Microbial Pathogenesis 1987 ; 3: 455-459

Distribution and properties of the mannose-resistant hemagglutinin produced bySalmonella species

Madelon C. HaluIa'' and B . A . D . Stocker

Department of Medical Microbiology, Stanford University School of Medicine, Stanford,CA 93405, U.S.A .

(Received August 12, 1987 ; accepted in revised form 17 September, 1987)

Halula, M . C. (Department of Medical Microbiology, Stanford University School of Medicine,Stanford, CA 93405, U .S.A.) B. A. D. Stocker. Distribution and properties of the mannose-resistant hemagglutinin produced by Salmonella species. Microbial Pathogenesis 1987 ; 3 : 455-459 .

Presence or absence of mannose-resistant hemagglutination (MRHA) or sheep erythrocytesby Salmonella species was found to be consistent within most serotypes tested and did notcorrelate with O-antigen . Broad-host-range serotypes including S. typhimurium and S . enteritidisproduced MRHA while host-specific serotypes including S. typhi, S. dublin, and S. gallinarumwere MRHA-negative. MRHA produced by S. typhimurium was soluble, heat-stable, and notinhibited by any of eleven carbohydrates tested . Further investigation of MRHA may provideinsight into mechanisms of Salmonella pathogenesis .

Key words : Mannose-resistant; hemagglutination ; Salmonella .

Introduction

Little is understood about the specific determinants which enable Salmonella typhi-murium to attach, colonize, and later cause diarrhea . A mannose-resistant hem-agglutinin (MRHA) of S. typhimurium first described by Jones and Richardson maymediate attachment of S . typhimurium to host cells. Descriptions of the MRHA activitydiffer significantly with respect to the range of species of erythrocytes agglutinated,the culture conditions reported to favor production of MRHA, and whether or notactivity was cell-associated .' 2

We have extended studies of the MRHA activity to other Salmonella serotypes aswell as S . typhimurium . The effects of culture conditions on production of MRHA andits physical characteristics by S . typhimurium strain SL51 66 was investigated .

Results

Five strains of S. typhimurium, each derived from a different wild-type isolate, weretested for ability to agglutinate red cells of eleven species . The strains tested included :SL1027, derived from LT2 ; 3 SL5166, derived from TML;' W118;' Q1 ; 5 and SL2201, a

'Present Address: Department of Microbiology and Immunology, Medical College of Virginia, VirginiaCommonwealth University, Richmond, VA 23298-0678, U .S.A .

0882-4010/87/060455+05 $03.00/0

© 1987 Academic Press Ltd

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Table 1 Mannose-resistant hemagglut-ination by salmonella serotypes

MR HA was tested as described in the text .

1 . 20 independent strains of S. typhimurium availablein the laboratory strain collection were examinedfor MRHA activity . These included derivatives ofW118, LT2, TML, FIRN and Q1 .

2 . 70 human clinical isolates of S . typhimurium wereobtained from the Enteric Disease Section,Microbial Disease Laboratory, Department of PublicHealth, Berkeley, California, and from Dr LucyTompkins, Department of Medicine, Stanford Uni-versity School of Medicine .

Fim (type-1 fimbriae) transductant of SL3201 .' Bacteria of each of the five strainscaused hemagglutination of sheep and goat erythrocytes, even in the presence of 1-methyl-mannoside, indicating "mannose-resistance". None of the five strains causedagglutination of erythrocytes of the other nine species tested . Screening of additionalstrains of S . typhimurium revealed that 14 of 15 laboratory strains and 64 or 70 recentclinical isolates were clearly MRHA-positive (Table 1 ) .

Results obtained for other Salmonella serotypes are shown in Table 1 . All isolatesof S . enteriditis, S . austin, S. agona, S. memphis, and S. virchow agglutinated sheepand goat erythrocytes . None of the S. dub/in, S. choleraesuis, S. typhi, S. paratyphi A,S. paratyphi B, S. paratyphi C, S. abortus equi, S. abortus ovis, S. arizonae, S .gallinarum or S . pullorum were hemagglutinating .

The source of erythrocytes was critical to the hemagglutination reaction . Fixed goaterythrocytes and three lots of fixed sheep erythrocytes obtained from Sigma ChemicalCo. were regularly hemagglutinated by S. typhimurium strains. By contrast, fresherythrocytes from two different sheep were not agglutinated, even after glutaraldehydefixation . Pre-treatment of erythrocytes with periodate, neuraminidase, or six proteases(see methods) had no effect on their agglutinability .S. typhimurium strain SL5166 (TML) was used to determine the effect of culture

conditions on MRHA production . MRHA was produced at all growth temperatures

M. C . Halula and B . A . D . Stocker

Serotype

No. MRHA positive0-antigenGroupNo. Isolates Tested

S. paratyphi A 0/2 A

S. typhimurium' 19/20 BS. typhimurium2 64/70 BS. abortus-equi 1 /3 BS. abortus-ovis 0/1 BS. agona 2/2 BS. paratyphi B 0/11 B

S. austin 1/1 C1S. choleraesuis 0/6 C1S. paratyphi C 0/1 C1S. virchow 2/2 C1

S. dublin 0/13 D1S. enteritidis 11/11 D1S. gallinarum 0/8 D1S. pullorum 0/8 D1S. typhi 0/10 D1

S. memphis 1/1 KS. arizonae 0/2

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tested, in complex and simple defined medium with or without the presence of glucose .MRHA was produced under anaerobic as well as aerobic growth conditions . Highestactivity was found in aerated cultures grown at 37 °C in BHI medium in logarithmicphase of growth .

Most M RHA activity was present in culture supernatants rather than cell-associated .BHI cultures of S. typhimurium SL5166 had MRHA titers of 4096 after 10 h ofincubation at 37°C . After centrifugation the culture supernatant likewise had titer of4096 while the pelleted bacteria resuspended to the original volume in PBS had titerof 8. MRHA activity associated with bacteria grown on solid medium was completelyremoved by washing twice with PBS ; activity was found in PBS used to wash thecells .

Attempts to inhibit the MRHA activity of supernatants with specific reagents havebeen unsuccessful . MRHA activity of S . typhimurium SL5166 culture supernatantswas not affected by the presence of mixed gangliosides or any of eleven sugars testedin the hemagglutination assay .

Undiluted BHI broth cultures did not hemagglutinate sheep erythrocytes ; when suchcultures were diluted 1 :16 or more in PBS, agglutination was observed . Addition ofas little as 1 % by volume of BHI broth to a M9-culture supernatant with a MRHA titerof 64 inhibited the agglutination reaction completely . BHI broth dialyzed against 1000volumes of PBS at 4°C for 48 h resulted in a 60% decrease in the inhibitory effect asmeasured by the volume of dialysed BHI needed to completely inhibit MRHA . Thedialysis membrane used had a molecular-weight cut-off of 12 kilodaltons (kDa) ; thisindicates at least 40% of the inhibition is due to component(s) in BHI with a molecularweight greater than about 12 kDa .

The presence of DTT, EGTA, or EDTA either in the agglutination reaction or duringstorage also had no effect on agglutination ; this suggests that Mg, 2 + Cat' andsulfhydryl groups do not play a role in hemagglutination . Treatment with 5 of 6proteases had no effect on MRHA activity ; pronase caused an eight fold reduction inM R HA activity .The receptor for MRHA on sheep erythrocytes has not been identified . Results of

this investigation suggest several surface components of the erythrocyte which arenot important in the agglutination reaction . Treatment of erythrocytes with sodiumperiodate does not eliminate the agglutination ; this suggests that carbohydrate residuesare not critical . If the receptor is carbohydrate in nature, it is not one of the eleventested for inhibition in this study . Treatment of erythrocytes with neuraminidase didnot affect the MRHA reaction suggesting sialic acid residues removed by this enzymeare also not required for agglutination . Protease treatment of erythrocytes also had noeffect on hemagglutination .

Discussion

In this study we report the results of screening Salmonella typhimurium strains for theability to agglutinate eleven species of erythrocyte . In the presence of a-methyl-mannoside the majority of S . typhimurium agglutinate sheep and goat erythrocytesbut none of the other nine species tested . Although previous reports of MRHAproduced by S . typhimurium have varied considerably in the species of erythrocyteagglutinated, sheep erythrocytes were consistently agglutinated in this and previousstudies.` At least 5 of 7 blood group systems recognized in sheep have homologs ingoats;' thus the agglutinin may recognize the same or a related surface component onthe two species of erythrocytes. A component(s) in BHI with a molecular weightgreater than 12 kDA does inhibit MRHA activity . This observation may partially explain

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differences in MRHA activity reported by other workers where BHI was used 12 andprovide a clue to the nature of the MR HA receptor .

MR HA activity is extremely resistant to heat and a variety of proteases, only pronaseamong 6 proteases tested had a significant effect on MRHA activity .

These results support previous findings' that MRHA activity is soluble and not cell-associated. Soluble adhesins have been implicated in the pathogenesis of several otherorganisms including afimbrial adhesins present on E. coli uropathogenic strains KS52 8and 8279 and soluble hemagglutinin produced by Vibrio cholerae" which may play arole in attachment to gut epithelium ."

Mannose-resistant hemagglutination of sheep erythrocytes is not limited to S .typhimurium (Table 1) . Production or lack of production of MRHA does not appearto be correlated with the type of 0-antigen or LPS produced and is generally consistentamong representatives of a serotype . In general, it appears that host-adapted speciessuch as S. pullorum, S. paratyphi B, and S. typhi, are MRHA negative while thosespecies which have a broad host range such as S. enteriditis and S. typhimurium seemto be MRHA positive . S. typhi, S. dublin, and S. choleraesuis are noted to causeinvasive disease in humans and are consistently MRHA negative . Those serotypeswhich are normally limited to the gastrointestinal tract in humans such as S .typhimurium and S. enteriditis, are consistently MRHA positive .

The results of this investigation suggest S. typhimurium MRHA is unique andinteresting. Further investigation of the nature of the hemagglutinin may yield valuableinformation about the role of this determinant in pathogenesis of S. typhimurium andperhaps other Salmonellae as well .

Materials and methods

S. typhimurium strains used (Table 1) were maintained on Dorset egg slants or in agar stabsat room temperature . The complex media used included L-broth and L-agar," Brain HeartInfusion (BHI, Difco), and blood agar base (CM55, Oxoid) . Minimal media used was M9 13containing either glucose, 5 g/l, or glycerol, 10 ml/I, and amino acids, 50 pg/ml as needed .

Hemagglutination was detected by the static settling method of Jones and Richardson' withthe following modifications. Glutaraldehyde-fixed erythrocytes of eleven species were obtainedfrom Sigma Chemical Co . (St. Louis, MO .) and reconstituted as suggested by the supplier .These included sheep, goat, human type-O, pig, horse, guinea-pig, rabbit, mouse, chickenturkey, and bovine . Fresh erythrocytes from two sheep were washed three times in Alsever'ssolution 14 and used as a 10% suspension (vol/vol) in phosphate buffered saline (PBS, 14) . Forother tests fresh sheep erythrocytes were glutaraldehyde-fixed ." To eliminate agglutinationmediated by type-1 fimbriae, all hemagglutination tests were done in the presence of x-methyl-mannoside, 20 g/I . Bacteria grown overnight at 37'C, on solid media were tested by resuspendingapproximately 109 CFU in 100 p I of PBS and adding 10 pI of reconstituted erythrocytes to thebacterial suspension in microtiter 'U' trays (Dynatech Laboratories, Inc ., Alexandria, VA ., Cat .no . 001-010-2301 ) . Results were recorded after settling incubation on ice for 2 h . A dispersedpattern of deposited erythrocytes indicated hemagglutination . A compact 'button' indicatedabsence of hemagglutination . To determine MRHA titer of broth cultures, 2-fold serial dilutionswere made in PBS .

To test the effect on agglutinability erythrocytes were pretreated as 1% suspensions withpronase, protease, protease IV, trypsin, papain (Sigma), proteinase K (Beckman) or V8 protease(Miles Laboratory) at 0 .5 mg/ml at 37'C for 24 h. Erythrocytes were also pre-treated as a 10%suspension with neuraminidase (Arthrobacter ureafaciens, Cal Biochem) (2 .5 pg/ml) for 3hours at 37'C . Erythrocytes were also pre-treated with sodium periodate, 0 .01 M, pH 5.0 at14'C in the dark for 2 h . Erythrocytes were washed three times with PBS following enzyme orperiodate treatment .

The following were tested for ability to inhibit MRHA activity of culture supernatants : mixedgangliosides (Supelco), 40 and 400 jig/ml ; Ethylene glycol -bis-(f3-aminoethyl ether) -N,N, N',N'-tetraacetic acid (EGTA), 1 mm ; ethylene diamine tetraacetic acid (EDTA), 1 mm ; or dithiothreitol

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(DTT), 1 mm . The following carbohydrates were tested for their ability to inhibit hernagglutinationat 20 mg/ml : glucose, a-methyl-mannoside, arabinose, fucose, fructose, N-acetyl-glucosamine,galactose, lactose, maltose, rhamnose, and xylose. M9-culture supernatants containing MRHAactivity were treated with pronase, protease IV, trypsin, papain (Sigma), proteinase K (Beckman)or V8 protease (Miles Laboratory) at 0 .5 mg/ml at 37 °C for 24 h .

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