Mutant Common

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0021-9193/78/01365348$02.00/0

Copyright X) 1978 AmericanSociety for Microbiology PrintedinU.S.A.

Enterobacterial Common Antigen in Mutant Strains of Salmonella

D. MANNEL,'P. H. MAKELA,2ANDH. MAYER"*

Max-Planck-InstitutfurImmunbiologie, D- 78 Freiburg i.Br., WestGermany,' and Central Health

Laboratory(StateSerumInstitute),Helsinki,

Finland2

Received for publication8May 1978

A good correlation was found to exist between the serologically determined presenceof enterobacterial commonantigen (ECA) and theamount of the rare sugar constituent D-mannosaminuronic acid. Strains classified by serological techniques as ECA+, ECA-, and ECA" were found to possess the expected amounts of mannosaminuronic acid in the ECA-enriched phenol-soluble PL-L fractions. This correlation providesstrong evidence onthe identityof the mannosaminuronic acid-glucosamine polymer with the ECAasdefined by Kunin(J.

Exp. Med. 118:565-586,1963).

The enterobacterial common antigen (ECA) describedby Kunin in1963 (8) isatypicalcom- ponent of enteric bacterial cell envelope. Until recently, the presence ofECAwasdetermined indirectly because thestructureandcomposition of the antigenwas notknown. Passivehemag- glutination (HA) has been the usual method for testing theECAcontentofrespectivestrains(9, 29). Immuneprecipitationinagargelsservedas an alternative detection method(6,20,28),and recently bacterial agglutination has also been used, althoughthe latter method is only appli- cable to enterobacterial R mutants (18). The recent isolation ofECA fromSalmonella montevideo SH94 and the identification of its chem- icalstructure as apolymer ofN-acetyl-D-gluco- samineandN-acetyl-D-mannosaminuronic acid (16, 17) made itpossibletoscreenanumber of strains for thepresenceorabsence ofECA. To prove the validity of this approach, we have tested a number of strains, classified by the passive HAassay as ECA-, ECA+, orECAt', for thepresenceofthese ECA constituents. The excellentcorrespondence between thetwometh- odssupportsthe resultsonthe chemicalnature of ECA described above, which are important prooffor theidentityofthe isolated ECA (12).

The strains used for this investigation are genetically related derivatives ofthree Sabno- nella serotypes (see Table 1); those mutants were obtained by slightly different procedures based on therecentlyacquired knowledgeof the genetic determinationsofECA (12).

Threetypesofmutantsdefectivein ECA have been detected in Salmonella and Escherichia coli(11, 13,26). Bothrffandrfe mutations block the synthesis of ECA. The rfegene function is alsorequired forthebiosynthesisofthe0-spe-

cific polysaccharide part of the cell wall lipopolysaccharide (LPS) in mostbutnotallsero- types.Salnonellaofgroup B(Oantigens4,12) donotrequire therfefunction for0-chainsyn- thesis:rfemutantsinthisgrouparesmooth(S), withcompleteLPS,butECA-.Bycontrast,rfe mutants in both S. montevideo (group C, 0 antigen 6, 7) and S. minnesota (group L, 0 antigen 21)arerough (R), unabletosynthesize completeLPS,and alsoECA-.

In addition, some gene or genes in the rfb cluster, which is the main determinant of the 0- specific polysaccharide, are required for ECA production ingroup BofSalmonella(14).Thus, S.typhimuriumwithalargepart of its

rfb

genes deletedorreplaced by^therfbgenesofgroupC organisms are ECA" bythe HA method. Al- though the immediate products of the rfe and the ECA-determining rib geneshave not been identified, strains with various combinations of rfe andrfbloci fromgroups BandC, respectively (abbreviated hereasB-rfe, etc.),behaveasifthe C-rfelocus would contain the information which ingroupBisdivided between theB-rfeand the B-rfbloci. Hybrid strains which are genotypi- cally C-rfe+ are also ECA+, irrespective ofthe rfb locus, whereas B-rfe+ strains are ECA+ in combinationwith

B-rfib

butnotwith

C-rfi-

(11, 14).

MATERIALS AND METHODS The bacteria used in this study were previously described S. montevideo, S.minnesota, and S. typhimurium strains representing ECA+, ECA-, and ECACe categories; their propertiesare described in Table 1.For extraction ofECA, cultures were grown inafermentor at370Cat a constant pH of 7.2. The detailedconditions for growth and the medium used have been described previously (25).

348

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Isolationandpurification ofECA. Theproce-

dureemployed^wasrecently described in detail (16).

Briefly,bacterial cellswereextractedbyacombined hotphenol-water extraction (27) and PCP (phenol- chloroform-petroleum ether) fractionation (5).After dialysis andlyophilization, theaqueousphase of the phenol-water extraction was treated with the PCP mixture. The LPS precipitated when somedrops of water were added tothe phenol phase, while ECA remained in solution andwassubsequentlyrecovered by extensive dialysis andlyophilization. This fraction

wasresuspendedinwaterand centrifugedat105,000

xgfor4h.Thelyophilizedsupernatantisenrichedin ECA andwasusedinseveraltestsasthePL-L fraction.

Further purification was achieved by column chro- matography on diethylaminoethyl-cellulose (DE 32, Whatman) with stepwise elution with0.5, 1.0,and1.6 M ammonium acetate-methanol buffer (1). ECA is elutedinthe middlefraction and, afterdialysis and/or electrodialysis (4),waslyophilized.

Analyticalmethods.Hydrolysis of the ECA^ma- terialwasusuallydoneinsealed ampouleswith4N HClat100°C for2h.Aminosugars wereseparated andidentified byhigh-voltageelectrophoresisonSS 2043a^paper(Schleicher & Schiill,Diiren, Germany) atafieldstrength of45V/cm inabuffercontaining pyridine, formic acid, acetic acid, and water (1:1.5:10.90,vol/vol)atpH2.8(21).

Thedrned chromatogramswerestainedwithsilver

nitrate-NaOH. Determinations oftotal acetylas well asof^0-andN-acetylwereperformedby the method of Frommeand Beilharz(3).

Serological methods. ECA antisera ^{were pre-} pared as described earlier (17, 29) by intravenous immunization ofNewZealandWhite rabbitsat4-day

intervals with increasingamounts (0.25, 0.5,and 1.0 ml) of either living or heat-treated (1000C for 1 h) bacterial suspensions (10'° cells perml) thoroughly washed withsaline. E.coliandShigeUa boydui type 3-(F3140)^wereusedasimmunogens(29).

ECA determination by HA. Indirect HA was

performed (22)withfresh humanerythrocytes(RBC) ofbloodgroupA;theywerethoroughlywashed with saline and suspended to a concentration of 0.5% in

saline.ForsensitizingRBCwithECA,supernatantsof heated cultures (1h, 10000) oramountsof 50jigof antigenicmaterialwereaddedto 5 ml of RBCsuspen-

sions.After incubationat370Cfor30min,theexcess

antigen^waswashedoff,and the sensitizedRBCwere

resuspendedinsalinetogivea0.5%suspension.The RBC suspension was added to series of antiserum dilutions in microtiter plates as described in detail previously (13, 22).

Supernatants ofECA+bacterial cultures couldsen-

sitize theRBCtogivemaximalHAtiters(about 5,000, corresponding to a serum dilution of 1:5,000) ^even when small volumes(0.01ml)wereadded to the 5-ml RBC suspension. In contrast, 1-ml volumes of the supernatants of heated ECA- strains left the titer

usuallybelow 10. However, sometimes intermediate reactions^wereseen,sothatthetiterremainedbelow the maximal valuewithlarger amountsof the sensitiz- ing supernatant. Such reactions were designated as

ECAb (11, 14).

HA inhibition.Tomeasuretheinhibitingcapacity of^asubstance, the HA inhibition(HAI)testwasused.

Theinhibitor wasdissolved in normalrabbitserum

(diluted 1:50 with phosphate-buffered saline). Serial dilutions(0.25pl)^ranging^from250 to 0.25ugof the inhibitorpermlwereincubated at370Cfor 1 h with TABLE 1. Salmonellastrains used

Strain No. ECA LPSa rfab rtb rfe rff Source or reference

S.montevideo group C SH94 + S 6,7 + + + + S.montevideo 129 (2)

SH3465 - S6,7 + + B+ + (11)

S. minnesota group L F1114 + S 21 + + + + (10)

F1119(R5) Trace RcP- P3785 + 3786d + (7, 10)

SH5641 + RcP- P3785 + + + Recombinant from

F1119(7)

F1122(R8) Trace RcP- P4277 + 3788d + (7, 10)

E?3787

SH5644 + RcP- P4277 + + + Recombinantfrom

E?3787 F1122; (7)

SH3786 Trace S 21 + + + 4257 (13)

SH5657 + S 21 + + + + Recombinant from

SH3786 (7)

S.typhimui groupB SH5150 - Ra + Deletion' + 4270 (14)

SH5177 Trace Ra + Deletion + + Recombinantfrom

SH5150 (7)

SH5178 - S4,12 + + + 4270 (7)

SH5179 + S 4,12 + + + + Recombinant from

SH5177 (7) LPScharacteristics: S,smooth,completeLPS with 0antigensasdenoted (6,7; 21;or4,12);R,rough incomplete LPS of chemotypes Ra,completecore,orRcP-, incompletecore(Liideritzetal.[10]).

'Alleles:+,wild-typefunctionalallele;P3785 etc., mutation3785incistronrfaP,etc.

'B+, Wild-typegenes from group B.

"Themutationsrfe-3786 andrfe-3788maybeidentical(7).

Deletion his-809coveringpartof thertband hisclusters.

VOL. 135,1978

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25

p1

of a serum dilution containing2to 3 HAunits.

Then 50

pl

of the sensitized RBCwasadded,and the plates were again incubatedat37°C for the same time.

The lowestinhibitorconcentration giving a total inhibition of HA wasrecorded after1 hat roomtem- perature.

Agargelprecipitations. Precipitationsin agargel wereperformed by the method ofOuchterlony (23);

immunoelectrophoresis insodium barbiturate buffer of pH 8.6 wasby themicrotechniqueofScheidegger etal. (24). Forsemiquantitativedetermination ofECA, theradial diffusiontestof Mancinietal.(15)wasused, substituting antiserum for the agargel buffer,1volume to 3volumes,respectively.Thediameter of the precipitation area wasmeasured after24h.

RESULTS

S. montevideo strains. In aprevious investigation (16) thechemistryofECA wasstudied by using as its source SH94, aS. montevideo wild-type strain which in preliminary experi- ments was shown to contain a rather large amount ofECA. We showed that ECA can be extracted fromfreeze-dried bacterial cells bya combination of thephenol-waterand PCPpro- cedures. ECA becomes finally enriched in the phenolphase, and thisphenol-soluble material can be isolated in amounts corresponding to 0.3% of thedryweightof the bacteria (16). The same extraction method was applied to theS.

montevideo derivative SH3465(Table1),which by theHAtesthad been characterizedasECA negative (12), and some (less than 0.2%) of phenol-solublematerialwas obtained. Thema- terial from the wild-type strain was a potent inhibitor (at <0.25 ,ug/ml) in the HAI test, whereas material from SH3465 was unable to inhibit theECA-specific HAreactionevenina veryhigh concentration (>250,ug/ml).

Equalamounts ofhydrolyzed PL-L material from both strainswere thenanalyzed by high- voltage electrophoresis. The material from SH94 containedlargeamountsofglucosamine (GlcN) and mannosaminuronic acid (ManNUA), whereas theSH3465 material showed only small amounts of GlcN (so little that it could have derived from contaminating LPS) and no ManNUA or itslactone(Fig. 1).

Second, we measured the acetyl content in thePL-Lfractions ofSH94andSH3465.In ECA fromS. montevideo SH94,allaminosugars have been shown to be N-acetylated, and the total acetyl content amounted to 13% of ECA dry weight (16). Table 2 shows a large difference between the strainsin the acetyl content, mainly duetoN-acetyl. These chemicalresultsparallel theserological findings: SH3465is ECAnegative inthe serological assay, and contains very few constituentstypicaltotheisolatedECA from S.

montevideo SH94(noManNUA, littleGlcNand N-acetyl).

a b c d

MUan IUi acrone

6

^r^GIhN

man NU

FIG. 1. High-voltagepaperelectrophoresisof hy- drolysates ofPL-L fractions oftwo S. montevideo strains. Lanesa andd,StandardsofGicandGlcN (10pgeach);laneb, PL-Lfraction oftheECA--strain S. montevideo SH3465(150pgof hydrolysate);lane c, PL-Lfraction ofthe ECA+strainofS. montevideo SH 94(150pigofhydrolysate). Conditions: 3kV, Ih, pH 2.8, Trevelyan staining.

S. minnesotarfeandrfmutants. S. min- nesotastrainswith knowngeneticdefectswere tested byHAfor theirabilitytocoat RBC for ECA-specific agglutination. Only rfe'

rfft

strains-thewild-type strainsF1114, aswellas the rfe' rff+ derivatives SH5641, SH5644, and SH5657 of rfe ^or

rff

^mutants (Table 1)-were positive in this test. The rfe and rffmutants showed an ECA ',e reaction (large amount of supematant needed tosensitizeRBC) (11, 12).

This series of S. minnesota mutants was extracted with the phenol-water-PCP procedure to yield the corresponding PL-L fractions.

Stainedhigh-voltage electropherograms ofhydrolyzed portions of these fractions clearly showedasubstance with themigrationvalueof the ManNUA lactone in all the ECA-positive strains (F1114, SH5641, SH5657). Trace

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351 amountsof this substancewere also detectable

in the rffmutant SH3786 but not in the rfe mutant strains F1122 and F1119. Additionally, all tested fractions also contained GlcNindif- ferentamounts; therfemutants (F1122, F1119) had relatively little of this amino sugar com-

pared with therffmutantSH3786. Nevertheless, in all of the rfeor rffmutantsthe amount of GlcNwasless than that intherfe'

rff

strains.

In Table 3 the PL-Lfractions obtained from the wildtype (F1114), therffmutant(SH3786), and the

rff'

recombinant (SH5657) are com-

pared for yield, serological activity in HA, in the HAI and precipitation reactions, and in their content ofManNUA. These data showa good correlation betweenall thesetests: the content of ManNUA ishigh in the strains serologically classifiedasECA+, and low (but detectable) in the

rff

mutantwhichisserologicallyECA'.

The PL-L from the rfe mutants (F1119 and F1122) and theirECA+

rfe'

recombinant derivatives (SH5641, SH5644) were subjected to a

semiquantitative ECAprecipitationassaybyra-

dial diffusion. Table 4 shows results obtained withequalamountsof the PL-L fractionscom-

pared with the PL-L fraction^fromS. montevideo SH94. Theprecipitationareasproduced by the ECA of the mutant strains are significantly smaller than theprecipitationareasof thecured

strain. The PL-Lfraction from S. montevideo SH94 ^seems to contain somewhat^more ECA- specific material than the fractions from the ECA+S.minnesotastrains.

TABLE 2. Comparisonoftheacetylcontentofthe PL-Lfractions fromthe S. montevideostrainsSH94

(ECA+)andSH3465(ECA-)

LyophilizedPL-Lmaterial(%) with:

PL-Lfrom

Totalacetyl O-acetyl N-acetyl

SH94,ECA+ 12.7 0.7 12.0

SH3465,ECA- 1.0 0.2 0.8

The PL-L fraction from one ofthese ECA+

recombinantstrains (S.minnesotaSH5641)^was purified further. The ECA activity waseluted with 1.0M ammoniumacetate-methanol buffer fromthediethylaminoethyl-cellulose columnas wasthecasewith ECA isolated from S. montevideo SH94 (16). The isolated materialwas com-

pared with the similarly purified ECA from S.

montevideo SH94 in immunoelectrophoresis.

Bothgave anidentical precipitationpatternwith the E. coli 014 antiserum(17).

The LPS didnotseemtointerferewith either theserologicalorchemicalassaysforECA. Both ofthe tested ECA+ and ECA- strains included smooth forms with complete LPS and rough forms with an incomplete core (chemotype RcP-) (see Table 1).

S typhmunurium rfand

rlb

deletionmu-

tants. As described by Miikela et al. (14), S.

typhimurium strains lacking certain rfb genes

dueto a chromosomal deletion insegments of the hisand the rfbgeneclustersgiveanECA'C reaction with the HAtest, suggesting that they

TABLE 4. Comparison ofthe PL-Lfractions from ECA+S. montevideo and various S.minnesota mutantsand their recombinants in thesingle radial

diffusion technique (15).

Strain

S. montevideoSH94, ECA+.

S.minnesotaF1122,ECAt..

S.minnesota F1119,ECAt.

S. minnesota SH5644, ECA+.

S. minnesotaSH5641, ECA+.

Precipitation areaa diam

(mm)

14.5 6.0 6.5 11.0 10.0

aThediameter of thewellwas5mm,receiving 5

;&g ofa0.1% solution of the PL-L fractions. Twenty- five percent of the barbiturate buffer ofthe 1%agar

gel was substituted by ECA antiserum prepared against S. boydii type 3- (F3140) heat-killed whole cells.The diameter of theprecipitationarea was mea-

suredafter 24 h.

TABLE 3. Comparisonofthe PL-Lfractions from ECA+andECA" smooth strainofSalmonella

Antiserum ti- Inhibitoryamt .t d

Strain Description ECA YieldofPL terobtainedin inHAIb Ag/ Preci ManNUAd

F1114 Wildtype + 35.5 5,120 7.8 + +

SH3786 rffdefective Trace 10.7 1,280 250 - Trace

SH5657 rff recombi- + 28.5 5,120 3.9 + +

nant from SH3786

aHA, IndirectECA-specifichemagglutination;the data indicate titer of the antiserum obtainedwhenRBCs (5 ml ofa0.5%suspension)werecoated with25I&gof the PL-L fractions. Antiserumwasasinfootnotec.

b

HAI

methodasinTable2.

cIE,Immunoelectrophoresis.ECAantiserumwasrabbit antiserumagainstheat-killed wholecellsof S.boydii type3-(F3140).

dThe presenceof ManNUAwasdeterminedbyhigh-voltageelectrophoresisatpH2.8.

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contain little ECA material. Upon subsequent transfer, thesestrains accumulate secondaryrff mutations andbecome ECA-.Recombinantsof the double mutant whose his and rfb regions have been replaced with functional his' rftb regions byconjugation still containthe rff^mutation; they^are smooth but ECA-. Therefore, the^newrffmutations by themselvesappearto prevent ECA synthesis,but donotaffect LPS.

To see whether the amount of chemically determined ECA would also in these strains correspond to their serologically determined ECA content, we isolated the PL-L fractions fromafamily of isogenic strains. Thegenotypes,

rftb

rff,

rfb+

rif, rfb

rffF,

and rfb

rff

were

derivedbyconjugation from SH5150, which^contains ^a his-809 deletion that extends into rfb regionaswell asarifmutation(14). The yield ofthe PL-L fractions, their serological activity in HA and HAI, and the ManNUA content determined byhigh-voltage electrophoresis are

shown in Table5. Twoof the strains showno

ECA activity and^noManNUA-theybothcon-

tain the ^rifmutation either alone ^or together with the rfb-deletion. Some GlcN is found in these strains as in all other ECA- strains, although in smalleramountsthaninECA+ strains.

Theyield of the PL-Lmaterial islow inall of the strins except the ECA+ rfb+ ^rfr strain SH5179. The ^small amount of PL-L material extracted from SH5177, the rfb

rff'

strain, is however active in both the HA and the HAI tests. This strain, therefore, contains trace amounts of ECA; correspondingly, some

ManNUA could be detected by high-voltage electrophoresis.

Again, LPS^assuch didnotinfluence theECA reactivity.Although therfbdeletion strainswere

rough, the rfb+ rffderivative ^was snooth but completelyECA-.

DISCUSSION

Theresults showedgoodcorrelation between the presence orabsence of ManNUA and the serologically determined ECA phenotype. The

correlationextendedtostrains inwhichthese-

rological behavior suggested the presence of trace amounts of ECA: they had reduced amounts of the ECA-containing material (the PL-L fraction), and this contained little ManNUA.Thisanalysis, togetherwithasimilar analysis comparing ECA+strains of Enterobac- teriaceaewithECA- strains of othergram-neg-

ativefamilies(12), providesstrongevidencesup-

portingtheidentityof theManNUA-GlcNpol-

ymerwith theECAasdefinedbyKunin(8).

ManNUA is a rare constituent of carbohy- drate structures in gram-negative bacteria; in the family ofEnterobacteriaceae, it has been foundonlyasconstituentof theK7and theK56 capsular polysaccharides of E. coli (19; H.-C.

Fleming, diploma work, UniversityofFreiburg, Freiburg, West Germany, 1972). Its detection

canserve,therefore,^as^agoodindicator for the

presence of ECA; however,these observations needtobeconfirnedby serological^assays.GlcN

orN-acetyldeterminations in the semipurified ECA material (thePL-Lfractions)ofourstrains agreedwith the ManNUA determination.They would be less conclusive alone, because GlcN andN-acetyl-D-glucosamineare alsopresentin other cellwallmaterials, most notablyin LPS whicheasilycontaminates the ECA material.

The correlation between thechemically and the serologically determined ECA content was

consistent for the three types ofmutants (rfb, rfe, andrffi possessing thealtered ECA genes.

Although the biochemical function of the first two is unknown, the rffgenes probably deter- mine enzymesof theN-acetyl-D-mannosaminuronic acid biosynthesis pathway (H. Nikaido, personal communication).Thusfar,wehaveno

direct information withregard tothosefactors thatproducetheECA' phenotype, although

we were abletocorrelate itspresence with the

presence of ^small amounts of ManNUA. The

ECAtcephenotypeof theS.minnesotarffmu-

tantSH3786(Table 4)couldeasilybeexplained byassuming that therifmutation isleakyand allowssomesynthesisofECA. Thesameexpla- TABLE 5. Comparison of PL-Lfractions obtained fromthe S.typhimuriumstrains

Strain_Strainrfbrfbrffrif LPSLPS YieldYield(%)a Titer obtained

~HA

^b in Inhibitory amt in^HAC(ug/nil) MaNAManNUAd

SH5120 Deletion' - R 3.9 10 250

SH5177 Deletion + R 3.5 640 125 Trace

SH5178 + - S 3.5 10 250 -

SH5179 + + S 18.5 2,560 62.5 +

aAmountrelated to thewater-phasematerial.

bHA,IndirectECA-specific hemagglutination; the data indicate titer of the antiserum obtained when RBC (a 0.5%suspension)werecoated with50ug of thePL-Lfractions per ml. ECA antiserum was rabbit antiserum against heat-killedwholecellsofS. boydii type 3- (F3140).

'Methodasin

Table

2.

dThe presenceof ManNUA was determined by high-voltage electrophoresis at pH 2.8.

'Deletionhis-809;seeTable1.

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ECA IN MUTANT STRAINS OF SALMONELLA 353 nationmayapplytothe ECA' phenotype of

the S. minnesotarfe mutantstrains F1122 and F1119: the rfemutation probablyoccursin the

same rfe allele in both these strains (7). How-

ever, thethird typeof ECA ^estrainswere S.

typhimurium

derivatives withacompletelack of certainrfbgenes,either becauseof chromosomal deletionorbecause of replacementbyrfbgenes

ofgroup C origin, ^as in the strain SH4146 described indetail by Miikelaand Mayer (11).In this strainit isunlikely thattraceECA production occurred because of an incompletely blockedgene.When thetracereactivitywasfirst discovered in these strains, we considered the possibilitythat itwasnotatrueECA reactivity at all but instead a serological cross-reaction given byarelatedcompound (11). The correlation of serologically determined ECA and ManNUA in these strains argues against the cross-reaction hypothesis. We suggestthat the ECAproductionin strains lackingtherfbfunc- tion^canbeattributedtoareplacementbyan as

yetunidentifiedgene.Suchafunction could, for example, beto turnonthe function of therffor

rfe,inthiscase groupCcould differfromgroup

Bbynotrequiringthis activation,orbycontain- ing the regulatory gene(s) in the rfe-rffgene

region.

ACKNOWLEDGME]NTS

We thankChristiane Widemann and Sirkku Waaralafor excellent technicalassistance. Wearegreatlyindebted toJack London, Bethesda, Md.,for hishelpinpreparingthemanu-

script.

We thanktheSigridJuselius Foundation and theFinnish Medical ResearchCouncil forgrantstosupportthisresearch.

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