INFECTION AND IMMUNITY, Apr. 1983, p. 236-244 Vol. 40, No. 10019-9567/83/040236-09$02.00/0Copyright 0) 1983, American Society for Microbiology

Effect of Lipopolysaccharide Mutations on the Pathogenesis ofExperimental Salmonella Gastroenteritis

CLIFFORD S. MINTZt* AND R. H. DEIBELDepartment ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received 8 September 1982/Accepted 19 January 1983

Lipopolysaccharide mutants of Salmonella typhimurium provoked diminishedamounts of fluid in rabbit ileal loops as compared with the response to the wildtype. The responses elicited by these mutants ranged from 0 to 60% of that causedby the parent strain. Two completely rough mutants and one leaky rough mutantwere chosen for further study. Purified lipopolysaccharide from the parent and themutant strains failed to stimulate fluid exsorption in ileal loop experiments.Histological studies revealed that the three lipopolysaccharide mutants were lessinvasive than wild type and were less able to generate an inflammatory reaction inthe rabbit ileum. A Salmonella enterotoxin was present in culture filtrates fromone rough mutant and the wild type; however, the rough mutant appeared toproduce less toxin. Enterotoxic activity was absent in culture filtrates from thetwo other rough mutants. These results suggest that reductions in both invasive-ness and the ability to produce Salmonella enterotoxin decreased the ability ofthese mutants to provoke fluid exsorption. Also, the results indicate thatlipopolysaccharide mutations can have a profound effect on the enteropathogenicproperties of S. typhimurium.

Salmonella-mediated gastroenteritis is char-acterized by diarrhea, abdominal pain, vomiting,and, in some instances, fever. The exact causeof the diarrhea associated with the disease isunknown. Until recently, it was generally be-lieved that the diarrheal response elicited bySalmonella was due largely to the ability ofthese organisms to invade the small intestinalmucosa (6, 7). The resultant inflammatory re-sponse could directly or indirectly stimulatefluid secretion (8, 9). However, in recent years,several enterotoxic factors produced by Salmo-nella spp. have been isolated and characterized(13, 15, 28, 29, 31, 32; D. M. Sedlock, Ph.D.thesis, University of Wisconsin, Madison,1979).The first of these factors was described by

Koupal and Deibel (15) and later by Sedlock etal. (32). Using the infant mouse and rabbit ilealloop assays, they were able to demonstrate theexistence of an enterotoxic component in fil-tered culture supematant fluids from S. typhi-murium and other Salmonella species. The Sal-monella enterotoxin was heat stable and proteinin nature, and the majority of enterotoxic activi-ty was associated with the outer membrane ofthe cell wall. A second factor has been described

t Present address: Department of Microbiology and Immu-nology, Oregon Health Sciences University, Portland, OR97201.

by Sandefur and Peterson (29). They isolatedfrom cultures of S. typhimurium a heat-labilefactor that produced a delayed skin reactionidentical to that produced by Vibrio choleraeenterotoxin. Like cholera toxin, the Salmonella-delayed permeability factor (now called Salmo-nella toxin) was shown to cause elongation ofChinese hamster ovary cells (13, 29).Taylor and Wilkins (37) found that rough

mutants derived from smooth enteropathogenicstrains of S. tennessee and S. anatum failed tostimulate fluid accumulation in rabbit ilealloops. Aside from this study, little attention hasbeen paid to the role of lipopolysaccharide(LPS) in the pathogenesis of Salmonella-mediat-ed gastroenteritis. Through the use of LPS mu-tants derived from an enterotoxigenic strain ofS. typhimurium, we hoped to gain a betterunderstanding of the relationship between LPS,invasiveness, and the Salmonella enterotoxin inthe pathogenesis of Salmonella-mediated diar-rheal disease.

MATERIALS AND METHODSStrain employed. The organism used in this study, S.

typhimurium PHL67342, was obtained from EleanorChristenson, Wisconsin State Laboratory of Hygiene,Madison, Wis., and has been shown to produce Sal-monella enterotoxin (31, 32). Stock cultures weregrown on nutrient agar slants, maintained at roomtemperature, and transferred every 2 to 3 months.

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LPS MUTATIONS AND SALMONELLA GASTROENTERITIS 237

Isolation of LPS mutants and phage testing. LPSmutants of S. typhimurium PHL67342 were isolated byselection with Felix-O and P22 phages as described byWilkinson et al. (41). These mutants, as well as thewild type, were tested for sensitivity or resistance to abattery of rough-specific and smooth-specific Salmo-nella phages (kindly supplied by B. A. D. Stocker,Stanford University, Stanford, Calif.) according to themethods of Wilkinson et al. (41).

Serological determinations. Somatic (0) antigenanalysis and flagellar (H) antigen analysis were per-formed by the U.S. Department of Agriculture Nation-al Veterinary Services in Ames, Iowa.

Isolation and purification of LPS. In all cases, LPSwas isolated from organisms grown in brain heartinfusion broth (BHI, Difco Laboratories, Detroit,Mich.) for 16 h at 37°C on a Gyrotory shaker (NewBrunswick Scientific Co., New Brunswick, N.J.) setat 100 rpm. After incubation, the cells were harvestedby centrifugation at 18,000 x g for 15 min at 4°C andwashed once with distilled water.LPS was isolated from strains PHL67342 and RM3

by using the hot phenol-water method (40). The phen-ol:chloroform:petroleum ether method was used toisolate LPS from mutants RM4 and RM8 (5). All LPSswere purified by repeated centrifugation at 100,000 xg.

Chemical composition of LPS. The estimations ofglucose, galactose, rhamnose, mannose, heptose, andN-acetylglucosamine were carried out by gas-liquidchromatography, using the alditol acetate derivitiza-tion method described by Albersheim et al. (1). Myo-inositol was added to all samples and served as aninternal standard. The sugar derivatives were analyzedon a Varian model 3770 gas chromatograph (VarianInstrument Group, Palo Alto, Calif.) equipped with aflame ionization detector. Two columns were used foranalysis. The first column (1.83 m by 2 mm) contained3% SP2340 on Supelcort 100 to 200 mesh. The secondcolumn (1.83 m by 2 mm) contained 3% OV275 onGas-Chrom Q 100 to 200 mesh, and it was primarilyused for the N-acetylglucosamine analysis.

2-Keto-3-deoxyoctonic acid (KDO) determinations.KDO determinations were made by using the thiobar-bituric acid methods of Osborn (25) and Warren (38).Standards for 2-deoxyribose were included to correctfor the interference due to 2-deoxysugars (38). Sam-ples and standards were read at 534 nm (absorbancemaximum of deoxysugars) and 552 nm (absorbancemaximum of KDO) in a Gilford spectrophotometermodel 250 (Gilford Instrument Laboratories, Inc.,Oberlin, Ohio).

Protein determinations. Protein determinations weremade according to the modified Lowry proceduredescribed by Markwell et al. (22). Bovine serumalbumin (Sigma Chemical Co., St. Louis, Mo.) wasused as a standard.Sodium dodecyl sulfate (SDS)-polyacrylamide gel

electrophoresis. The discontinuous gel system de-scribed by Palva and Makela was used for LPS analy-sis (26). LPS samples (100 ,ug) were suspended inbuffer (pH 7.8) which contained 1.0o SDS, 1.0o 2-mercaptoethanol, 0.02 M Tris, 0.008 M sodium phos-phate, and 9% glycerol, with 0.2% phenol red added asthe tracking dye. The samples were solubilized byheating at 100°C for 5 min. Electrophoresis was run atconstant current (12.5 mA) for approximately 5.5 h.

After electrophoresis, the gels were stained for carbo-hydrate by using the periodic acid-Schiff proceduredescribed by Fairbanks et al. (4).

Rabbit ileal loop assay. The rabbit ileal loop assaydescribed by Sedlock and Deibel (31), with a 1%solution of N-acetyl-L-cysteine (Sigma) as a mucolyticwash, was used in this study. Unless otherwise indi-cated, the sample volume was 2 ml and the incubationtime of the assay was 8 h. Sterile BHI broth was usedas a negative control, whereas the positive controlconsisted of a 16 h aerobically grown (16 h at 37°C)BHI broth culture of S. typhimurium PHL67342. Loopratios were calculated as the volume of fluid (millili-ters)/length of intestine (centimeters), and only datafrom those rabbits which responded appropriately tothe controls were recorded.

Histological examination of rabbit small intestinaltissue. Tissue from rabbit ileal loops exposed to aerobi-cally grown BHI broth cultures (16 h at 37°C) of eitherS. typhimurium PHL67342 or LPS mutants was re-moved and fixed in 10%o neutral Formalin. The fixedtissue was sectioned in paraffin and stained withhematoxylin-eosin or Brown and Brenn stain. Controlsections were prepared from rabbit ileal loops inocu-lated with phosphate-buffered saline (PBS), pH 7.4, orsterile BHI broth. The stained sections were examinedfor inflammation and the presence of S. typhimuriumby two individuals who did not have any prior knowl-edge of the experimental conditions employed.

Recovery of S. typhimurium PHL67342 and LPSmutants from rabbit ileal loops. Rabbit ileal loops wereconstructed as previously described by Sedlock andDeibel (31). A 2-ml volume of an aerobically grown (16h at 37°C) BHI broth culture of the test organism wasinoculated into a loop; 7.5 h later, the animal wasanesthetized and the intestine was externalized. Then2 ml of the initial inoculum (kept at 4°C) was injectedinto a second loop that was constructed during theoriginal surgical procedure. After 30 min, the animalwas sacrificed by an ether overdose. The 0.5- and 8-hloops were excised, the fluid in the loops was drainedand its volume was measured, and the length of theintestinal segment was determined. The excised tissuewas then weighed and homogenized in 10 ml of sterilePBS, pH 7.4. The tissue homogenates and correspond-ing fluid from each loop were serially diluted in PBSand quantitatively plated in duplicate on MacConkeyagar plates. After 24 h of incubation at 37°C, viablebacteria in each preparation were estimated. Repre-sentative colonies were picked and identified as S.typhimurium, using the H-antigen analysis. In all ex-periments, aerobically grown (16 h at 37°C) BHI brothcultures of the parent strain were included as positivecontrols, and sterile BHI broths served as the negativecontrols.

RESULTSLPS mutants. Sixteen spontaneous mutants

resistant to either Felix-O or P22 phage wereisolated. These mutants were tested for sensitiv-ity to smooth-specific phages (lyse only smoothstrains), smooth-rough specific phages (lysesmooth and some rough strains), or rough-spe-cific phages (lyse only rough strains). All of theisolates exhibited phage sensitivity patterns

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238 MINTZ AND DEIBEL

characteristic of rough strains (18, 41). On thebasis of their phage sensitivity patterns, themutants were placed into six distinct groups.Somatic antigen analysis revealed that 14 of the16 isolates did not have any detectable 0-anti-genic material present at the cell surface. Two ofthe isolates appeared to produce intact 0 anti-gen, as indicated by positive slide agglutinationtests.One mutant from each of the phage groups

was selected for further study. The phage sensi-tivity patterns and cultural characteristics ofeach of these mutants are shown in Tables 1 and2. Each of the six rough mutants exhibitedbiochemical profiles identical to that of strainPHL67342 when tested in the API 20 Enterobac-teriaceae system (API, 20E, Analabs Products,Plainview, N.Y.). Also, all six mutants ferment-ed galactose and were motile.

Rabbit ileal loop response to LPS mutants of S.typhimurium PHL67342. Whole culture prepara-tions of all mutants except RM4 provoked fluidexsorption in the rabbit ileum. However, therabbit ileal loop response to each of the mutantswas significantly less than the response elicitedby BHI whole culture preparations of the parentstrain PHL67342 (Table 3). Most notably, wholeculture preparations of strain RM4 failed tostimulate a secretory response in rabbit ilealloops. The inability of strain RM4 to stimulatefluid exsorption in the rabbit ileum was observedafter both 8 and 24 h of incubation.

Characterization and chemical composition ofPHL67342 and rough mutant LPS. On the basisof rabbit ileal loop responses, phage sensitivitypatterns, and cultural characteristics, rough mu-tants RM3, RM4, and RM8 were selected forfurther study.

Partially purified LPS preparations from all ofthe organisms contained trace amounts of pro-tein (1 to 3% [wt/wt]) and were essentially freefrom nucleic acid contamination, as determinedby gas-liquid chromatography. To ensure that no

enterotoxic activity was associated with any ofthe LPS preparations, 0.5 mg (dry weight) of theLPS from strains PHL67342, RM8, RM4, andRM3 was tested in the rabbit ileal loop assay. Noenterotoxic activity was detected in any of theLPS preparations tested. Attempts to test great-er amounts of LPS in rabbit ileal loops usuallyresulted in the premature death of the rabbits.The LPS of strain PHL67342, RM3, RM4, and

RM8 was initially characterized by using SDS-polyacrylamide gel electrophoresis. After elec-trophoresis, periodic acid-Schiff-stained prepa-rations of the tract of LPS from strainsPHL67342 and RM3 showed two bands: a com-pact fast-moving band and a large, diffuse, slow-moving band (Fig. 1). The fast-moving band hasbeen shown to correspond to LPS core devoid of0-antigenic material (14). The second, moreslowly moving band most likely represents com-pleted LPS molecules that contain differentnumbers of repeating units in the 0-side chains(11, 14, 26). The presence of numerous LPSmolecules with 0-side chains of different lengthsmay account for the smeared, diffuse appear-ance of the slow-moving band. The electropho-retic patterns of PHL67342 and RM3 LPS arecharacteristic of LPS produced by smoothstrains of S. typhimurium (14, 23). However,close examination of the gels revealed that RM3produced significantly more core material thandid PHL67342 (Fig. 1). This result suggestedthat RM3 LPS was somewhat different fromwild-type PHL67342 LPS.

Similarly treated preparations of RM4 andRM8 LPS showed only the fast-moving band(Fig. 1). This electrophoretic pattern is charac-teristic of LPS produced by rough strains of S.typhimurium (11, 14, 23).The carbohydrate composition of PHL67342,

RM3, RM4, and RM8 LPS was estimated bygas-liquid chromatography (Table 4). PHL67342LPS contained the usual component of core-specific sugars and large amounts of the 0-

TABLE 1. Phage sensitivity patterns of S. typhimurium PHL67342 and LPS mutantsSensitivitya to the following phage:

Smooth-Strain Smooth-specific rough Rough-specific

specificP22 P22h 9NA Felix-O 6SR 4X174 Br2 Br6O Ffm C21 P221

PHL67342 + + + +RM1 - - - - + _ _ + + _ _RM2 - + - - + - - + + - -RM3 - - - - + + + + + - _RM4 - - - - + - + + +RM8 - - - - + - + + ± - -RM17 - - - - + - - + + - -

a +, Sensitive; -, resistant; ±, thinning or plaques evident.

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TABLE 2. Cultural and colonial characteristics ofS. typhimurium PHL67342 and LPS mutants

Strain Colonial Auto- 0 Hmorphology' agglutination" antigen antigenPHL67342 Smooth - + +RM1 Rough + - +RM2 Rough + - +RM3 Intermediate - + +RM4 Rough + - +RM8 Rough + - +RM17 Rough + + +

aAppearance on nutrient agar.b Static brain heart infusion broth culture grown at

37°C for 24 h.

specific sugars, rhamnose and mannose. This isrepresentative of LPS produced by smoothstrains of S. typhimurium (12). Strain RM3,which exhibited a rough phage sensitivity pat-tern, produced LPS which contained smallamounts of 0-specific sugars and abundantamounts of core-specific sugars, especially hep-tose. Rough mutants of S. typhimurium produceLPS enriched for heptose (10). In view of thesefindings, it appeared that RM3 was a leakymutant which produced both rough and smoothLPS. The abundant quantities of core-specificsugars and the absence of 0-specific sugars inRM4 and RM8 LPS confirmed the rough pheno-type of these mutants.

Histological examination of rabbit intestinaltissue after exposure to S. typhimuriumPHL67342, RM3, RM4, and RM8. The resultsfrom rabbit ileal loop experiments with roughmutants RM3, RM4, and RM8 showed that eachof the mutants elicited quantitative differences inthe rabbit ileal loop response. Also, on the basisof phage sensitivity patterns and sugar analysis,there appeared to be differences in the composi-tion of the LPS produced by each of the mu-

TABLE 3. Rabbit ileal loop response to LPSmutants of S. typhimurium PHL67342

Strain tested Fluid accumulation"

RM4...... 0.0 (5)CRM1...... 0.15 +0.1 (9)RM17...... 0.34 0.05 (5)RM3...... 0.35 ± 0.06 (3)RM2...... 0.38 ± 0.2 (4)RM8...... 0.47 ± 0.18 (6)PHL67342b .. .... 0.75 ± 0.08 (32)

a Fluid accumulation represents the average ilealloop ratio (milliliters to centimeters) ± standard errorof the mean. The numbers in parentheses indicate thenumber of tests performed.

b Wild-type organism included for comparison.c The same result was observed after 24 h of incuba-

tion in the rabbit ileum.

24

FIG. 1. SDS-polyacrylamide gel electrophoresis ofextracted LPS preparations. LPS (100 ,ug) was addedto each lane. The gel was stained for carbohydrate bythe periodic acid-Shiff reagent method. Lane 1,PHL67342 LPS; lane 2, RM3 LPS; lane 3, RM4 LPS;and lane 4, RM8 LPS.

tants. The possibility existed that a change in theLPS structure would result in a change in inva-sive potential and fluid exsorption. Thus, aninvestigation of the effect of the various mutantson the histology of the rabbit ileum was under-taken.

Control sections, obtained from rabbit ilealloops inoculated with BHI broth or PBS, exhib-ited normal intestinal villi. However, the surgi-cal procedure used to construct the loops gener-ated a mild inflammatory response, as evidencedby the presence of polymorphonuclear leuko-cytes (PMN) in the submucosal regions.

After 8 h of incubation in the rabbit intestine,whole culture preparations of strain PHL67342generated an acute inflammatory reaction char-acterized by marked edema and a cellular infil-tration of the lamina propria and submucosalregions. Significant numbers of PMN were ob-served in the lamina propria and submucosalregions as well as in the lacteals of the villithemselves. In general, villous morphology ap-peared somewhat disrupted. A significant accu-mulation of mucus containing PMN, lympho-cytes, and plasma cells was observed in thelumen. An examination of the Brown and Brennstained tissue sections revealed the presence ofgram-negative bacteria (presumably S. typhi-murium) in the lacteals of the villi. Also, largenumbers of gram-negative bacteria were ob-served in the lumen associated with the mucus.Whole culture preparations of strain RM3

caused a low-grade inflammatory response char-acterized by slight edema and the presence of

t1. t._!

*.Y: ..|N T*: . .... .. ::.

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TABLE 4. Sugar composition of PHL67342, RM3, RM4, and RM8 LPSsMol% of sugar"

LPS from:Abe Rha Man Gal Gic GlcNac Hep KDOb

PHL67342 NDC 20.6 23.5 29.4 11.8 8.8 5.9 1.6RM3 ND 2.9 8.0 32.4 16.2 16.1 24.2 3.6RM4 ND 0.0 0.0 30.0 26.6 16.6 27.0 3.7RM8 ND 0.0 0.0 28.9 27.5 14.5 28.9 5.3

a Abe, abequose; Rha, rhamnose; Man, mannose; Gal, galactose; Glu, glucose; GlcNac, N-acetylglu-cosamine; Hep, heptose; KDO, 2-keto-3-deoxyoctonic acid.

b KDO values are expressed as percentage of total LPS.c ND, Not determined.

small numbers of PMN in the submucosal re-gions. The villous morphology appeared intact.Mucus containing gram-negative bacteria and afew PMN was observed in the lumen. No bacte-ria were seen in the mucosal or submucosalregions of the intestine.Whole culture preparations of strain RM8

caused a slight inflammatory response similar tothat observed in the control sections. There wasslight edema, and a few PMN could be seen inthe lamina propria and submucosal regions. Vil-lous morphology appeared intact. Some mucus,containing a few gram-negative organisms andPMN, was seen in the lumen. No bacteria wereseen in the mucosal or submucosal regions of theintestine.Whole culture preparations of strain RM4

generated a low-grade inflammatory reactionequal to that observed in the control sections.Some PMN were seen in the lamina propriaaround the crypts and in the submucosal re-gions, and no edema was evident. Gram-nega-tive bacteria were seen in the lumen but not inthe mucosal or submucosal regions of the intes-tine, and some mucus was present, but it did notcontain any PMN.The results of this histological study suggest

that live whole culture preparations of strainPHL67342 are capable of causing an acute in-flammatory reaction in rabbit ileal loops after 8 hof exposure. The presence of gram-negativebacteria in the mucosal regions of PHL67342-infected intestinal tissue confirms the invasivenature of this strain. Strain RM3, which pro-duces small amounts of complete LPS, is capa-ble of causing a mild inflammatory reaction inrabbit ileal loops which is greater than thatgenerated by the surgical procedure. However,the inflammation caused by RM3 is somewhatless than that produced by the parent strainPHL67342. Strain RM8, which produces LPSdevoid of 0-side chains, produced an inflamma-tory reaction slightly greater than or equal tothat observed in control loops. Strain RM4,which also produces rough LPS, produced aninflammatory reaction equivalent to that gener-

ated by the surgical procedure in the construc-tion of the loops. In both cases, the inflamma-tion caused by rough mutants RM4 and RM8was much less than that observed in RM3- andPHL67342-infected rabbit ileal loops.

Survival of strains PHL67342, RM3, RM4, andRM8 in rabbit ileal loops. Rough strains of S.typhimurium have been shown to be more sus-ceptible than smooth strains to the action ofvarious antibiotics, dyes, and bile salts (30).Also, rough mutants have been found to be moresusceptible to phagocytosis by PMN and macro-phages in vitro as compared with correspondingwild-type strains (34). The possibility existedthat the reduced inflammatory response elicitedby rough mutants RM3, RM4, and RM8 resultedfrom the inability or reduced ability of theseorganisms to survive in the rabbit small intes-tine. To test this hypothesis, rabbit ileal loops(constructed in the same animal) were exposedfor 0.5 and 8 h to whole culture preparations ofeither PHL67342, RM3, RM4, or RM8. Afterexposure, both loops were excised, and thenumber of viable bacteria associated with theintestinal tissue and fluid from each loop wasdetermined.There was no significant difference in the

number of Salmonella cells recovered from the0.5- or the 8-h loops (fluid and tissue) for any ofthe strains tested (data not shown). The resultsfrom these experiments showed that rough mu-tants RM3, RM4, and RM8 could survive andpersist in the rabbit small intestine as well asstrain PHL67342. Morever, there was no signifi-cant difference in the number of Salmonella cellsrecovered per gram of intestinal tissue fromeither the 0.5- or 8-h loops for any of the strainstested (data not shown). This suggests that thesemutants do not have altered adhesive propertiesand that they can associate with and survive onthe rabbit small intestinal mucosa as well as thewild type can. The results from these experi-ments show that an increased susceptibility ofRM3, RM4, and RM8 to phagocytosis or bilesalts or to any other component of the intestinalmilieu was not responsible for the reduced in-

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LPS MUTATIONS AND SALMONELLA GASTROENTERITIS 241

flammatory responses elicited by these orga-nisms.

Production of SalmoneUa enterotoxin by strainsPHL67342, RM3, RM4, and RM8. As mentionedpreviously, S. typhimurium PHL67342 has beenshown to produce an enterotoxin capable ofstimulating fluid exsorption in rabbit ileal loops(31). A loss or reduction in the amount of thisfactor could serve to explain the altered rabbitileal loop response elicited by rough mutantsRM3, RM4, and RM8. Therefore, it was ofinterest to examine the production of the entero-toxin by strain PHL67342 and by correspondingLPS mutants.

In these experiments, strains PHL67342,RM3, RM4, and RM8 were grown in BHI brothfor 16 h at 37°C on a gyratory shaker. The cellswere removed by centrifugation at 18,000 x gfor 20 min at 4°C. The resultant supernatantfluids were filtered through 0.45-,um filters (Mil-lipore Corp., Bedford, Mass.) and tested imme-diately for enterotoxic activity in rabbit ilealloops.

Enterotoxic activity was detected in sterileculture supernatant fluids from both PHL67342and RM3, although the response was diminishedwith the mutant (Table 5). No enterotoxic activi-ty was found in sterile culture filtrates fromrough mutants RM4 or RM8.

DISCUSSIONThe Salmonella LPS can be divided into three

structurally distinct regions (19, 35). The outer-most region consists of 0-side chains which arecomposed of repeating units of oligosaccharidesthat contain specific sugars in specific linkages.These 0-side chains are linked to region 2, thecore oligosaccharide, which is in turn linkedthrough a ketosidic linkage to lipid A (region 3).The 0-antigenic portion of LPS has long been

known to be an important virulence determinantin the pathogenesis of systemic salmonellosis(i.e., enteric fever [21, 27]). Rough forms thathave lost the 0-specific side chains of their LPSsare much less virulent than smooth strains (3,

TABLE 5. Rabbit ileal loop response to sterilizedculture supernatant fluids from S. typhimurium

PHL67342, RM3, RM4, and RM8Strain Fluid accumulation'

PHL67342 ...................... 0.33 ± 0.12 (8)RM3 ........................ 0.26 ± 0.14 (8)RM4 ........................... 0.0 (4)RM8 ........................... 0.0 (8)

(AOC

1Di

Structure

Man-Abe

Rho

IGlS In

Mon-AbeRho

Gal

GIcI

IGolI

GocifGlc 7T

HepIHeplI(KDO)3

LipidA

Chernotype Mutant closs((wild pe)

Sr4

Ra

R,b,

Rb2Rb3

A..RC -Rd2A.

< rfc

rfaL(rfbrfaKrfoJrfa(R-res2)rfaHgalEga/U(rfoGrfaFrfoCrfoDrrfoE

FIG. 2. Structure ofLPS produced by S. typhimur-ium and LPS mutants. Abbreviations: Abe, abequose;Man, D-mannose; Rha, L-rhamnose; Gal, D-galactose;GlcNac, N-acetyl-D-glucosamine; Glc, D-glucose;Hep, L-glycerol-D-manno-heptose; KDO, 2-keto-3-deoxyoctonic acid.

20, 24). The loss of 0 antigen can result fromdefects in 0-antigen biosynthesis and assembly(rJb and rfc mutants, respectively) or from theproduction of incomplete core structures (rfamutants, [35]). An attempt to restate visually theLPS structure and chemotype with correspond-ing mutant types or classes is shown in Fig. 2.On the basis of phage sensitivity patterns (18,

41), colonial and cultural characteristics, andsugar composition of LPS, it was determinedthat RM3, RM4, and RM8 produce LPS ofchemotype Rb (Fig. 2). Since methylation analy-sis of the LPSs from these mutants was notperformed, it was difficult to differentiate amongthe three subclasses of the Rb chemotype. How-ever, it is likely that differences in the LPSsproduced by RM3, RM4, and RM8 do exist sinceeach of these mutants exhibited a slightly differ-ent phage sensitivity pattern.

Strains RM4 and RM8 appear to be complete-ly rough rfa mutants, whereas strain RM3 is aleaky rfa mutant which produces small amountsof smooth LPS. The presence of N-acetylglu-cosamine in RM4 and RM8 LPS is somewhatpuzzling. N-Acetylglucosamine is usually foundin strains of S. typhimurium that produce either

a1 Fluid accumulation represents the average ilealloop ratio (milliliters/centimeters) ± standard error ofthe mean. The numbers in parentheses represent thenumber of tests performed.

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smooth LPS or LPS of the Ra chemotype.Holme et al. (12) reported the presence of N-acetylglucosamine in LPS extracted from mu-tants of S. typhimurium belonging to chemo-types Rb, Rc, and Rd. These authors suggestedthat the presence of large amounts of glucos-amine in lipid A can complicate the determina-tion of N-acetylglucosamine in LPS prepara-tions. The removal of lipid A by mild acidhydrolysis before gas-liquid chromatographyanalysis would facilitate a more accurate estima-tion of the N-acetylglucosamine content of LPSpreparations obtained in this study. The resist-ance of mutants RM4 and RM8 to Felix-O phageprovides additional evidence that these orga-nisms produce LPS ofchemotype Rb rather thanRa, since N-acetylglucosamin'e has been foundto be an integral part of the receptor site for thisphage (17).

Partially purified LPS preparations fromstrains RM3, RM4, RM8, and PHL67342 failedto stimulate fluid accumulation in rabbit ilealloops. These results are in agreement with thefindings of Koupal and Deibel (15) and Sedlock(Ph.D. thesis), who found that LPS isolatedfrom both clinical and laboratory strains of S.enteriditis and S. typhimurium did not causefluid accumulation in either the infant mouse orrabbit ileal loop assay. It is likely that LPS is notresponsible for the fluid loss associated withSalmonella-mediated gastroenteritis.The reduced inflammatory responses generat-

ed by rough mutants RM3, RM4, and RM8apparently did not result from an inability ofthese organisms to associate with or survive onthe rabbit intestinal mucosa. Indeed, it appearsthat the reduced inflammatory reactions elicitedby rough mutants RM3, RM4, and RM8 resultedprimarily from a reduction in the ability of theseorganisms to invade the rabbit small intestinalepithelium. The absence of detectable gram-negative bacteria in the intestinal mucosa ofrabbit ileal loops exposed to whole culture prep-arations of strain RM3, RM4, or RM8 substanti-ate this hypothesis. In contrast, salmonellaewere readily observed in the lacteals of the villiin intestinal tissue obtained from PHL67342-infected rabbit ileal loops. Thus, it appears thatLPS mutants of S. typhimurium PHL67342,which either lack or produce reduced amountsof 0-antigenic material, are less invasive thanthe smooth parent.The results of the present histological study

are in agreement with the findings of Tannock etal. (36), who found that S. typhimurium roughmutants with defects in the core region of theirLPS were much less invasive than smoothstrains in the ileum of specific-pathogen-freemice. It appears that some relationship existsbetween the chemical composition and structure

of LPS and invasiveness in S. typhimurium.Several investigators have suggested that mu-

cosal invasion followed by acute inflammation isessential for fluid secretion in Salmonella-medi-ated gastroenteritis (6-8). The localized inflam-matory reaction is thought to trigger the releaseof prostaglandins, which in turn induce intestinalsecretion (6, 8). The ability of rough mutantRM8 to cause fluid accumulation in the absenceof a significant inflammatory reaction suggeststhat invasion is not essential for fluid secretion.Moreover, a number of strains of S. typhimur-ium which invade the intestinal mucosa ahdcause an acute inflammatory reaction do notstimulate intestinal secretion in rabbit ileal loops(7). These results suggest that some bacterialfactor(s), presumably the Salmonella enterotox-in, is in part responsible for the fluid secretioncharacteristic of Salmonella gastroenteritis.However, some role for the invasive processcannot be ruled out since most of the roughmutants tested in this study were capable ofgenerating a mild inflammatory reaction, in addi-tion to fluid accumulation, in the rabbit ileum.The absence of enterotoxic activity in culture

supernatant fluids from rough mutants RM4 andRM8, which produce LPS of chemotype Rb,suggests that smooth intact LPS is necessary forthe extracellular expression of Salmonella en-terotoxin. The ability of strain RM3 to producedetectable extracellular amounts of the entero-toxin may result from the production of smallamounts of smooth LPS by this organism. Inter-estingly, the amount of enterotoxin synthesizedby strain RM3 appears to be less than thatproduced by strain PHL67342. This differencecould result from the quantitative difference inthe amount of smooth LPS produced by the twoorganisms.

Sedlock (Ph.D. thesis) found that a substantialamount of the enterotoxic activity exhibited bystrain PHL67342 remained cell associated. Asimilar finding was reported by Kunkel andRobertson (16) for enterotoxigenic Escherichiacoli that produce the heat-labile toxin. The abili-ty of rough mutant RM8 to stimulate fluid accu-mulation in rabbit ileal loops, in the absence ofsignificant inflammation and enterotoxic activityin culture supernatant fluids, suggests that thisorganism is still capable of producing the cell-associated form of Salmonella enterotoxin.Smooth LPS may be required for the release ofthe enterotoxin from the cell.

It is unlikely that strain RM4 produces abiologically active form of the enterotoxin sinceculture supernatant fluids, as well as wholeculture preparations of this mutant, do not stim-ulate fluid accumulation in rabbit ileal loops.The exact reason for the loss of activity by roughmutant RM4 is unknown. Several investigators

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LPS MUTATIONS AND SALMONELLA GASTROENTERITIS 243

(2, 33) have reported a loss of outer membraneproteins associated with LPS mutations in S.typhimurium. The lack of enterotoxic activityexhibited by rough mutant RM4 may have re-sulted from the loss of the enterotoxin from theouter membrane of this organism. However, thisis unlikely, since there is no significant differ-ence in the protein composition of outer mem-brane preparations from wild-type strainPHL67342 and rough mutant RM4 (data notshown). Gmeiner and Schlecht (10) have recent-ly suggested that proteins in the outer membraneof S. typhimurium undergo conformationalchanges or different packing, depending uponthe sugar composition of the core oligosaccha-ride of the LPS. Conformational changes or arearrangement of the enterotoxin in the outermembrane of rough mutant RM4 or both couldexplain the loss of enterotoxic activity exhibitedby this organism.

It is evident from the work presented in thisstudy that LPS mutations have a profound effecton the enteropathogenic properties of S. typhi-murium PHL67342. Each of the LPS mutantsexamined in this study elicited reduced rabbitileal loop responses when compared with strainPHL67342. In all cases tested, the reduction inrabbit ileal loop responsiveness was accompa-nied by a reduction in both the invasiveness andthe production of Salmonella enterotoxin bythese organisms. Therefore, it is difficult tocomment on which of these two factors is pri-marily responsible for the fluid secretion charac-teristic of Salmonella-mediated gastroenteritis.However, it is becoming increasingly apparentfrom the work presented in this study, as well asfrom the work of others (39; Sedlock, Ph.D.thesis), that both invasiveness and the produc-tion of Salmonella enterotoxin contribute to thediarrhea associated with this disease. Moreover,it appears that intact LPS is necessary for themaximum expression of virulence in entero-pathogenic strains of Salmonella.

ACKNOWLEDGMENTS

We are grateful to D. T. Berman and R. Weltman for theirinterpretation of the histological sections. We thank R. Sew-ard for his help in the rabbit ileal loop survival studies and A.Singh for his excellent technical help. The technical assistanceof J. Bradshaw-Rouse, I. Moriyon, and T. Henkin is gratefullyacknowledged.

This research was supported by the College of Agriculturaland Life Sciences, University ofWisconsin, Madison, and bythe Graduate School Research Committee, University ofWisconsin, Madison.

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