INFECTION AND IMMUNITY, Apr. 1993, p. 1442-14460019-9567/93/041442-05$02.00/0Copyright © 1993, American Society for Microbiology

Superoxide Dismutase and the Resistance of Escherichia colito Phagocytic Killing by Human Neutrophils

ERZSEBET PAPP-SZABO, CLAIRE L. SUTHERLAND, AND P. DAVID JOSEPHY*

Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistryand Biochemistry, University of Guelph, Guelph, Canada NIG 2W1

Received 19 October 1992/Accepted 25 January 1993

Transformation ofEscherichia coli K-12-derived strains with a plasmid carrying the genetic determinants forsynthesis of lipopolysaccharide 0 antigen by ShigeUla dysenteriae allows the construction of phenotypicallysmooth derivatives. We show that such E. coli K-12 derivatives are highly resistant to killing by human serum.

Isogenic wild-type and sodB mutant (Fe superoxide dismutase-deficient) strains were constructed. The resultsof experiments on phagocytic killing of these strains by human neutrophils are reported. We observed no

difference between the sensitivities of wild-type and sodB mutant strains to phagocytic killing, in contrast to theresults reported by other researchers who used species other than E. coli or strains other than K-12.

Human neutrophils phagocytose and kill invading micro-organisms (29). When neutrophils are challenged with opso-nized bacterial cells, or other antigenic stimuli, they undergoa respiratory burst of greatly increased oxygen consumption(30). Oxygen is reduced to superoxide via a specializedrespiratory chain, which transfers electrons to the respira-tory burst NADPH oxidase (27). Phagocytosed bacteria arekilled by the toxic action of the products of the respiratoryburst; these products include the superoxide anion, hydro-gen peroxide, hydroxyl radical, and oxidized halogen spe-cies, such as HOCl (1). The critical immunological role of therespiratory burst response is supported by the observationthat patients with inherited chronic granulomatous disease,whose neutrophils are incapable of producing the burst, are

at great risk of death from bacterial infection (28).The defenses of Eschenchia coli against oxidative stress

have been studied in detail in recent years (9, 31, 39). Manyenzymes play a role in preventing and repairing damage byoxidants. E. coli possesses two genes encoding superoxidedismutases, enzymes which catalyze the conversion of su-

peroxide radicals into hydrogen peroxide and molecularoxygen. Two genes, sodA and sodB, encode the manganese-containing and iron-containing enzymes, respectively (5). Ahybrid heterodimer form is also produced (26). The Mnsuperoxide dismutase is markedly induced following expo-sure of E. coli to redox-cycling agents; this response iscontrolled by the regulatory soxR and soxS genes (18). Incontrast, the Fe superoxide dismutase is constitutive (26). Arecent report (21) suggests that there are functional differ-ences between the Mn- and Fe-containing enzymes, in termsof their abilities to protect specific intracellular targets (DNAand 6-phosphogluconate dehydratase).Hydrogen peroxide is converted to water and oxygen by

the action of catalase-hydroperoxidase. Catalase HPI isencoded by katG (42) and is induced by exposure of cells tohydrogen peroxide, under control of the regulatory geneoxyR (38). HPII is encoded by the structural gene katE andregulated by katF (33, 39). Fang et al. have recently reportedthat a katF mutant of Salmonella typhimurium is less viru-lent in mice than is an isogenic wild-type strain (11).Mutants unable to produce either form of superoxide

* Corresponding author.

dismutase are inc'apable of growing aerobically on minimalmedium (5). However, single mutants (either sodA sodB+ or

sodA+ sodB) grow normally under these conditions. Mu-tants devoid of superoxide dismutase (sodA4 sodB) andmutants devoid of catalase (katG katE) show increasedsensitivity to killing by chemical oxidants (12, 32). Again,single mutants are much less sensitive than double mutants.Ma and Eaton (22) have recently shown that the protective

action of bacterial catalase against hydrogen peroxide tox-icity is manifested only at high bacterial cell concentrations.Since the bacterial cell wall is freely permeable to hydrogenperoxide, isolated E. coli cells are unable to generate an

inside-outside concentration gradient of this oxidant. How-ever, colonies of catalase-producing cells (or dense suspen-sion cultures) can exert a mass action effect and clearhydrogen peroxide from solutions (8); this effect protects thecells against hydrogen peroxide toxicity.

Since exposure of enteric bacteria to redox-cycling agents(e.g., paraquat, menadione, and plumbagin) or hydrogenperoxide leads to induction of oxidative stress defenseenzymes, exposure of the bacteria to the products of therespiratory burst might also cause such induction (19). Whilethis has not been shown directly, S. typhimurium cells, uponexposure to macrophages, respond by increasing the synthe-sis of several dozen proteins, including the heat-shockprotein GroEL (4, 14). Can bacterial enzymes contribute tobacterial defense against phagocytic killing? Our goal is totest this possibility by measuring the susceptibility to killingof genetically well-characterized strains with null mutationsin relevant genes. A direct approach to evaluating the rolesof superoxide dismutase and catalase in bacterial resistanceto phagocytic killing is to test the sensitivity of isogenicstrains, wild type or those with mutations in the genes forthese enzymes. However, laboratory strains of E. coli de-rived from strain K-12, which have been used in mostgenetic constructions involving these genes, are phenotypi-cally rough (i.e., do not synthesize the complete 0 antigen).These strains are highly sensitive to killing by serum, sincethe 0 antigen plays a major role in bacterial resistance tocomplement (25). Opsonization with serum is necessary foreffective phagocytic killing, and serum sensitivity makessuch experiments difficult or impossible. Experiments are

typically performed with virulent, serum-resistant clinical

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SUPEROXIDE DISMUTASE AND E. COLI RESISTANCE TO KILLING 1443

isolate strains (35). However, the introduction of the desiredmutant alleles into such strains may be difficult.Sturm and Timmis (40) constructed a plasmid, pSS37,

which incorporates the genetic determinants for synthesis ofthe lipopolysaccharide 0 antigen by Shigella dysenteriae.When E. coli K-12 strains are transformed with this plasmid,they attach the S. dysenteriae 0 antigen to the K-12 coreoligosaccharide (10, 34). Such 0-antigen-producing, smoothE. coli strains might be expected to be serum resistant. Inthis study, we showed this to be true and applied thisapproach to the construction of smooth, isogenic wild-typeand sodB E. coli K-12 strains. The results of experiments onphagocytic killing of these strains by human neutrophils arereported.

MATERLILS AND METHODS

Bacterial strains, plasmid, and phage. GC4468 (F- AlacU169 rpsL) and QC773 [GC4468 1(sodB-kan)1-A2 Km'] werethe gift of D. Touati (5). Plasmid pSS37 was isolated fromstrain CS1859, the gift of Carl Schnaitman. Phage Ffm wasobtained from C. Whitfield, Department of Microbiology,University of Guelph.Medium and growth conditions. The bacteria were rou-

tinely grown in Luria-Bertani (LB) broth at 37°C withshaking at 120 rpm in the presence of streptomycin (10,ug/ml; nontransformed strains) or chloramphenicol (25 ,ug/ml; transformed strains) to an optical density at 600 nm ofapproximately 1.0.

Transformation. Both wild-type GC4468 (control) andsodB QC773 strains were transformed with pSS37 by elec-troporation (Bio-Rad Gene Pulser), and transformed cellswere selected by growth on LB plates with 25 ,ug ofchloramphenicol per ml. Putative (chloramphenicol-resis-tant) transformants were tested for resistance to lysis byphage Ffm. A phage-resistant, serum-resistant (see below)clone was selected for both strains transformed with theplasmid and used in subsequent experiments. These strainsare designated DJ4468 and DJ773, corresponding to theTouati laboratory numbering.

Plasmid pSS37 was isolated from the transformed strainsby the rapid-boiling method (20) and analyzed by agarose gelelectrophoresis.

Superoxide dismutase activities were analyzed by poly-acrylamide gel electrophoresis and activity staining withnitroblue tetrazolium, following the published method (3).Serum resistance. The bacterial preparations were done as

described below for the phagocytic killing assay. Bacteria(approximately 2 x 106 cells per ml, final concentration) andserum (0, 1, 3, or 5%, final concentrations) were combined inKRP+ (Krebs original Ringer phosphate) buffer at a totalvolume of 1 ml. (KRP+ buffer was prepared as describedpreviously [7], except that the CaCl2 concentration waslowered from 3.3 to 2.2 mM. Divalent cation [Ca2' andMg2+] solutions were autoclaved separately and then mixedwith the buffer.) Samples were withdrawn and diluted, asdescribed below for the phagocytic killing assay. Bacteriawere overlayed in top agar onto LB plates. The plates wereincubated overnight, and the colonies were counted.

Materials for neutrophil phagocytic killing assay. Freshhuman blood samples were obtained, with informed consent,from healthy adult volunteers with various blood types.Blood was collected in Vacutainers containing EDTA (forneutrophil preparation) or without additives (for serum prep-aration). KRP- buffer is KRP+ buffer without Ca2+ andMg2+.

Neutrophil preparation. The methods for separation onHistopaque 1119/Histopaque 1077 followed the Sigma Diag-nostics procedure no. 1119 (February 1990), except that thecells were washed with KRP- buffer and recovered bycentrifugation at 250 x g. The cells were resuspended in theassay buffer (KRP+ buffer containing 0.1% gelatin and0.16% glucose) at about 3 x 106 cells per ml. Cell yield wasdetermined by hemocytometer count. Viability was deter-mined by staining with trypan blue, as described previously(37), and was greater than 90%.

Preparation of bacteria. Bacteria were grown overnight at37°C, washed twice with KRP- buffer, and diluted into assaybuffer at about 8 x 106 cells per ml. (Bacterial cell concen-trations were estimated on the basis of the previouslymeasured relationship between the optical density at 600 nmand CFU per milliliter for each strain.)

Phagocytic killing assay. The killing assay was carried outby exposing bacteria simultaneously to fresh autologousserum (5%, final concentration) and neutrophils. The follow-ing were combined, in the order given: serum, 50 p,l;neutrophil preparation, 106 cells (300 p.l); KRP+ buffer (450,ul); and bacterial suspension, 1.6 x 106 cells (200 ,ul).Controls contained buffer in place of the neutrophil prepa-ration.Three blank tubes and three assay tubes were run for each

strain tested. The assay was initiated by adding bacteria, atzero time. Tubes were rotated in LabQuake rotators, in a37°C incubator. Samples (40 to 50 ,ul) were withdrawn atintervals up to 2 h. The aliquots were added to 3 ml of chilled(4°C) lysis buffer (1 mM sodium phosphate [pH 7.4] plus0.1% gelatin) and then allowed to stand at room temperaturefor at least 20 min. The tubes were vortexed thoroughly;aliquots (25 ,ul) were withdrawn, mixed with 2.0 ml of topagar at 45°C, vortexed, and overlayed onto LB plates. Theplates were incubated overnight.For the experiment comparing preopsonization with our

standard assay (see Discussion), bacteria were suspended infresh 5% serum for 25 min at 37°C, with rotation, at aconcentration of 107 cells per ml, before the addition ofneutrophils. For the phagocytic killing, bacteria and neutro-phils were suspended at a concentration of 2 x 106 cells perml, and the serum concentration was brought back to 5% inthe assay mix.

Colonies were counted with an image analysis system, byusing a Pulnix TM7-CN video camera and BioScan Optimassoftware, on an IBM-compatible 486 computer (Infrascan,Inc., Richmond, British Columbia, Canada).Data analysis of phagocytic killing assay. For each time

point, the percent survival value was determined, relative to100% at time zero, for the given strain. (The number ofcolonies per plate at zero time ranged from 500 to 800.Log(S) values were calculated. For each time, 12 measure-ments were averaged (three tubes per experiment, fourexperiments.) The data represent the means + standarderrors of these log(S) values.

RESULTS

Strain construction. Chloramphenicol-resistant coloniesobtained following transformation of GC4468 or QC773 withplasmid pSS37 were tested for resistance to lysis by phageFfm. More than 90% of the tested clones were phageresistant. One clone from each transformation was selectedfor use; the transformed strains are designated DJ4468(sodB+) and DJ773 (sodB).We confirmed the presence of the pSS37 plasmid in both

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1444 PAPP-SZABO ET AL.

A B C DE

FIG. 1. Superoxide dismutase activity-stained gels. Gels wererun and stained following the method of Beauchamp and Fridovich(3) (see the text). Lanes: A, Cu,Zn superoxide dismutase (frombovine erythrocytes; Sigma Chemical Co.), 0.5 jLg (control) (indi-cated by the horizontal line on the left); B, GC4468, 178 ,ug ofprotein; C, DJ4468, 250 ,ug; D, QC773, 295 ,ug; E, DJ773, 271 ,ug.The horizontal lines on the right indicate Mn superoxide dismutase(top), hybrid superoxide dismutase (middle), and Fe superoxidedismutase (bottom).

0.0(

-0.6

00r-4

-1.0

strains DJ4468 and DJ773 by plasmid minipreparation andagarose gel electrophoresis (data not shown). The sodBphenotypes were confirmed by polyacrylamide gel electro-phoresis and activity staining (Fig. 1).

Strain GC4468 grew normally in the absence of serum butwas rapidly killed in its presence. Survival was reduced toless than 1% within an hour at serum concentrations as lowas 1%. (Data for 5% serum concentrations are shown in Fig.2.) In contrast, DJ4468 (transformed GC4468) was serumresistant; at a 5% serum concentration, the cell numberdropped slightly at early time points but then increasedagain, demonstrating bacterial growth (Fig. 2). The results

1000

100

*!

000

0.1

FIG. 2.are descriland dottecsymbols,shown; ei

three plat(

0 30 60 90 120

Time (min.) -FIG. 3. Phagocytic killing of E. coli strains by human neutro-

phils. Circles and solid lines, DJ4468; squares and dotted lines,DJ773. Open symbols, bacteria plus serum only (controls); filledsymbols, bacteria plus serum and neutrophils. The results shown arethe averages from four independent experiments, each performed intriplicate, and are the means + standard errors of log(S) values.

for the sodB strains, DJ773 and QC773, were analogous tothose for the corresponding sodB+ strains (data not shown).

Phagocytic killing by neutrophils. The phagocytic killingassay was carried out for both the sodB+ DJ4468 and sodBDJ773 strains, in the presence (closed symbols) or absence(open symbols) of human neutrophils (Fig. 3). The controlincubations without neutrophils confirm the serum resis-tance of these strains (above).The neutrophil-dependent killing of both strains varied

substantially from one experiment (i.e., blood donor) to thenext; survival at 120 min varied from 3 to 30%. Neverthe-less, in each individual experiment, there was virtually nodifference between the susceptibilities to killing of the sodBand sodB+ cells. In the averaged data (Fig. 3), the error barsfor the two strains overlap at all time points.

DISCUSSION

We have shown that the transformation of E. coli K-12\ derivative strains with pSS37 yields smooth, serum-resistant4 j strains, which are suitable for use in neutrophil-dependentl killing assays. This technique should make the examination

of the contribution of various gene products, characterizedin K-12 strains, to bacterial survival following phagocytosis

.____.___.___.___.___.____ .___ .___ .___.__ feasible.030 60 90 120 Several previous studies, using either genetic or biochem-

ical approaches, have addressed the role of superoxideTime (mijn.) -. dismutase and catalase in protecting bacterial cells.

Ssit . in. T o Yost and Fridovich manipulated Fe-containing superoxidebed in the text. Triangles and solid lines, GC4468; circles dismutase levels in E. coli B cells by growth in eitherdlines, DJ4468. Open symbols, controls (no serum); filled iron-supplemented or iron-deficient medium and studied the5% serum. The mean results of a typical experiment are survival of the bacteria following exposure to whole humanrror bars represent the standard deviation of results for blood (43). Iron-deficient bacteria were more susceptible toes per point. phagocytic killing. A little later, Mandell (23) examined the

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pathogenicity (in mice) of a series of Staphylococcus aureus

strains (clinical isolates). Pathogenicity correlated with thecatalase content but was independent of the superoxidedismutase content among these strains. A low-catalase ri-fampin-induced mutant strain was more sensitive to phago-cytic killing by human neutrophils. Mandell concluded thatcatalase, but not superoxide dismutase, protected the bac-teria against phagocytic killing.

Schwartz et al. (35) used exposure to paraquat to stimulateincreased superoxide dismutase and catalase activities in E.coli ATCC 25922. These investigators did not observe in-creased bacterial resistance to killing by human neutrophils,following such induction, and concluded that these enzymes

"may have access to their substrates only after the surfaceof the organism has already been irreversibly damaged."Oxidative stress causes inhibition of membrane-associatedfunctions, such as lactose uptake (13). However, the inter-pretation of the results of Schwartz et al. (35) is not straight-forward, because of the complexity of the action of paraquaton E. coli. In addition to superoxide dismutase and catalase,paraquat induces very many proteins and enzymes (at least33 [17]), including the DNA repair enzyme endonuclease IV(6); of course, paraquat is itself toxic to E. coli (24).These early studies compared bacteria subjected to differ-

ent growth conditions or chemical agents or of ill-definedgenetic backgrounds (clinical isolates). Probably, all thesestudies were confounded by the multiple differences among

the various strains or conditions examined.Recently, Franzon et al. introduced a new approach to

testing the relationship between superoxide dismutase andsensitivity to phagocytic killing (15). Transposon insertionnull mutations in E. coli superoxide dismutase and catalasegenes were transduced into a smooth, pathogenic strain ofShigella fle-xneri. These investigators reported that a sodBmutant of S. flexneri, constructed by this method, was

"extremely sensitive to killing by phagocytes when com-

pared to the wild-type parent." After 20 min of exposure ofopsonized bacteria to human polymorphonuclear leuko-cytes, survival of the wild-type cells was more than 10%,while survival of the sodB cells was only about 0.1%. ThesodB mutant was also reported to be much less pathogenicthan the wild type, as studied with a rabbit ileal loopinfection assay. A katFG catalase-deficient mutant also hadincreased sensitivity to killing. This report, like the muchearlier work of Yost and Fridovich (43), suggested that Fesuperoxide dismutase plays an important role in protectingthe bacterial cell against killing.

Brucella abortus possesses both an Mn and a Cu,Znsuperoxide dismutase. The occurrence of the latter form ofthe enzyme is noteworthy, since the Cu,Zn form is typicallyeukaryotic and has only been found in a few species ofbacteria. Tatum et al. (41) have constructed null mutations ofthe Cu,Zn sod gene in this species and compared the survivalof such mutants with that of their parental strains in HeLacells and in a mouse macrophage-like cell line. In bothsystems, no major differences in survival were observed. Incontrast, in a long-term in vivo assay (persistence of bacteriain the spleens of mice inoculated intraperitoneally), thewild-type cells showed greater viability than the Cu,Zn sodmutant cells. "Although we have not directly demonstratedimpaired survival within macrophages, the sharp reductionin the splenic levels of the Cu,Zn sod mutant over 10-26days post-infection suggests that [Cu,Zn superoxide dismu-tase] contributes to the increased survival of B. abortuswithin murine macrophages during the early stages of infec-tion."

Virulent strains of Nocardia asteroides produce a secretedand surface-associated superoxide dismutase. Pretreatmentof these bacteria with a monoclonal antibody specific for thisenzyme reduced the number of organisms subsequentlyrecovered from mice infected by injection into the tail vein(2). This suggests that the surface-associated superoxidedismutase contributes to bacterial virulence. In summary,various studies have reached markedly different conclusionsregarding the role of superoxide dismutases in bacterialresistance to phagocytic killing.We did not observe any difference in survival between

wild-type and sodB mutant strains. This result is consistentwith that of Schwartz et al. (35), who observed no effect onsurvival when E. coli superoxide dismutase was inducedwith paraquat before challenge with neutrophils. The Mnsuperoxide dismutase (sodA gene product) should still beinducible in our sodB strain, DJ773.However, our results contrast with those of Franzon et al.

with S. flexneri (15). Several differences between the presentexperiments and those of Franzon et al. must be noted.First, we have studied E. coli rather than S. flexneii. ThesodA gene may play a more important role in E. coli than itdoes in S. fle-xneri and mask the effect of the sodB genotype.In this regard, we note that Franzon et al. reported that"several attempts to isolate S. flexneri sodA transductantsproved unsuccessful. The reasons for this remain unclear"(15). However, these two species are so closely related thatthe validity of the generic status of Shigella spp. has beenquestioned (36), so it seems unlikely that interspecies differ-ences are responsible for the discrepant findings. The exper-imental protocols differ in some respects. Franzon et al.preopsonized bacteria with 5% serum concentrations priorto interaction with neutrophils, whereas we used simulta-neous opsonization. The latter approach resembles moreclosely the in vivo situation. In an additional experiment, wetested the effect of preopsonization (16) (see Materials andMethods). Strains DJ4468 and DJ773 were compared. Withboth strains, the initial killing was greater than that observedwith the simultaneous opsonization method, but there wasno obvious difference between the two strains (data notshown). Therefore, we do not believe that this aspect of theexperimental protocols accounts for the difference betweenthe previous report and our observations.We used dilute phosphate-O.1% gelatin lysis buffer, rather

than 1% deoxycholate (15). Franzon et al. did not report theresults of control experiments without neutrophils. We com-pared the survival of both DJ4468 and DJ773 exposed to 5%serum alone and then treated with either dilute phosphate-0.1% gelatin lysis buffer or 1% deoxycholate. For bothstrains, deoxycholate caused substantial cell killing even atzero time of incubation with serum (data not shown). There-fore, we did not attempt to perform phagocytic killing assaysusing such a lysis solution.

In conclusion, we observed no effect of the sodB gene onthe sensitivity of E. coli to phagocytic killing. Furtherstudies of sodA and double mutant strains are now inprogress.

ACKNOWLEDGMENTS

We thank Carl Schnaitman, Department of Microbiology, ArizonaState University, and David Speert, Department of InfectiousDiseases, University of British Columbia, for helpful discussions.We thank Margaret Berry and Premila Sathasivam for taking theblood samples. John Snider and Danny McManus carried out theplasmid preparation and electrophoresis. Lillian DeBruin ran theSOD activity gels.

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This research was supported by a grant from the Natural Sciencesand Engineering Research Council (NSERC) of Canada. C.L.S. wasan NSERC summer student awardee for 1992.

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