JOURNAL OF BACrERIOLOGY, Mar., 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 3Printed in U.S.A.

Isolation and Characterization of the Ki (L) Antigenof Escherichia coli'

ROGER BOLA*OS2 AND CHARLES W. DEWITITDepartment of Microbiology, School of Medicine, Tulane University, New Orleans, Louisiana

Received for publication 30 September 1965

ABsTRAcrBOLANOS, ROGER (Tulane University, New Orleans, La.), AND CHARLES W.

DEWIrr. Isolation and characterization of the Kl (L) antigen of Escherichia coli.J. Bacteriol. 91:987-996. 1966.-An acidic polysaccharide with the serologicalproperties of the Kl (L) antigen has been isolated from Escherichia coli 02:K1:H4by means of phenol-water extraction, fractionation with hexadecyltrimethylam-monium bromide (Cetavlon), filtration through Sephadex G-200, and chroma-tography on anion-exchange cellulose columns. Nucleic acid and protein contentwere reduced to a nondetectable level. There is no contamination with 0 antigen.The active material appears in two different positions in the Cetavlon fractiona-tion, each with a slightly different serological specificity, as followed by the inhibi-tion of passive hemagglutination. By paper chromatography, the polysaccharidemoiety of the 0 antigenic fraction is composed of glucose, galactose, hexosamineand rhamnose. The absence of colanic acid in either Kl fraction was not proven,although its participation in our assay system, as well as the participation of thecommon or cross-reacting antigen, was ruled out by serological means.

Escherichia coli has been recognized for sometime as the causal agent of human infantile di-arrhea, and is also known to cause various acuteinflammatory infections, often fatal, in humansand animals. Attempts to find a correlation ofvirulence with the presence of cell wall lipopoly-saccharide (24, 27), or sialic acid-containing com-pounds found in some E. coli strains (10), havenot been successful. It has been shown, however,that there is a direct relation of mouse lethality tocertain types of K antigens, the L (labile) typebeing most closely associated with virulence (16),and in particular Ki, K5, and K44 apparentlyplaying a very important role in the host-parasiterelationship (10).

In the earlier experiments by Kauffmann (16)on the role of the L-type antigen, better protec-

1 This work is part of a dissertation submitted bythe senior author to the Graduate School of TulaneUniversity, in partial fulfillment of the requirementsfor the degree of Doctor of Philosophy. A prelimi-nary report was presented at the 64th Annual Meet-ing of the American Society for Microbiology inWashington, D.C., May, 1964.

2 Part of this work was done during the tenure of aFellowship from the Visiting Research ScientistProgram under the auspices of the National Academyof Sciences. Present address: Department of Micro-biology, University of Costa Rica, San Jose, CostaRica.

tion was obtained in mice passively immunizedwith antiserum directed against the encapsulatedstrain than with antiserum against the smoothnonencapsulated strain. No protection could bedemonstrated after active immunization, how-ever. More recently Jenkin and Palmer (14) haveshown that the rate of clearance of E. coli fromthe blood and peritoneal cavity of infected mice isdirectly proportional to the concentration of anti-body directed against surface components of thebacteria, the effect being an enhancement of thephagocytic capabilities of the host defensive cells.Although this antibody is called "specific," itcan be evoked by stimulation with apparently un-related antigens (lipopolysaccharide preparedfrom different organisms), and it has not been de-termined whether it is directed against the somatic(0) antigen, the capsular (K) antigen, or theflagellar (H) antigen, all of which are external tothe cell wall. In fact, there are almost no recentreports of studies on the role ofK antigen and itshomologous antibody in the host-parasite rela-tionship of E. coli.

Envelope materials in the Enterobacteriaceaehave been found, in general, to be acidic poly-saccharides (34). Attempts to define chemicallythe envelope materials of E. coli have led toidentification of several very interesting and re-lated substances. Barry and Goebel (2), studying

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BOLAN4OS ANDDEWITJT

a colicinogenic strain of E. coli (K235-01 :K1),isolated colominic acid, a homopolymer of N-acetylneuraminic acid (1), from the supernatantfluid of the culture medium. This substance, or avery similar one, has been found in several otherserotypes of E. coli as well as in Salmonella djkartaand S. dahlem (3). No serological role has beendemonstrated for colominic acid (3), despite thefact that a substance containing neuraminic acidis responsible for the haptenic activity of Neisseriameningitidis group C polysaccharide (31). DeWittand Rowe (4) and DeWitt and Zell (5) isolatedN-acetylneuraminic acid and N,7-0-diacetylneu-raminic acid from the cell surface of some sero-types (Ki and K5). Despite the fact that thesecompounds were shown to be associated with theenvelope material, and their relationship with Kantigen was suggested, no role in the determinantgroup was demonstrated. Recently, Goebel (11)obtained an acidic polysaccharide, colanic acid,from E. coli K235 L+0 (m) responsible for themucoid characteristics of the strain and presentas a capsule which interferes with the serologicalactivity of the 0 antigen. It proved to be antigenicin rabbits and serologically distinct from Ki anti-gen. Qrskov et al. (21) isolated A and B types ofK antigens from E. coli strains 08 :K42(A) :NMand 0141 :K85ab -> bc(B) :H4. The polysaccha-rides were shown to contain K antigenic specificityand to be distinct from 0 antigen and fromcolanic acid by both chemical and serologicalcriteria. The sugar composition was, in general,simple hexoses and methyl pentoses, amino sug-ars, and hexuronic acid.We present here evidence of the isolation of ma-

terial containing the serological activity of one ofthe L type of K antigens of E. coli (02 :K1 :H4)which differs chemically and serologically fromthe 0 antigen and from colanic acid.

MATERALS AND METHODS

The E. coli strain (E-107; received from The Up-john Co., Kalamazoo, Mich.) used in this study wasisolated from a human systemic infection and hasbeen found by us to have a mouse LD5o of <10 or-ganisms per mouse. It is serotype 02:Kl:H4, but, asit has been propagated exclusively on solid media,flagellar antigen development is suppressed and noanti-H antibody is present in our antisera when testedagainst a motile culture. A smooth, translucentvariant has been isolated which lacks the capsularantigen but retains the somatic antigen and is referredto as K(-). Two E. coli strains which produce colanicacid, K235 L+O(m) and K12(m), were kindly sup-plied by W. Goebel.

Anti-O and anti-OK sera were prepared in rabbitsagainst infusion broth cultures and were standardizedby bacterial agglutination according to Edwards andEwing (7). Anti-K sera were prepared by adsorption

of the anti-OK sera with heated (100 C, 60 min) K-positive cells or live K-negative cells. Both gave identi-cal results in the serological tests used. An anti-O 14serum, kindly sent to us by E. Neter, was used in theassay of common or cross-reacting antigen (18, 33).

Passive hemagglutination (HA) was performed withsheep erythrocytes sensitized with untreated bacterialextracts in the case of Kl antigens or with extractspretreated by exposure to sodium hydroxide (20) inthe case of 0 antigenic material. (The term "antigen"will be used throughout because one fraction isolatedearly in the investigation was found to be antigenic inrabbits. Fractions obtained later have not been ex-amined for antigenicity.) The optimal concentrationof sensitizing KI antigen was determined each timeby using twofold dilutions of antigen and assayingfor maximal titer against a standard anti-Kl serum.Optimal concentrations were remarkably constant,ranging from 5 to 10 ,ug per ml of a 2.5% suspension oferythrocytes. Sensitized erythrocytes (0.1 ml of a1.0% suspension) were added to serial twofold dilu-tions of antiserum (0.4 ml), and the test was read afterincubation for 1 hr at 37 C and then overnight at 5 C.Hemagglutination inhibition was read as the leastamount of material which on preincubation at 37 Cfor 30 min with 0.2 ml of antiserum dilution com-pletely inhibited 3 to 4 hemagglutination (HA) units.The procedure used for extraction of the Ki anti-

gen was modified from that of Qrskov et al. (21).Briefly, cells grown in chemically defined medium(22) were extracted with 45% aqueous phenol (32).The water-soluble portion was submitted to fractionalprecipitation with Cetavlon (hexadecyltrimethyl-ammonium bromide) according to Scott (26). Theentire procedure is presented in detail in Fig. 1.

As can be seen, the usual dialysis step was replacedby filtration through Sephadex G-50 (8) obtainedfrom Pharmacia Fine Chemicals, Inc., Rochester,N.Y. Lyophilization was replaced by precipitationfrom cold ethyl alcohol. Both methods were intro-duced because of earlier loss of Kl activity duringthese steps. Purified materials have been found to beslightly less labile. To obtain dry fractions, the alcoholprecipitates were washed twice in acetone, suspendedin diethyl ether, centrifuged, and dried in air. Frac-tions were free from Cetavlon ( <1 ,ug of quaternaryammonium ion) as measured by the reaction of Foghet al. (9).The number of fractions and yield of each obtained

by the Cetavlon fractionation varied among the dif-ferent experiments. In general, the more concentratedstarting material yielded more fractions. However,those precipitates appearing at very close NaCl molari-ties were found to be similar in serological activityand chemical composition and were, therefore, pooled.

Total carbohydrate was determined by the anthronereaction (13) with glucose as standard, and was con-sidered to represent polysaccharide content. Nucleicacid was estimated by differential adsorption at 260to 280 mMA, and was confirmed by the orcinolreaction of Nejbaum (25). Uronic acids wereestimated with the carbazole reaction of Dische(6) with glucuronic acid standards, and pro-tein was determined with the Lowry modification of

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KI (L) ANTIGEN OF E. COLI

acetone-dried cells

45%. aqueous phenol 60 C, 5 min

1,000 X gfor 10 min at 5 C

aqueous phase

two extractions with1 vol of diethyl ether

aqueous phase

10 vol of ethyl alcohol, 5 C overnight

ether phasediscard

precipitate redissolvedin water or saline

Sephadex G-50

effluent-Vi(large molecules)

10 vol of ethyl alcohol, 5 C overnight(trace of sodium acetate)

supernatant fluiddiscar-d

effluent Vo + Vi(small molecules)

discard

supernatant fluiddiscard

precipitate CRUDE ANTIGEN (mixture of 0 and K)

0.15 M NaCl

excess Cetavlon

supernatant fluidI

I vol of water

supernatant fluid precipitate("O" ANTIGEN) (0.075 M fraction)

precipitate

0.5 M NaCl

Iinsoluble soluble fractionfraction excess Cetavlon

0.1%o Cetavlon inwater added dropwise

precipitates collected as they appear

FIG. 1. Fractionation scheme for preparation of KJ (L) antigen. All Cetavlon precipitates were washed twicewith NaCI solutions of the same molarity as the precipitating solution, dissolved in I M NaCl, and precipitated threetimesfrom ethyl alcohol to remove Cetavlon. The occurrence ofthe fraction insoluble in 0.5 M NaCl is not constant.

the phenol reaction (15) with crystalline ovalbuminas a standard. Total sialic acids were measured by theresorcinol reaction (28), and free sialic acids with thethiobarbituric acid assay of Warren (30).From optical density readings obtained with crsytal-

line N-acetylneuraminic acid (NANA), and bacterialpolysaccharide free from sialic acid, simultaneousequations were derived which yielded: micromoles ofsialic acid equals absorbancy at 580 m,u (0.618) minusabsorbancy at 450 m,u (0.114) for use with the resor-cinol assay. From density readings with NANA and

crystalline 2-deoxyribose was obtained: micromoles ofsialic acid equals absorbancy at 549 m,u (0.102)minus absorbancy at 532 my (0.06) for use with thethiobarbituric acid assay.

Acid hydrolysis was in 2 N H2SO4 at 100 C for 90min; the solution was neutralized with BaCO3, andthe resulting BaSO4 was removed by centrifugation.

Paper chromatography was by the descending fronttechnique with the use of Whatman no. 1 paper andpyridine-ethyl acetate-acetic acid-water (5:5:1:3) orn-butanol-pyridine-water (6:4:3) as solvents. Papers

phenol phase+

cell debrisdiscard

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BOLANOS AND DEWIFT

were dried and developed with the O-aminobiphenylreagent of Timell et al. (29).

ECTEOLA-cellulose columns were prepared ac-cording to Ringertz and Reichard (23) with elution bystepwise increments of NaCl concentration from 0.1to 1.0 M.

RESULTS

The preferred assay ofK antigen and thus con-trol of antigen isolation procedures would havebeen bacterial agglutination inhibition with K( +)cells and anti-K antiserum. Crude K antigen, ob-tained by extraction of 02:Kl cells with aqueousphenol (Fig. 1), was shown by rabbit immuniza-tion to contain both 0 and K antigens. As wasexpected, anti-K titers of the standard antiserawere found to be as much as seven twofold dilu-tions higher by passive hemagglutination (PHA)with the crude antigen as compared with bacterialagglutination (BA), with a corresponding increasein sensitivity of the inhibition assay of antigen.Although a drastic reduction in the bacterial cellconcentration (optical density at 660 m,u, 0.3;0.05 ml per 0.5 ml of total volume) resulted in anincrease in BA serum titer so that it equaled thePHA titer, the method was not sufficiently re-producible. Use of inhibition of PHA required amore careful analysis of the specificities of theanti-K serum; the results of these tests are pre-sented in Tables 1 and 2.

In Table 1 it is shown that the anti-0 activityof the parent anti-02:Kl serum is removed byadsorption with 02 bacterial cells to yield an anti-serum with good anti-Kl activity when assayedby either BA or PHA. Both of these antisera. as

well as anti-02 serum, fail to agglutinate cells ofeither of two colanic acid-producing strains,K12(m) or K235 L + O(m), assuring us thatanticolanic acid antibody is not interfering. Fur-ther, although the anti-014 serum shows a minorcross reaction with 02-sensitized erythrocytes, itis completely negative against KI-sensitizederythrocytes, thus indicating that the common or

Kunin antigen-antibody system is not interfering.In Table 2, these results are confirmed by the

complete adsorption of anti-Kl antibody, as

measured either by BA or PHA, by living 02:K1cells or erythrocytes sensitized with Kl antigen,but not by 02 cells or K12(m) cells.When crude antigen (Fig. 1) was fractionally

precipitated as Cetavlon complexes from solutionsof decreasing electrolyte concentration, numerousfractions were collected, the number of whichvaried among different experiments. Early experi-ments yielded only one fraction (that precipi-tating at 0.19 M NaCl) positive for K antigen onthe basis of PHA inhibition with the crude K anti-gen-anti-Kl serum system. Two additional frac-tions (0.28 and 0.075 M) were capable of sensi-tizing erythrocytes without pretreatment by heator alkall. Erythrocytes so sensitized were agglu-tinated only in anti-Kl serum, and each hemag-glutination reaction was inhibited by either frac-tion.The results of two such experiments, one re-

porting inhibitory levels as dry weight, the otheras total carbohydrate, are given in Table 3. In-hibitory levels of the 0.28 and 0.075 M fractionswere 0.12 and 0.016 ,ug (as carbohydrate), whentested in their homologous PHA systems, 0.50

TABLE 1. Bacterial agglutination and hemagglutination titers of various antisera

Bacterial agglutination Hemagglutination*

Antiserum Reciprocal of serum dilution vs. Esclterichia coli strains-________-_________ -________ ______ -Isolated Lipo-

02:Ki) K antigen polysac- None02: 02:K(-) (100 C, K12(m) K235 L + 0 (m) charide

Kl(+ 60 min)

Anti-02: K1 160 5,120 5,120 0 t 0 800-3, 200 16,000 0Anti-Kl 80 0 0 0 0 800-3,200 0 0Anti-02 0 10,240 5,120 0 0 01 32,000 0Anti-014 0 160 0

Antigen Ki 02 Colanic Colanic Kl 02revealed acid acid

* Hemagglutination was determined with the indicated sensitizing fractions. Titers with isolated Klantigen depend on whether the crude antigen or one of the Cetavlon fractions (Fig. 1) was used forerythrocyte sensitization. Lipopolysaccharide was prepared by centrifugation (30,000 X g for 4 hr) of aWestphal-type lipopolysaccharide (15) followed by treatment with NaOH according to Neter et al. (20).

t Zero indicates <1:10.I Cells sensitized with crude antigen yield moderate titers here due to residual 0 antigen. Cells sensi-

tized with purified fractions are uniformly negative.

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Kl (L) ANTIGEN OF E. COLI

and 0.06 ,ug in the reciprocal heterologous sys-tems, and > 50 ,ug when crude antigen was usedfor red cell sensitization. In each case, the erythro-cytes sensitized with the 0.075 M fraction weremore susceptible to inhibition than were the cellssensitized with the 0.28 M fraction.These results were confirmed by experiments in

which anti-Ki serum was adsorbed with erythro-cytes optimally sensitized with either the 0.28 orthe 0.075 M fraction and the residual homologousand heterologous titers were determined (Table4).

Later, more careful fractionation of crude anti-gen by Cetavlon precipitation showed that, al-though precipitates were obtained at about 0.19M NaCl, they no longer displayed any serologicalactivity. Further, an artificial mixture of the 0.28and 0.075 M fractions was capable of inhibitingthe erythrocyte-crude antigen versus anti-KIserum system, suggesting that the 0.19 M fractionobtained earlier was the result of incompleteseparation of the 0.28 and 0.075 M activities. Theoccurrence of the 0.075 M fraction and its sero-logical specificity remained constant. The 0.28 Mfraction could be separated into several smallerfractions, appearing at closely related NaCl con-centrations but showing the same serologicalspecificity.The results of such an experiment are given in

Table 5, and include the chemical and serologicalanalyses of the fractions. Those precipitates whichusually appear at NaCl concentrations above 0.4M are insoluble and contain a brown pigment.Those fractions precipitating at about 0.3 and0.075 M NaCl show Kl specificity, the 0 specific-ity appearing in the supernatant fluid of the 0.075M NaCl precipitation. It was not possible to relatedirectly or simply the serological specificity to anyone of the major biochemical components (car-

bohydrate, protein, or nucleic acid), or to sialicacid. Fractions 5, 6, and 7 have KI activity andare characterized by a high nucleic acid content;fractions 3 and 4 are also mainly nucleic acid yethave no KI activity. Also, fractions 5 and 6, whichcontain the main portion of the Ki antigen pre-cipitating in this region, show an increased carbo-hydrate content. This relationship is not sup-ported by fractions 9, 10, and 11, however. Al-though all fractions were negative for uronicacids, their absence cannot be assumed because ofthe high interference of simple sugars in the carba-zole reaction (6).

Further attempts to purify the 0.28 M (pool offractions 5, 6, 7, and 8 of Table 5) and 0.075 M(fraction 12, Table 5) fractions were made by useof gel filtration and ion-exchange chromatog-raphy. Elution patterns from G-75, G-100, andG-200 Sephadex were similar in that the Kl ac-tivity of both fractions appeared in the excludedvolume, indicating a molecular size in excess of

TABLE 2. Specificity of the standard anti-KIserum as shown by antibody adsorption

Reciprocal of serumdilution

AdsorbingmaterialBacterial Hemaggluti-

agglutination a(tioe(02: Kl cells) (cruden

None..................... 80 80002:Kl cells................. 0* 002 cells (100 C, 60 min) 80 800K12(m) cellsl. 800Sheep erythrocytes sensi-

tized with crude antigen... 0 0

* Zero indicates <1:10.

TABLE 3. Serological specificity ofK antigenic fractions by hemagglutination inhibition

Fraction used for inhibition

A,ntiserumn | Erythrocyte-sensitizing Crude antigen 0.28 M 0.075 M Supernatant

Dry wt CHO Dry wt CHO Dry wt CHO CHO(JAg) 0,g) (JAg) (JAg) (JAg) (JAg) (Gg)

Anti-Ki Crude antigen 1. 56* 0.15 > 100 >50t >100 >50t > 500.28 M 0.30 2.0 0.12 12.5 0.5 >500.075 M 0.30 0.10 0.5 0.06 0.05 0.016 >50

Anti-02 Lipopolysaccharide 00.06 > 50 > 100 > 50 0.05

* Minimal inhibitory amounts vs. 3 to 4 hemagglutination units of antiserum.t A mixture of 2 ,ug of 0.28 M fraction and 0.16 Lg of 0.075 M fraction (as total carbohydrate) inhibited

the standard anti-Ki antiserum and crude antigen sensitized erythrocyte hemagglutination system. Noend point was determined.

t See Table 1.

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BOLANOS AND DEWIlT

200,000. Typical experiments with the use ofSephadex G-200 with each Ki antigenic fractionare presented in Fig. 2. It should be noted that thescale for the two experiments is different, as aboutfour times as much material was recovered fromthe 0.28 M fraction as from the 0.075 M fraction.There was considerable increase in specific Klactivity of both fractions. Most of the nucleic acidand about 50% of the protein was separated fromthe KI activity of both fractions by this method,with a resultant increase in specific serological ac-tivity.

Sialic acid behaved differently in the two frac-tions. In the 0.28 M fraction, the Kl activity wasnot separated from this compound. Further, aconstant proportion of resorcinol and thiobar-bituric acid-positive material (15% free sialicacid) was obtained. In the 0.075 M fraction, how-ever, most of the resorcinol-positive material wasseparated from the Kl activity and that not sepa-rated showed a variable percentage of free sialicacid (8 to 77%). The polysaccharide peak is quitehomogeneous in both fractions, appearing mainlyin the excluded or large molecule effluent. Theresults of these experiments indicate a very similargross composition for the two Ki antigenic frac-tions, i.e., a high carbohydrate content with lowamounts of associated protein, nucleic acid, andsialic acid. It is possible that a better separation ofthese components, particularly of the sialic acidfrom the 0.075 M fraction, could be obtained bythe use of longer columns (8).

Preliminary experiments have suggested that

ion-exchange column chromatography may beuseful for further purification. Cation-exchangeresins (Dowex-50) do not bind the Kl activity,but are excellent for removal of traces of de-tergent after the Cetavlon fractionation. Anionexchangers in the chloride or acetate form effec-tively retain Ki activity, but elution methods triedwith Dowex-2 (NaCl, sodium acetate, pH 5.5acetate buffer) have either been unsuccessful orhave resulted in Kl inactivation. ECTEOLA-cellulose in the chloride form retained the 0.075M fraction, and upon elution with increasingchloride ion concentration gave a complete sepa-ration of the carbohydrate and Kl activity (0.4M NaCl) from the nucleic acid (0.8 M NaCl).

TABLE 4. Serological specificity of KJantigenic fractions by adsorption

of hemagglutinins*

Fraction on Fraction used for adsorptionerythrocytes forhemagglutination None 0.075 x 0.28 X

0.075M 800 100 <1000.28 M 1,600 800 200

* A 1-ml amount of a 1:50 dilution of anti-Klserum was adsorbed once at 37 C for 30 min and asecond time at 4 C for 18 hr with 0.2 ml of packederythrocytes optimally sensitized with the indi-cated fraction. Adsorbed serum was then assayedin doubling dilution in the appropriate hemagglu-tination system.

TABLE 5. Characterization of Cetavlon precipitates of aqueous phenol extracts of 02:KI cellsa

Fraction no. NaCl concn Protein (mg) Ribonudeic Sialic acid (mg) Carbohydrate Serologicalacid (mg) (mg) activityb

lc 7.5 70 10 107 02:K12 0.5Md3 0.4 M 0.5 10 0.4 1.7 -4 0.36 M 0.15 6.0 0.23 1.2 _5 0.33 M 0.4 30 0.63 3 KI6 0.32 M 0.14 4.0 0.33 2 Kl7 0.30 M 0.13 2.3 0.15 0.4 Kl8 0.28 M 0.04 0.13 0.6 0.06 KI9 0.24 M 0.05 0.03 0.28 0.0410 0.20 M 0.01 0.02 0.35 0.05 _11 0.15 M 0.14 0.1 0.37 1.012 0.075 M 0.13 2.0 1.13 1.0 Kl13 Supernatant fluid 3.0 16 4.2 90 02

a The extract used was 1.0 g (dry wt) or 250 mg (carbohydrate) of aqueous-phenol extract from 7.0 g(dry wt) of acetone-dried cells.

b Hemagglutination inhibition. Erythrocytes sensitized with NaOH-treated lipopolysaccharide forassay of 0 antigen and with either 0.075 or 0.28 M fraction for assay of K antigen. Both the latter testsgave similar results.

Starting material (crude antigen).d Insoluble in water or organic solvents.

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Ki (L) ANTIGEN OF E. COLI

Samples of the 0.075 M supematant fluid (0antigen), 0.20 to 0.19 M precipitate (colanic acid),and the 0.075 and 0.28 M precipitates were sub-mitted to acid hydrolysis and paper chroma-tography. The identification of components istentative, and is based on coincident mobilities ofknown and unkncwn in two solvent systems. Theresults (Table 6) show identity of our 0.19 to 0.20M fraction with colanic acid (11). The 0 antigenappears identical with chemotype VIII of Kauff-mann et al. (17), and is thus compatible with sero-type 02. No reliable results were obtained fromthe examination of Kl active fractions, duemainly to the low yield, and possibly also to theinterference of high levels of sialic acid in the re-covery of other sugars after hydrolysis (12).Chromatographic patterns varied in different ex-periments, with the exccqtion of the consistentabsence of rhamnose and uronic acids, which dis-tinguishes the material from 0 antigen and colanicacid, respectively. Large-scale preparations forfurther fractionation are in progress.Crude Kl antigen was exposed to different tem-

peratures and hydrogen ion concentrations, andthe residual activity was measured by hemagglu-tination inhibition. It showed no decrease in

0.2QM FRACTION

,2

.1

0

.4

.3

.2.

.*

.05

.K PROTEIN

Q075M FPACTION

iPROTEIN

o.i-

SIALIC ACID.05

1_0

0.1.

NUCLEIC.2 - ACID

.1.

II1

._.| CARBOW4DRATEA.

5 ± 15 D+ Vi FRACTION

51ALIC- ACID

FRCE\Ta_r

.0 NUCLEIC ACI D

k.

CARBOYDRATE

4 Is8vi FQACTION

FIG. 2. Fractionation of the two KJ active Cetavlonpreparations by Sephadex G-200. Columns preparedwith a height-diameter ratio of 10:1.

hemagglutination inhibitory activity after 2 weeksin aqueous solution at either 5 C or room tem-perature (22 to 26 C). At temperatures abovethis, however, there was a gradual loss of activity,the speed of inactivation being proportional tothe temperature (Fig. 3).When held in phosphate buffers of different pH

at a constant temperature of 5 C, crude Ki anti-gen was stable after 4 days in thepH range of 5 to8. At hydrogen ion concentrations below or abovethis range, however, there was a rapid loss inserological activity (Fig. 4). This lability to tem-perature and pH extremes is in agreement withthe known behavior of L-type antigens when stud-ied in their natural state as part of the E. coli cap-sule.

DIscussIoNThe results presented in this paper show that it

is possible to partially purify a material containingthe serological characteristics of the Kl (L) anti-

TABLE 6. Component sugars of 0 antigenand colanic acid

0.075 M 019Sugar sup,er- 02 0.19- Colanicnatant antigen 0.20t acid

Glucose .............. + + + +Galactose ............ + + + +Fucose + +Rhamnose............ + +Glucosamine ......... + +Galactosamine ....... + +Glucuronic acid + +

o S-22°c, da4sD 37°c,hoursxlOa 6°C, bours* 1000c, mtnutes

:V 75

50

VI

p4 2

+

+

L 3 5 7 16

TimeFIG. 3. Effect of temperature on Kl as measured by

hemagglutination inhibition. Crude antigen; constantpH 7.2.

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-L TOT^L

FREE0

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BOLANOS AND DEWITT

I.' 4 J% c

>75*.4-0

04

0

FIG. 4. Effecthemagglutinationtemperature of 5

gen of E. coli.tained, each O0sodium chloridhexadecyltrimeone apparentlylogical specificition inhibition.KI antigen ma;or it may be an

in which partiations has occuiimmune sera rr

all directed ageyet showing s:specificity of ththat such diffetwo Ki fracti(the fact that nethe crude antishibit complete]adsorbed anti-]possibility. Withat we have ias it appears iprepared matetivity, containiland protein thaThe polysac

purified materiwith quaternar,Cetavlon andactive fractionsgen as measure

with lipopolysaccharide-sensitized erythrocytes,this test being capable of measuring as little as

__________________ 00.02 jig (20). Isolation of Ki-specific material freefrom 0 antigen now allows us to study the roleof this product in virulence, and such studies are

o pU5-I 8 in progress.o pW1 3.3 The serological analysis of the adsorbedA W9 anti-Ki serum assures us that we are not measur-A P 7"3 ing colanic acid, rather than Ki hapten; however,

we cannot disregard the possible presence of theformer as a contaminant of our isolates, and mustassume temporarily that part of our polysaccha-ride is colanic acid. Not only does colanic acidprecipitate with Cetavlon at the approximateNaCl molarity at which we obtain one of the Klfractions (11, 21), but also even those E. colistrains not obviously mucoid do produce small

20 40 GO / |' quantities of this substance (11, 19).Two biochemical entities apparently remain

Time Cn14ours associated with the KI activity: carbohydrate andofpH on K .activity as measured by a small amount of protein. The continual direct

t inhibition. Crude antigen; constant association of the polysaccharide peak and theC. peak of KI activity suggests that it contains the

Ki hapten. Protein is probably not involvedTwo different fractions were ob- directly in KI activity. We have on occasion ob-

ne precipitating at a characteristic tained fully active fractions with no detectablele concentration in the presence of protein, and further there is no direct relationshipthylammonium bromide and each between protein concentration and Kl inhibitorydisplaying a slightly different sero- activity.ity as measured by hemagglutina- .We believe that sialic acid deserves further con-

. This difference may be real and sideration, despite the fact that neither colominicy be composed of two specificities, acid (3) nor sialyl compounds released from theartifact of the isolation procedure, cell surface (5) show any serological activity in

l degradation of one or both frac- the Ki system. When the results of Sephadexrred. It is well known that hyper- filtration of the 0.075 M fraction (Fig. 2) are care-

ny contain a gamut of antibodies, fully analyzed, it can be seen that the major por-,iayntain determiantiboup tion of the sialic acid is delayed in elution from,mall differences in the size and the column, being separated from Kl activity,

e combining site (15). It is possible and that the ratio of free to total sialic acid variesrences are being measured in the throughout the elution series. It is possible thatDns presented here. Furthermore, the slow-moving portion of sialic acid corre-

ither of the isolated fractions, nor sponds to colominic acid, which is reported ton i dialyze slowly (2), and thus would be of a molecu-gen containig both, iS able to in- .'

ly bacterial agglutination by the lar size small enough to diffuse into G-200 Sepha-Ky bantierum, suppotsithiationythe dex. That portion of the sialyl compound that

ssle cannot be separated from Kl activity may bee cannot,at any rate,concludey ysocante atheK an.ten of E.ncl linked electrostatically to the active material as a

inlatedtheapsule; angen of hveo result of the extraction procedure, and thus be ain the capsule; however, we have contaminant rather than an integral part of Ki-rial with significant specifc ac-rialwhsnsantigen. This is supported by the high ratio ofng far less nucleic and sialic acids thiobarbituric acid positive material in the earlyin our crude extract. eluates of this experiment. However, it is alsocharide moiety of the partially possible that sialic acid constitutes a small butial is acidic in that it forms salts integral part of the serologically active molecule,y ammonium compounds such as and thus might play a role in the haptenic group.anion-exchange resins. Both Kl The lability of the K (L) antigen in naturewere obtained free from 0 anti- when submitted to high temperatures and ex-

Id by hemagglutination inhibition tremes in pH (7, 16) agrees with the results found

994 J. BACTERIOL.

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Ki (L) ANTIGEN OF E. COLI

for the fractions isolated here. This suggests thatan extraction technique which uses elevatedtemperature or acid hydrolysis, or both, is notthe most suitable.

ACKNOWLEDGMENTSWe are grateful to Walter F. Goebel of the Rocke-

feller Institute of Medical Research and to ErwinNeter of the Children's Hospital of Buffalo, N.Y., forsupplying cultures and antisera. We also wish to thankKatherine McCarron and E. Benes for their assist-ance in carrying out some experiments.

This investigation was supported by Public HealthService research grant AI-04624 from the NationalInstitute of Allergy and Infectious Diseases.

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