 illustrates thisby showing an increase in the inhibiiton of sulfiteoxidation with increasing amounts of purifiedbovine SOD added to the riboflavin medium. Inaddition, cell extracts (partially purified) fromL. monocytogenes A4413 demonstrated a similarpattern of inhibition when added in concentra-tions 50-fold greater than the purified SOD.Approximately 10% greater inhibition wasachieved with bovine SOD than with SOD ofthe bacterial cell extract.Effect of intact cells on sulfite oxidation.

The influence of whole cells on sulfite oxidationwas examined to determine whether the systemmight be applied for estimation of intact-cell

SOD activity. Figure 6 shows the effects ofstrains 34-S and 1370 on sulfite oxidation interms of percentages of inhibition of oxygenuptake in the riboflavin medium when it con-tained the various samples of viable organisms.The lowest rate of sulfite oxidation was causedby strain 1370, which also possesses the highestspecific activity of SOD in cell extracts. Nooxygen uptake due to endogenous respirationwas observed at the cell densities employed.

DISCUSSIONSOD has been postulated as a virulence factor

in pathogenic anaerobes (26). It appears that theSOD content of anaerobes varies with their ox-ygen tolerance, and such aerotolerant anaerobespossess the greatest pathogenic potential. Ananalogous situation may exist with respect tofacultative organisms which tolerate the greatlyincreased oxygen tension, in the form of super-oxide, produced locally by phagocytic cells. SODactivities of the various L. monocytogenesstrains showed little variation except when thecatalase-positive strains were compared with thetwo catalase-negative strains. Catalase-negativeListeria strains demonstrated significantlygreater SOD activities than the catalase-positivestrains. Marked correlation of SOD activitieswith virulence could not be observed among thecatalase-positive strains, although the avirulentstrain 9037-7 did contain the lowest activity.

960

800 ,-EM

640 E

rE

480 ,M

320 a

160

2 4

Time (minutes)FIG. 4. Measurement of sulfite oxidation and ef-

fects of purified bovine SOD. Reactants (the ribo-flavin medium) were illuminated and the rate ofsulfite oxidation with and without addition of SODwas monitored by use of an oxygen electrode. Resultsshown are from a representative experiment.

INFECT. IMMUN.

4D0-

4-

.D

wEL

Zm4-CL

9IN

SUPEROXIDE DISMUTASE OF LISTERIA 869

50 100*Purified SOD (ng/ml)

10I

2.5 5.0o A4413 cell extract (ug/mi)

FIG. 5. Inhibition of sulfite oxidation by bovineSOD and partially purified L. monocytogenes (strainA4413) cell extract. Percentage of inhibition was cal-culated from the rate of oxygen uptake in the ribo-flavin medium with no enzyme added. Data shownare mean values of at least two independent deter-minations.

Consideration of relative catalase activities incombination with SOD activities of these strainssuggests that virulence may involve the orga-nisms' ability to achieve the proper balance ofcatalase and SOD. Previous studies in this lab-oratory (J. H. Jackson, Ph.D. thesis, IndianaState University, Terre Haute, 1969) employingthree of the strains used in the present studyshowed maximal catalase activity to be associ-ated with strain JHH. Strain A4413 releasedapproximately 50% ofthe total volume of oxygenfrom H202 observed for strain JHH, and 9037-7released less than 25% of this amount. The twovirulent strains, A4413 and JHH, contain rela-tively high catalase and relatively high SODactivities. The strains of low virulence, 9037-7,1370, and A2419, contain relatively low catalaseand relatively low SOD activities or totally lackcatalase while possessing a high content of SOD.The presence of iron in DLM cultures mark-

edly influenced SOD activities. Excess iron (100/g/ml) in combination with aeration by shakingthe culture flasks caused a twofold increase in

the SOD activity of strain A4413. The role ofiron in enhancing SOD activity could involveutilization of the element as the metal compo-nent in the enzyme or incorporation of the ele-ment in other respiratory enzymes leading toinduction ofSOD. Development ofa cytochromechain could be dependent on excess iron in thegrowth medium and the resultant increased res-piratory activity responsible for release of super-oxide anions. SOD-like activity of metal ionsand non-SOD iron complexes has been reportedby Halliwell (15). Although EDTA effectivelyprevents interference at low iron concentrations,it was anticipated that the highest concentrationof iron (100 Ag/ml) added to DLM might resultin some nonspecific SOD activity. This appar-ently was not the case, since heat-treated cellextracts containing the same iron concentrationdemonstrated no SOD activity.

Gel electrophorsis of Listeria crude cell ex-tracts generally revealed only one band of SODactivity. Two slightly separated bands of activitywere occasionally observed with Listeria, butonly after storage of the extracts at -20oC for

Ca

6%,Eq0

2-

100 300Dry cell weight ( ug/ml )

FIG. 6. Effects of L. monocytogenes 1370 and 34-Son sulfite oxidation in terms ofpercentages of inhi-bition of oxygen uptake. Control rates of oxygen up-take were obtained by measuring oxygen consump-tion in the riboflavin medium in thepresence ofheat-killed cells. Data represent mean values from at leasttwo independent determinations.

VOL. 23, 1979

870 WELCH ET AL.

extended periods of time. It is assumed thatsome change in the size or conformation of theprotein occurred during freezing to account forthe different mobilities observed in the gels.Bacterial SOD isozymes have been observed inE. coli (8, 16, 31), Pseudomonas ovalis (30),Mycobacterium phlei (10), and Rhodopseudo-monas spheroides (21). The existence of at leasttwo bands ofSOD activity in E. coli representingSOD isozymes was confirmed during this study(unpublished data). Mycobacterium smegmatis(17) and Bacillus megaterium (3) have beenreported to contain only one form of the enzyme.

L. monocytogenes cells were fractionated todetermine the cellular location of SOD. Initialattempts to form spheroplasts by lysozymetreatment were unsuccessful. Many of thestrains were resistant to lysozyme under theexperimental conditions employed. Strain 1370was found to be susceptible to lysozyme treat-ment, provided Mg2" was added to the incuba-tion mixture soon after exposure to lysozyme.The use ofEDTA or absence ofmagnesium saltscaused rapid lysis of spheroplasts, even in thepresence of 0.5 M surcrose. Ghosh and Murray(14) observed similar effects with various otherstrains. Magnesium, in addition to sucrose, ap-parently is necessary to stabilize the sphero-plasts.

Fractionation procedures were employed toseparate cytoplasmic, periplasmic, and mem-brane-bound proteins. Gel electrophoresis ofsamples from each preparation demonstratedproteins unique to each fraction. During experi-ments in which premature lysis of the sphero-plasts occurred, definite contamination of theperiplasmic fraction from the cytoplasm wasevident from examination of the gels.The greatest percentage ofSOD activity (62%)

was found in the periplasmic fraction of Listeriashake cultures grown in tryptose broth. Whencells were grown identically in DLM, 70% of thetotal activity appeared in the membrane frac-tion. Low percentages of total activity (17 to21%) were found in cytoplasmic fractions fromcells grown in either medium.There may not be a great difference between

membrane-bound and periplasmic enzymes, atleast in terms of functional characteristics. Theenzyme may be loosely positioned in the outerregion of the plasma membrane, which wouldaccount for variability in location insofar as theperiplasm and solubilized membrane fractionsare concerned. This suggests that the enzymemay be available to interact with superoxidegenerated external to the cell. These results arein contrast to the SOD localization in E. coli,which has recently been shown to be in thecytoplasm or cell matrix (8). Earlier work (31)

suggested that the manganese-containing SODwas located in the cytoplasm and protected thecell against its own flux of superoxide, whereasthe iron-containing SOD was located in the per-iplasm and protected the cell against externalsources of superoxide such as increased partialpressures of oxygen and polymorphonuclear leu-kocyte metabolism. Reexamination (8) revealedthat the isozymes are in the cell matrix and thatthere appears to be an additional third isozyme.However, the cells were treated with a diazo-nium reagent which selectively inactivated onlyenzymes external to the plasma membrane andresulted in no reduction of SOD activity. Al-though this indicated that the isozymes werelocated within the limits of the membrane, itwas inconclusive concerning membrane-boundforms, since the diazonium reagent presumablydid not penetrate and inactivate internal mem-brane-associated enzymes.

Survival of Listeria in a superoxide-generat-ing medium was examined to determine theability of Listeria to cope with an environmentcontaining superoxide. Results were markedlydifferent when the virulent catalase-positivestrain (A4413) and the low-virulence catalase-negative strain 1370 were exposed to superoxideat different pH values. In contrast to 1370, A4413demonstrated resistance to killing at pH 7. How-ever, at pH 10 strain A4413 lost viability soonerthan strain 1370. This may be explained on thebasis of relative concentrations of superoxideand hydrogen peroxide at different pH values.According to McCord and Salin (24), in a systemsuch as the one employed in the present study(autoxidation of riboflavin), the ratio of super-oxide to H202 varies with pH. Thus, a relativelygreater amount of H202 would be formed at pH7, which would implicate catalase as an impor-tant factor in survival. On the other hand,greater survival of strain 1370 at pH 10 than atpH 7, or as compared to A4413 at pH 10, may beexplained by its high content of SOD and lackof necessity for catalase at the higher pH. If oneconsiders pH as a determinant in the relativeconcentration of superoxide, the phagocytic vac-uole, which has a low pH, would be expected tocontain a significantly greater amount of perox-ide relative to the steady-state level of superox-ide. The question may then arise whether havingSOD would be any advantage at all to the intra-cellular pathogen for survival. It seems thatthere would be an obvious requirement for SOD,even at low pH, for rapid enzymatic dismutationof superoxide to prevent reactions with H202yielding the hydroxyl radical (24), and for pre-venting direct formation of singlet oxygen. Thisoxygen species has been proposed as the agentresponsible for phagocyte-mediated microbici-

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SUPEROXIDE DISMUTASE OF LISTERIA 871

dal activity. Allen and co-workers (2) have elu-cidated the role of superoxide as an intermediatein the chemiluminescent response of phagocytesand have demonstrated that the quantitation oflight production, as a result of singlet oxygenproduction, can be correlated with bacterial kill-ing (1).

Further evidence for accessibility of ListeriaSOD to exogenously generated superoxide wasobtained by assaying SOD activity in viablecells. Comparison of SOD activity of intact cellsand cell extracts of strains 1370, 9037-7, and 34-S gave approximately the same patterns of sul-fite oxidation inhibition. No previous studieshave demonstrated SOD activity of intact bac-terial cells, although Tyler (27) described a po-larographic technique for assays of particulateSOD in rat liver homogenates. He also employedsulfite oxidation as an indicator of superoxideconcentrations. An enzymatic source of super-oxide (xanthine oxidase) was used rather thanphotochemical generation of free radicals. At-tempts to use xanthine oxidase as the source ofsuperoxide in Listeria whole-cell SOD assaysproved unsuccessful as a result of inhibition ofxanthine oxidase by the intact cells. Both viableand heat-killed Listeria cells appeared to inter-fere with xanthine oxidase-mediated generationof superoxide, although no inhibition of xanthineoxidase activity was observed in the spectropho-tometric assay involving the reduction of cyto-chrome c. The riboflavin method was found tobe suitable, provided the correct ratio of sulfiteto riboflavin was contained in the reaction mix-ture. The concentration of riboflavin employedwas necessarily low (8 x 1O-' M). At higherconcentrations, without proportional adjust-ments of the sulfite concentration, sulfite oxi-dation was found to be less sensitive in inhibitionby SOD. McCord and Fridovich (23) observedsimilar effects when studying dimethylsulfoxide-induced sulfite oxidation.Adjustment of the sulfite-riboflavin concen-

tration provided a system which was useful fordetection of 5 to 100 ng of SOD per ml, asdetermined by addition of the purified bovineenzyme. The reversal of sulfite oxidation inhi-bition by addition of greater concentrations ofbovine SOD was also observed when an excessof Listeria cell extract was added. The expla-nation for this effect is not known, but at leasttwo possibilities exist. Sulfite oxidation occurredspontaneously when EDTA was omitted fromreaction mixtures. If one assumes that tracemetals catalyzed this rapid oxidation, it is feasi-ble that the metal component of SOD can actsimilarly when the enzyme concentration isgreat enough to overcome the effects of EDTA.A more likely possibility, perhaps, is the forma-

tion of large amounts of hydrogen peroxidewhich either directly oxidize the sulfite or reactwith superoxide to form the hydroxyl radical(*OH).

It was not unexpected that iron would bedetected in association with Listeria SOD sincethe stimulatory effect of supplemental iron inthe growth medium on SOD activity was ob-served. Likewise, if one assumes that SOD con-tributes to the pathogenicity of Listeria, an iron-containing SOD could help explain questionsinvolving the mechanism of the virulence-en-hancing effect of iron (25).The failure of this in vitro study to demon-

strate an obvious correlation between virulentstrains of L. monocytogenes and high levels ofSOD may be explained by the failure of in vitroexperimental systems to take into account manypossible factors influencing susceptibility to dis-ease. For example, although mouse pathogenic-ity studies show strain 9037-7 normally to be anavirulent strain, its LD50 can be lowered 5 logunits by treatment of mice with iron (25). Highlevels of SOD activity in vitro may not accu-rately reflect the potential for SOD activity invivo, since the enzyme appears to be an iron-containing protein in Listeria. Whereas a "free"source of iron usually exists in culture media,the organisms are required to compete for ironwith the host under conditions of in vivo growth(28). Strong competition for iron normally existsin the host because ofthe presence ofthe bindingproteins transferrin and lactoferrin. Pathogensmust encounter transferrin as a serum compo-nent and must compete with lactoferrin in secre-tions and as a component of phagocytic cellmembrane and lysosomal contents. The associ-ation constant of these binding proteins for ironis 103 or more (18). In addition to the bindingproteins lactoferin and transferrin, iron may besequestered in the form of ferritin by cells of thereticuloendothelial system. Results of unpub-lished studies in this laboratory demonstratedthat L. monocytogenes was able to obtain ironfrom ferritin when ferritin was included in thegrowth medium. It is possible that virulence inListeria is dependent on activities of SOD, orsome other iron-containing compound, only tothe extent that the organisms can successfullycompete with the host for available iron.

ACKNOWLEDGMENTThis investigation was supported by Public Health Service

grant AI-10075 from the National Institute of Allergy andInfectious Diseases to C.P.S.

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