INFECTION AND IMMUNITY, Mar. 1983, p. 1392-14020019-9567/83/031392-1 1$02.00/0Copyright © 1983, American Society for Microbiology

Vol. 39, No. 3

Alterations in the Pathogenicity of Escherichia coli K-12 AfterTransfer of Plasmid and Chromosomal Genes from Shigella

flexneriPHILIPPE J. SANSONETTI,'t THOMAS L. HALE,' GUSTAVE J. DAMMIN,2 CHRISTINE KAPFER,'

HUGH H. COLLINS, JR.,' AND SAMUEL B. FORMAL'*Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012,' and

Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, Boston,Massachusetts 021152

Received 8 October 1982/Accepted 20 December 1982

A 140-megadalton plasmid (pWR110), which has previously been associatedwith virulence in Shigella flexneri, was transferred to Escherichia coli K-12.Segments of S. flexneri chromosomal material were then transferred to theplasmid-bearing K-12 strains. The virulence of these transconjugant hybrids was

assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test.A K-12 strain which harbored only pWR110 invaded HeLa cells, producedminimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test.Plasmid-containing K-12 hybrids which had incorporated various shigella chromo-somal regions gave differential reactions in the rabbit ileal loops and in the Serenytest. Analysis of these transconjugants indicated that three regions were linkedwith virulent phenotypes. These included the his region (when the genes responsi-ble for 0-antigen synthesis were cotransferred) and the kcp locus (linked to thelac-gal region). Either of these chromosomal regions was sufficient to allowinvasion of the rabbit ileal mucosa. In addition to both of these regions, anothershigella chromosomal segment linked to the arg and mtl loci was necessary forfluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus,derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transferof a large plasmid and three chromosomal segments from S. flexneri, appeared tocontain the necessary determinants for full pathogenicity in a variety of laboratorymodels.

The Escherichia and Shigella genera are soclosely related that they cannot be distinguishedon the basis of whole polynucleotide hybridiza-tion (1), and they can only be differentiatedbiochemically on the basis of a few reactions.The extensive genetic homology of these generafacilitates the conjugal transfer and integrationof chromosomal material from Escherichia coliHfr strains to Shigellaflexneri. Previous studieshave used this classical genetic technique toidentify areas of the Shigella chromosome whichare linked to virulence genes. For example, S.flexneri loses the ability to evoke keratoconjunc-tivitis in the guinea pig eye (Sereny test) whenthe purine E (purE) region of E. coli K-12replaces a homologous chromosomal region invirulent strains. Thus, the shigella keratocon-junctivitis provocation locus (kcp) is defined byits linkage to the purE region (5). Similarly, S.flexneri hybrids which have received the xy-

t Present address: Institut Pasteur, Paris, France.

lose(xyl)-rhamnose (rha) region from E. coli K-12 fail to cause disease when fed to starved,opiated guinea pigs (9) or to monkeys (7). Thesehybrid strains retain the capacity to invade co-lonic epithelial cells, a hallmark of virulent shi-gellae (17, 25), but they are unable to survive inthe mucosa (9). An avirulent colonial variant ofS. flexneri has been restored to virulence by theconjugal transfer of a glycerol kinase locus(glpK) from a virulent S. flexneri donor (15), andin another case, virulence has been restored bythe transfer of the maltose locus (maiB) from anavirulent E. coli K-12 (10). These studies indi-cate that several chromosomal loci must func-tion in concert or in sequence for expression ofthe virulent phenotype in shigellae.

Since virtually all available chromosomalmarkers, including those encoding shigella 0antigens and the purE locus, have been trans-ferred to an avirulent E. coli K-12 strain withoutany apparent alteration in pathogenicity (6),extrachromosomal loci are also necessary forthe expression of virulence. A virulence-associ-

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ated plasmid has recently been demonstrated inall serotypes of S. flexneri (22). Variants whichhave lost this 140-megadalton (Mdal) plasmidare uniformly avirulent, and virulence is re-stored upon reacquisition of the plasmid (22).Development of techniques for transfer of theTn5-labeled S. flexneri serotype 5 plasmid(pWR110) to E. coli K-12 has facilitated thestudy of conjugally transferred regions of theshigella chromosome as expressed in a K-12recipient which harbors the S. flexneri 140-Mdalplasmid. Thus, for the first time, we have beenable to study a continuum of virulence pheno-types as expressed in an E. coli K-12 back-ground and assayed in a variety of laboratorymodels. These studies indicate that the 140-Mdalplasmid of S. flexneri serotype 5 encodes deter-minants necessary for invasion of epithelial cellsin vitro, while chromosomal loci linked to thehistidine locus (his), the arginine (arg)-mannitol(mto region, and purE locus are required for fullexpression of virulence in animal models.

MATERIALS AND METHODSStrains. E. coli K-12 strain 395-1, a nalidixic acid-

resistant mutant of strain AB-1133, was used as therecipient in these experiments. This strain was resist-ant to 50 ,ug of nalidixic acid per ml. Three donorstrains were used. S. flexneri 2a Hfr strain 256 hasbeen previously described (4). It was prepared by themethod of F-linked terminal marker selection, and itefficiently transfers chromosomal genes in crosseswith E. coli recipients with a polarity of pro-arg-gal-lac-F. E. coli K-12 Hfr donor strain Hfr H-6 has alsobeen described before (5). This strain is a derivative ofHfr Hayes into which the shigella purE-kcp locus wasintroduced by transduction with phage P1 vir. Whenstrain Hfr H-6 was used as a donor, selections weremade for lactose utilization, and those hybrids whichalso received Gal' as a nonselected marker wereassumed to have inherited the S. flexneri kcp locus. Inmost instances, recombinants which had acquired theLac' and Gal' markers also inherited the Pro' andLeu+ traits from E. coli Hfr H-6. S. flexneri lb strainM25-8A served as the donor strain for the 140-MdalTn5-tagged plasmid (pWR110) which is identified bykanamycin resistance (Kanr). Strain M25-8A, whichhas been described previously (22), harbors a cointe-grate molecule of pWR110 and the mobilizing plasmidR64drdll, which encodes tetracycline and streptomy-cin resistance (Tetr and Strr). The characteristics ofthese strains are summarized in Table 1 and Fig. 1.Media and cultural conditions. Bacteria were rou-

tinely grown in Penassay broth (Difco Laboratories),in brain heart infusion broth, or on Trypticase soy agar(BBL Microbiology Systems). The composition ofminimal medium used for selection and scoring ofrecombinants has been described previously (4). Ami-no acids and other growth factors were added to theminimal medium to a final concentration of 50 ,ug/ml,and carbohydrates were added to a final concentrationof 0.5%. Antibiotics were used at the following con-centrations (,ug/ml): kanamycin, 20; tetracycline, 20;nalidixic acid, 50; streptomycin, 500. Fermentative

characters were scored by culturing in purple brothbase supplemented with the appropriate carbohydrateat a concentration of 0.5%. Tests for lysine decarbox-ylase (LDC) and indole production (tnaA) were per-formed by standard diagnostic procedures. The LDCmarker gene has not yet been mapped. Nevertheless,it is a useful characteristic to distinguish shigellae fromE. coli, and as noted below, transfer of the LDC-negative allele from S. flexneri to E. coli K-12 may beassociated with the transfer of certain virulence traits.Mating procedures. All donor and recipient strains

were grown at 37°C for 18 h in Penassay broth. Tofacilitate transfer of chromosomal genes, 1.0 ml ofdonor and 3.0 ml of recipient were mixed, incubatedfor various time periods, and washed twice in saline,and appropriate dilutions were plated on the selectivemedium. Equal amounts of the donor and recipientstrains were plated separately as controls. Recombi-nants were purified twice on the selective medium andscored for nonselected markers.

Transfer of the 140-Mdal plasmid was accomplishedby adding 0.1 ml of the donor and recipient strains to10 ml of Penassay broth and incubating for 18 h. Themating mixture was washed and plated on minimalmedium containing tetracycline. The transconjugantswere purified, and those which concomitantly re-ceived the Kanr marker were used for further study.

Plasmid DNA isolation and agarose gel electrophore-sis. Bacterial cells cultured in brain heart infusionbroth overnight were washed twice in TE buffer (0.05M Tris, 0.02 M disodium EDTA [pH 8]) and resus-pended in TE buffer (100 mg of bacteria per 0.5 ml).Plasmid DNA was prepared by the procedure of Casseet al. (2) and examined by electrophoresis in 0.7%agarose slab gels as described previously (16).

Serological tests. The 0-antigenic specificity oftransconjugants was determined by slide agglutinationtests with antisera which recognized S. flexneri groupfactors 3,4 or type antigens I and II. Unadsorbed E.coli K-12 strain 395-1 antiserum was also used toidentify unmodified recipient strains.

Gel electrophoresis of LPS. The Westphal procedure(26) was used to extract lipopolysaccharide (LPS), and2 jig of LPS from each strain was loaded on sodiumdodecyl sulfate-polyacrylamide gels prepared accord-ing to Laemmli (18). The running gel was 13% acryl-amide, and the polysaccharide bands were visualizedby a silver staining procedure (24).

Infection of HeLa cells. Nonconfluent monolayers ofHeLa cells were inoculated with bacteria and incubat-ed for 2 h at 37°C. The procedure for infection of tissueculture cells has been described previously (13).Giemsa-stained preparations were examined micro-scopically, and the proportion of infected cells wasdetermined.

Sereny test. The Sereny test to produce keratocon-junctivitis in guinea pigs has been described previously(17).

Rabbit ileal loop test. This procedure has also beendescribed previously (8). Ileal loops (10 cm) wereprepared in 3- to 5-lb (ca. 1.36- to 2.27-kg) anesthesizedrabbits, and these were inoculated with 1.0-ml vol-umes containing 109 cells of the test organisms. Therabbits were killed 18 h later, and the loops wereexamined for fluid accumulation. Volume-to-lengthratios were determined, and portions of the loop werefixed in 10% buffered Formalin. These fixed speci-

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TABLE 1. Characteristics of parenteral strainsStrain Species pro thr leu arg his thi nad

395-la E. coli K-12 - - - - - - +Hfr H-6d E. coli K-12 + + + + + + +256e S. flexneri 2a + + + + + +M25-8A (pWR110-R64drdll)1 S. flexneri lb + + + + + +

a 395-1 served as the recipient strain.b R, Resistance.S, Susceptibility.

d Hfr H-6 has the purE-kcp locus of S. flexneri 2a (4).e 256 served as the donor of chromosomal DNA (3).1 M25-8A(pWR11O-R64drdll) served as the donor of the 140-Mdal S. flexneri plasmid (19).

mens were processed by standard procedures andexamined microscopically for histological alterations,and the severity of lesions was scored as outlined inTable 2. Each culture was tested in a minimum of threedifferent rabbits, and the scores recorded in the tablesrepresent an average of these animals. Scores ofgreater than 1.0 signified an inflammatory lesion, with5.0 representing the severe lesion produced by wild-type S. flexneri. Controls had scores of less than 1.0.

RESULTSRole of plasmid pWR110. The effect of plasmid

pWR110 on the pathogenicity of E. coli K-12strain 395-1 was first examined. S. flexneri lbplasmid donor strain M25-8A(pWR110-R64drdll) was mated with the recipient E. colistrain 395-1, and Tetr Kanr transconjugants wereselected. The frequency of transfer was greaterthan 10-6. Due to frequent dissociation of thecointegrate plasmid, only 4 of 32 selected Tetrclones were also Kanr; however, 3 of the TetrKanr transconjugants had acquired the ability toinvade HeLa cells. Like the rough 395-1 parentstrain (Fig. 2A), these transconjugants tena-ciously adhered to HeLa cell monolayers, butcareful focusing revealed packets of organismsin the cytoplasm of cells exposed to the invasivetransconjugants (Fig. 2B). These ordered ar-rangements of bacteria, which were alwaysfound in the focal plane of the nucleolus, weresimilar to those observed in the cytoplasm ofHeLa cells infected with an invasive shigellastrain (12-14, 17). One of the transconjugantswhich invaded HeLa cell monolayers (7262-1-10) was tested in the Sereny test and failed tocause a positive reaction. It also failed to causefluid secretion when inoculated into ileal loopsin six different rabbits, but tissue specimensfrom these loops had a histological alterationscore of 1.5, a mild but definite reaction (Table3).Another experiment demonstrating the impor-

tance of pWR110 used 395-1 hybrid strain 443-2-1 which had received the his' and mtl+ shigellachromosomal loci from S. flexneri Hfr 256 andthe lac+-gal+ region from E. coli Hfr H-6. This

strain was negative in all virulence assays, butupon mating with M25-8A(pWR110-R64drdl1),two of seven Tetr and Kanr transconjugantswere able to invade HeLa cells. One of these(strain 443-2-1-Pl) evoked a 3.5 histological re-action in the rabbit ileal mucosa and caused apositive Sereny test.

It should be noted that in all experiments avariable and unpredictable preparation of cloneswhich inherited the Tetr and Kanr markers in-vaded HeLa cells. Agarose gel electrophoresisof plasmid preparations from the noninvasiveclones indicated that some no longer harboredan autonomous plasmid while others had ac-quired a plasmid with a mobility similar to thepWR110-R64drdll cointegrate molecule foundin invasive transconjugants. The R64drdll mo-bilizing plasmid had no effect on the invasive-ness of 395-1 when it was transferred withoutpWR110.

Role of the his and kcp regions. The experi-ments summarized in Table 3 were designed toinvestigate the his and kcp chromosomal regionswhich have been associated with the expressionof the group factor 3,4 somatic antigen of S.flexneri 2a (4) and keratoconjunctivitis provoca-tion (5), respectively. Strain 7172-1-5 was con-structed by mating E. coli K-12 strain 395-1 withS. flexneri Hfr 256 and selecting for arginineindependence in the 395-1 recipient. PlasmidpWR110-R64drdll was introduced into strain7172-1-5, and the plasmid-containing transconju-gant was designated 7180-5-1. The latter straininvaded HeLa cells, scored 1.5 in the rabbit ilealloop, and failed to produce a Sereny reaction.Strain 7180-5-1 was backcrossed with S. flexneri2a donor strain 256, and selections were madefor histidine independence. Two of these hy-brids, 7185-2-2 and 7185-2-12, were chosen forfurther studies. Strain 7185-2-2 had coinheritedthe S. flexneri 2a group factor 3,4 antigenicspecificity, while strain 7185-2-12 did not. Bothstrains invaded HeLa cells, as expected, andneither was positive in the Sereny test. Whenthe strains were tested in the rabbit ileal loops,

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TABLE 1-Continued

tnaA malB rha mtl fuc gal lac LDC str nal kan tet

+ + + - + - - + Rb R SC S+ + + + + + + + S S S S- - - + - + + - S S S S- - + + + - - - R R R R

strain 7185-2-2 produced a definite focal inflam-matory reaction (score of 3.0) in the laminapropria. In contrast, the tissues from loops in-jected with strain 7185-2-12 (which was serologi-cally negative for group antigen 3,4) differedonly slightly from controls (score of 1.5). Toconfirm previous observations on the impor-tance of the kcp locus in the Sereny reaction (5),strain 7185-2-2 was mated with E. coli K-12 HfrH-6, and a Lac' Gal' recombinant (strain 7194-2-3) was found to cause keratoconjunctivitis. Aswill be shown below, the S. flexneri arg+ locusis associated with fluid production in inflamedileal loops. Thus, strain 7194-2-3 (which wasarg+) evoked a score of 5.0 (Fig. 3A and B) andcaused fluid production in the rabbit ileal loopmodel.A second series of experiments was begun by

crossing the kcp+ donor strain Hfr H-6 with theplasmid-containing transconjugant strain 7262-1-10 and selecting for Lac' recombinants. A Lac'recombinant (strain 7275-1-6) which had inherit-ed the ability to utilize galactose and the kcp+locus as nonselected markers was backcrossedwith S. flexneri 2a donor strain 256, and selec-tions were made for arginine independence. The

Hfr H-6

\&

ty1_ ~ ~

FIG. 1. Chromosomal map of E. coli K-12. Lociinclude pro (proline), leu (leucine), malB (maltose),arg (arginine), rha (rhamnose), tnaA (tryptophanase),mtl (mannitol), xyl (xylose), fuc (fucose), his (histi-dine), gal (galactose), purE (purine), and lac (lactose).

selected and nonselected markers of strain 7312-1-18, which resulted from the latter mating, arepresented in Table 3. Even though it carried thekcp locus, strain 7312-1-18 failed to cause kera-toconjunctivitis in the guinea pig. Strain 7324-2-1was prepared by crossing S. flexneri chromo-somal donor strain 256 with strain 7312-1-18 andselecting for histidine independence. In additionto being His', strain 7324-2-1 agglutinated ingroup factor 3,4 antiserum and evoked a positiveSereny test.Demonstration of LPS O-polysaccharide side

chain expression. The results of in vivo virulenceassays with strains 7185-2-2 and 7324-2-1showed the importance of the his' chromosomalregion for full expression of the virulent pheno-type. The serological detection of group factor3,4 somatic antigen in E. coli K-12 hybridswhich had acquired the his locus from S.flexneriHfr 256 indicated that these hybrids had alsoacquired linked loci necessary for O-polysaccha-ride side chain expression. This was confirmedby electrophoresis of LPS preparations in sodi-um dodecyl sulfate-polyacrylamide gels. Thepolysaccharide component of LPS was visual-ized by silver staining. Figure 4 indicates thatthe 395-1 E. coli K-12 recipient contained onlythe polysaccharide core unit conjugated to lipidA (11); i.e., the strain was "rough." This LPS

TABLE 2. Scoring system for histologicalalterations in the rabbit ileal loop

Score'Abnormality

1 2 3 4 5

PMNsC in lamina propria ± + + + +Edema of the villi + + + + +Mucus depletion - villus ± + + + +Mucus depletion - crypt - ± ± + +Reduced villus/crypt ratio - - ± + +Lymphocytes in epithelium + ± + + +PMN exudate in submucosa - ± + + +PMN exudate in lumen - - ± + +PMNs in epithelium - - ± + +Bacteria in epithelium - - + + +Bacteria in lamina propria - - - + +Ulceration of the mucosa - - - + +

a-, Absent; +, variable or focal; +, present.b Wild-type S. flexneri causes a grade 5 reaction.' PMNs, Polymorphonuclear leukocytes.

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FIG. 2. HeLa cells exposed to bacteria for 2 h, washed four times, and stained by the Giemsa method. x910.(A) E. coli strain 395-1 adhered randomly to the monolayer and to the plastic substrate. (B) Transconjugant strain7262-1-10, harboring pWR110, adhered randomly but also appeared in intracytoplasmic pockets. (C) Hybridstrain 7185-2-2, which expressed the S. flexneri 2a group factor 3,4 somatic antigen, was nonadherent butretained the invasive phenotype.

structure was not altered in the plasmid-contain-ing transconjugant strain 7262-1-10. In contrast,hybrid strain 7185-2-2, which had acquired histi-dine independence and the group factor 3,4antigen after mating with S. flexneri Hfr 256(Table 3), expressed approximately 30 regularlyspaced polysaccharide-containing bands. Thesebands represent the heterogeneous 0-polysac-charide side chain lengths characteristic of asmooth LPS phenotype (11, 20). It should benoted that the apparent molecular weights ofthese LPS molecules varied slightly from thoseof the donor strain (which expressed the type IIantigen) shown in lane C. Chain lengths alsodiffered slightly in LPS extracted from His'kcp+ strain 7324-2-1. The adhesive properties ofstrains 395-1 and 7262-1-10, in the HeLa cellassay, were not observed in hybrid strains suchas 7185-2-2 which expressed the 3,4 somatic

antigen from S. flexneri (Fig. 2C). Apparently,the full complement of hydrophilic 0-polysac-charide side chains expressed by 7185-2-2 (Fig.4) affected the adhesiveness of this strain andallowed removal of extracellular organismswhen the monolayers were washed with anaqueous salt solution (13).

Role of the arg-mtl region. The next series ofgenetic experiments (Table 4) were performed toobtain some indication of the importance of thearg+ chromosomal region in virulence. Strainswere constructed by mating strain 7262-1-10with either S. flexneri 2a Hfr strain 256 or E. coliK-12 strain Hfr H-6 and selecting histidine inde-pendence or lactose utilization, respectively.Strain 7275-1-6 acquired the ability to utilizelactose and galactose from E. coli K-12 Hfr H-6and was assumed to be kcp'. In addition, itinherited the E. coli Pro' and Leu+ traits as

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GENE TRANSFER FROM S. FLEXNERI TO E. COLI 1397

TABLE 3. Importance of the shigella 140-Mdal plasmid (pWR110) and the his' or kcpt chromosomalregions to the pathogenicity of E. coli K-12 hybrids

Strain Shigella loci inherited 0-antigen Activity in laboratory modelsphenotype TCI" Mph F' Serenv

Expt 17172-1-5 (pro' leu+)' (argt rha- mtlt LDC-') R _ NDK ND ND7180-5-1 (pro' leu+) (arg+ rha- mtl+ LDC-) R + 1.5 - -

pWR1107185-2-12 (prot leut) (argt rha- tnaA intl+ R + 1.5

LDC-) pWR110 (his+)7185-2-2 (prot leut) (arg+ rha- tnaA- mntlt 3.4 + 3.0

LDC-) pWR11O (hist)7194-2-3 (argt rha- tnaA mtl+ LDC-) pWR11O 3.4 + 5.0 + +

(hist) (kcpt)

Expt 27262-1-10 pWR11O R + 1.7 - -7275-1-6" pWR110 (kcp+) R + 4.0 - -7312-1-

pWR110 (kept) (malB- arg+ rha- R + NDtnaA- mtl+ LDC-)

7324-2-1Ih pWR110 (kcpt) (malB- argt rha 3.4 + ND ND +tnaA- mtl+ LDC-) (hist)

"Tissue culture invasion.b Mucosal pathology in rabbit ileal loop (see Table 2).Fluid production in rabbit ileal loop.

"Chromosomal linkage groups in order of conjugal transfer frome Order of transfer unknown.*R, Rough.g Not done.h pro' and leu+ loci from E. co/i Hfr H-6 (see text).

nonselected markers. Strain 7290-1-19 inherited nitol utili;the shigella Hist trait from donor strain 256 and eny test (expressed the 3,4 group factor antigen. As non- caused fluselected traits, it gained the shigella Leut and of four hPro' markers and expressed the type II somatic dence we]antigen which has been associated with the pro' and threelocus. Hybrid strain 7293-2-2 was prepared by loop. Inspbackcrossing strain 7290-1-19 with E. coli strain that thoseHfr H-6 and selecting for lactose and galactose vitis receiutilization, i.e., the kcp shigella chromosomal which prnregion. This strain had lost the type II antigen, tive shigeindicating that the prot shigella locus had been Unfortunmreplaced by the prot E. coli Hfr H-6 region, and assigned -

it is likely that the leu+ shigella locus was also and it willreplaced with the leu+ E. coli region. All of characterithese strains (7275-1-6, 7290-1-19, and 7293-2-2)invaded HeLa cells, and all produced inflamma-tory reactions in rabbit loops; however, none The prccaused keratoconjunctivitis or induced fluid se- the activilcretion in ligated rabbit ileal loops. altered in

Strain 7293-2-2 was remated with S. flexneri virulence2a Hfr strain 256, and selections were made for gella gen(arginine independence or mannitol utilization. were conExperiments 7291 and 7300 were selected on region intmedia lacking arginine, and experiment 7306 ly acquirewas selected for mannitol utilization. The results 2-1 evaluaof these experiments are also summarized in in a genetTable 4. One of three hybrids selected for man- mtl+, and

S. flexneri 2a strain 256.

zation caused a delayed positive Ser-'positive in 5 days), and none of theselid secretion in the rabbit ileal loop. Allybrids selected for arginine indepen-re positive in the Sereny test (Fig. 3C),of these induced fluid in the rabbit ileal:ection of unselected markers indicatedstrains which caused keratoconjuncti-ived the rha- shigella allele, and thoseoduced fluid received the LDC-nega-Ila allele in addition to the rha allele.ately, the LDC locus has not yet beena position on the E. coli genetic map,I be important for future work that thisistic be mapped.

DISCUSSIONesent investigation clearly shows thatity of E. coli K-12 strain 395-1 can ben several models used to assess theof shigellae. Four regions of the Shi-ome were evaluated, and the strainsstructed to allow insertion of eacho an E. coli hybrid which had previous-d three other regions. Thus, strain 443-ated the effect of the plasmid pWR110tic background containing kcpt, argt -Ihist; strain 7194-2-3 (Table 3) evaluat-

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GITOM C1EXNER3 TO E. COLI 1399

ed kcp+ in a background containing arg+-mtl+,his', and pWR110; strain 7324-2-1 (Table 3)evaluated his' in a background containingpWR110, kcp , and arg+-mtl+; and strains fromexperiments 7300 and 7306 (Table 4) evaluatedarg+-mtl+ in a background containing pWR110,his+, and kcp+. Testing of these hybrids in theligated rabbit ileal loop and in the Sereny testshowed that all four shigella exogenote regionswere necessary for mucosal invasion and fluidaccumulation.

In constructing these strains, a number ofinteresting observations were made. For exam-ple, transconjugants, such as 7262-1-10 (Tables 3and 4), which contained the plasmid but nochromosomal markers, had the capacity to in-vade HeLa cells and to produce a mild lesion inthe rabbit ileal loop (score of 1.5), but they failedto give a positive reaction in the Sereny test. It isunclear why all transconjugants which harboredthe cointegrate pWR110-R64drdll plasmid werenot invasive in the HeLa cell model. It is possi-ble that deletions in pWR110, which were toosmall to be detected in agarose gels, were re-sponsible for loss of the virulent phenotype. Thevirulence determinants which the 140-Mdal plas-mid encodes are unknown, but recently, wehave demonstrated that minicells containingpWR110 incorporate radiolabeled amino acidsinto some outer membrane polypeptides (T. L.Hale, P. J. Sansonetti, P. A. Schad, S. Austin,and S. B. Formal, Abstr. Annu. Meet. Am. Soc.Microbiol. 1982, B108, p. 36). It is possible thatsome of these plasmid-encoded polypeptidesfunction as receptors modifying the bacterialsurface. These receptors could induce endocyto-sis of invasive organisms by sequentially bindingto determinants on HeLa cells or on intestinalepithelial cells.

Evidence of the importance of the group 3,4antigen in pathogenesis came from histologicalevaluation of rabbit ileal loops infected withplasmid-bearing hybrid strains 7185-2-2 and7185-2-12 (Table 3). Both strains inherited thehis+ shigella chromosomal locus. Strain 7185-2-2 also expressed the group factor 3,4 antigen,multiplied in the intestinal epithelium, and pro-

duced a moderate inflammatory reaction in theintestinal mucosa (score of 3.0). Strain 7185-2-12did not express the 0 antigen and caused only amild reaction (score of 1.5). Another example ofthe importance of the 3,4 group factor antigen inpathogenicity came from experiments withstrain 7312-1-18 (Table 3). This strain inheritedthe shigella kcp+ locus and markers in the arg-mtl region but remained His- and did not ex-press the shigella 3,4 group antigen. Strain 7312-1-18 did not cause keratoconjunctivitis, butupon acquisition of the ability to express the 3,4antigen, it produced a positive Sereny test(strain 7324-2-1; Table 3).

Silver-stained polyacrylamide gels of LPS ex-tracts showed that the acquisition ofpWR110 bystrain 7262-1-10 did not add any O-polysaccha-ride side chains to the rough E. coli K-12 recipi-ent, but the addition of the his' region from theshigella chromosome was associated with theexpression of group 3,4 antigen and the biosyn-thesis of approximately 30 side chain repeatunits. When the pattern of LPS molecules fromstrains 7185-2-2 and 7324-2-1 was compared withthat of the his' region donor strain 256, slightdifferences in molecular weight were found.Since the hybrid strains expressed group 3,4antigenic specificity, the N-acetylglucosamine-(1-2) -rhamnosyl- (l1~2) -rhamnosyl-(l1 > 3)-rhamnose primary chain structure was con-served (23); however, these hybrids lacked thetype II antigen which consists of a secondaryside chain of ot-glucosyl residues (7). Apparentlysuch modifications are detectable in sodiumdodecyl sulfate-polyacrylamide gels. A his' hy-brid which had acquired the kcp+ locus (strain7324-2-1) also synthesized LPS molecules ofslightly different electrophoretic mobility. None-theless, a survey of isogenic kcp+ and kcp-strains indicated that the ability to induce kera-toconjunctivitis was not associated with modifi-cations in the electrophoretic mobility of LPSmolecules (data not shown).

E. coli Hfr H-6 was chosen as the donor of thepurE-kcp+ region because the amount of shigellagenome which was transferred could be limited.When this region was transferred to E. coli K-12

FIG. 3. (A) Rabbit ileum 7194-2-3 (Table 3). The epithelium is interrupted and infiltrated with polymorphonu-clear leukocytes and lymphocytes. There is edema of the lamina propria, especially subjacent to the epithelium,and polymorphonuclear leukocytes are prominent. Bacteria are noted in the epithelium and in the subjacentspace (see arrow). The histological alteration score is 4.0. Giemsa strain; magnification, x800. (B) Rabbit ileum7194-2-3. This field is adjacent to that in (A). The higher magnification shows bacteria in superficial and deepportions of the lamina propria which is edematous and infiltrated by polymorphonuclear leukocytes (see arrows).Giemsa strain; magnification, x2,000. (C) Guinea pig cornea 7300-1-5 (Table 4). The epithelium on the left isedematous but intact and on the right is interrupted by a corneal ulcer in which there are clusters of bacteria (seearrow on right). The base of the ulcer consists of polymorphonuclear leukocytes. Bacteria in epithelial cellsadjacent to the ulcer (see arrow on left) have propagated but have not yet injured them. The pattern is that of apositive Sereny test. Giemsa stain; magnification, x2,000.

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A B C D EA.f .:

*' 44 '. ..-

AD* .

_ ..

s

FIG. 4. Silver-stained sodium dodecyl sulfate-polyacrylamide gel showing electrophoretic pattern ofLPS extracted from (lane A) E. coli K-12 strain 395-1,(lane B) hybrid strain 7262-1-10, (lane C) S. flexneri 2astrain 256, (lane D) hybrid strain 7185-2-2, and (lane E)hybrid strain 7324-2-1. Arrows which mark LPS mole-cules with 20 O-polysaccharide repeat units illustrateelectrophoretic heterogeneity among these strains.

strain 395-1 which contained pWR110 (strain7262-1-10), the resulting hybrid (strain 7275-1-6)produced a marked inflammatory reaction(score of 4.0) in the rabbit ileal loop (Table 4).

This was an unexpected observation, for previ-ous results with strains 7180-6-1 and 7185-2-12had indicated that an organism must possesssomatic antigens in order to elicit an inflamma-tory response. This was not the case with strain7275-1-6, which neither contained the his' shi-gella locus nor expressed the shigella groupantigen 3,4. Apparently the kcp+ locus aloneenhanced the survival of a rough, invasive E.coli hybrid long enough to evoke a significanthost response. The inheritance of the kcp+ locuswas also a factor in determining whether a straincould cause a positive Sereny test. A plasmid-containing E. coli K-12 hybrid with up to one-

half of the shigella chromosome (7185-2-2; Table3) did not produce keratoconjunctivitis, but itbecame Sereny positive after acquiring thepurE-kcp+ locus from E. coli K-12 strain Hfr H-6 (7194-2-3; Table 3). The possession of plasmidpWR110 and the purE-kcp+ and the 3,4 groupantigen loci was not sufficient, however, toconfer the ability to produce a positive Serenytest. Genes in the arg-mtl region were alsonecessary. Although the number of hybridsstudied was small, inheritance of the arg+ andrha- shigella alleles appeared to be associatedwith the capacity to cause keratoconjunctivitis,while the shigella mtl+ and rha- loci were

associated with fluid production (Table 4). Itshould be emphasized that the exact size ofexogenote incorporated by hybrids which were

selected for arginine utilization is unknown.Chromosomal segments from malB (located at

TABLE 4. Importance of the arg+-mtl+ shigella chromosomal region to the pathogenicity of E. coli K-1:2hybrids

Activity in laboratoryStrain Shigella loci inherited p-antigen models

TCI" MP" F' Sereny

7262-1-10 pWR110 R' + 1.5 - -7275-1-6e pWR110 (kcpt) R- + 4.0 - -

7290-1-19 pWR110 (pro+ leu+)1 (his+) 3,4 11 + 3.0 - -

7293-2-2e pWR110 (his') (kcp') 3,4 + 2.0 - -

7306-1-2e pWR110 (his+) (kcp+) (tnaA- mtl+) 3,4 + 1.5 - -

7306-1-3e pWR110 (his+) (kcp+) (malB- tnaA- mtl+) 3,4 + 3.5 - -

7306-1-8e pWR110 (his+) (kcp+) (rha- tnaA- mtl+) 3,4 + 3.5 - +g

7300-1-15e pWR110 (his+) (kcp+) (malB- arg+ rha- tnaA- mtl+) 3,4 + 4.0 - +

7291-1A-10e pWR110 (his+) (kcp+) (malB- arg+ rha- mtl+ LDC-") 3,4 + 4.5 + +7300-1-2e pWR11O (his+) (kcp+) (malB- arg+ rha- mtl+ LDC-) 3,4 + 4.5 + +7300-1-5e pWR110 (his+) (kcp+) (arg+ rha- tnaA- mtl+ LDC-) 3,4 + 4.0 + +

a Tissue culture invasion.b Mucosal pathology in rabbit ileal loop.Fluid production in rabbit ileal loop.

d R, Rough.e pro+ and leu+ loci from E. coli Hfr H-6 (see text).J1Chromosomal linkage groups in order of conjugal transfer from S. flexneri 2a strain 256.9 Delayed reaction.h Order of transfer unknown.

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GENE TRANSFER FROM S. FLEXNERI TO E. COLI 1401

90 min on the E. coli chromosome) to fuc (60min) could have been incorporated in virulenttransconjugants. However, unselected distalmarkers are not incorporated at a high frequencyin Shigella x E. coli hybrids (3), so the apparentlinkage of the 10-min arg-mtl chromosomal seg-ment with virulence may be significant. Experi-ments involving transduction of limited seg-ments of the shigella arg-mtl region can now beattempted by selecting markers such as arg+,rha-, and LDC- which have shown an associa-tion with the virulent phenotype.The present studies confirm previous observa-

tions concerning the role of the his'-linkedsomatic antigen (19), the 140-Mdal plasmid (21,22), the kep locus (5), and the arg-mtl (9) regionin the virulence of S. flexneri. Beyond this, thestrains which were constructed may prove ofvalue in correlating bacterial virulence determi-nants with biological activities. For instance, theHeLa cell-invasive transconjugant strain 7262-1-10, which harbors pWR110 and no detectableshigella chromosomal material, could offer ad-vantages in studying the mechanism of epithelialcell penetration by shigella-like organisms. Fur-thermore, the role of shiga-like toxin in fluidproduction might be confirmed by demonstrat-ing the presence of toxin in fluid-inducing hy-brids and its absence in non-fluid-inducingstrains.The results reported here have raised ques-

tions concerning the Sereny test. In the past wehave considered this test to be the laboratoryindicator to screen E. coli for the ability toinvade the intestinal mucosa, and certainly it is avaluable tool. We have constructed strains,however, which do not produce keratoconjunc-tivitis but which do invade the rabbit intestineand cause an inflammatory response. If theseorganisms can be prepared in the laboratory bysimple mating and selection procedures, itseems reasonable to assume that strains withsimilar characteristics can exist in nature.Should this be the case, such organisms could beetiological agents in some forms of inflammatorybowel disease which are presently not diag-nosed. The HeLa cell assay can be used todetect such organisms, and a study is in progressto examine this possibility.

It must be stressed that in this study only theclassical techniques of mating and selectionwere used to alter the biological activity of E.coli K-12. Procedures encompassing the field ofrecombinant DNA technology were neither usednor required. Strains of virulent, shigella-like E.coli already exist in nature. There is no reason todoubt that there are other strains with the inter-mediate properties of pathogenicity similar tothose which we have constructed. The lattercould obviously represent stages in evolution.

Finally, the present work adds confirmatorydata to the concept that the pathogenicity ofshigellae is determined by a multiplicity of genesfunctioning in concert or in sequence to conferfull virulence.

ACKNOWLEDGMENTS

We thank 0. Washington, S. Austin, S. Bondarew, and R.Constantineau for excellent technical assistance and J. C.Guidas for her help in preparation of the manuscript.

P.J.S. received a National Research Council Fellowshipwhile conducting this work. G.J.D. was supported by the U.S.Army Medical Research and Development Command contractno. DA 49-193-MD-2530.

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