Vol. 44, No. 2INFECTION AND IMMUNITY, May 1984, p. 274-2810019-9567/84/050274-08$02.00/0Copyright © 1984, American Society for Microbiology

Mechanism of Interaction of Salmonella and Schistosoma SpeciesRANDA F. MELHEM1t AND PHILIP T. LoVERDE2*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 49907,1 and Department of Microbiology,State University ofNew York, Buffalo, New York 142142

Received 17 October 1983/Accepted 23 January 1984

In endemic areas where Salmonella and Schistosoma species co-occur, several lines of evidence suggesta synergistic bacteria-parasite interaction that results in a protracted course for the salmonella infection thathas proven difficult to diagnose and therapeutically remedy. In an in vitro system using a pilus-negative anda pilus-producing transductant strain of Salmonella typhimurium we show that pili are the ligands forbacterial adherence to the schistosome surface tegument. Antipili antibodies produced in rabbits againstpurified pili, purified and digested to monovalent (Fab) fragments, blocked the association of Salmonella sp.to the surface tegument of Schistosoma sp., further demonstrating that pili are the appendages necessary forbacteria-parasite surface interaction. The use of carbohydrates, lectins, and enzymes demonstrated that thebacteria-parasite surface interaction was specific, mediated by pili that specifically recognize and bind tomannose-like receptors, probably glycolipids, on the surface of the worms. We suggest that prolongedsalmonellosis in schistosome-infected patients is due to an association of Salmonella sp. with theschistosome worms themselves and further that the schistosome worms provide a multiplication focus forthese bacteria in the portal mesenteric system, with a persisting bacteremia following.

The association of gram-negative bacteria and helminthparasites has been reported previously (2, 4, 9, 44, 48), theco-occurrence of Salmonella sp. and the blood fluke Schisto-soma sp. in humans being especially noteworthy (15, 16, 29,45, 46). In endemic areas where Salmonella and Schisto-soma sp. co-occur, clinical reports indicate a synergisticbacteria-parasite interaction such that patients with dualinfections show atypical symptoms of typhoid fever. That is,these patients have a history of intermittent fever and chills.The stool and often urine cultures are salmonella negative,which makes the disease difficult to diagnose. However,these patients are frequently bacteremic. The infection,which runs a protracted course, is difficult to treat. In duallyinfected patients, chemotherapy of Salmonella sp. in somecases is not effective until the schistosome infection istreated (26, 30, 31).The clinical evidence suggests that the bacteria-parasite

association may provide for persistence of the Salmonellainfection. In fact, Young et al. (49) removed adult Shisto-soma mansoni from the bloodstream of a patient withchronic salmonellosis and demonstrated a direct associationby culture and immunofluorescence of Salmonella paratyphiA with the surface tegument of S. mansoni. Experimentalstudies have shown that in animals infected with Salmonellaand Schistosoma sp., bacteria could be recovered fromvarious organs in greater numbers for longer periods of timecompared to controls infected only with salmonella (27, 37,38). Other laboratory studies have shown that Salmonellasp. can colonize the intestine and surface tegument ofSchistosoma sp. (24, 49). Using mutants of Salmonellatyphimurium and scanning electron microscopy, we impli-cated pili in the association of Salmonella sp. with theschistosome surface tegument (24), as others did subse-quently (5).

In this report, using a pilus-negative and pilus-producingtransductant strain of Salmonella sp. and monovalent anti-

* Corresponding author.t Present address: Department of Biological Sciences, American

University in Beirut, Beirut, Lebanon.

pili antibodies, we demonstrate that pili are the ligands bywhich salmonellae adhere to the surface tegument of Schis-tosoma sp. Using several carbohydrates, lectins, and en-zymes we demonstrate further that the association of Salmo-nella and Schistosoma sp. is specific in that the bacterial pilibind with mannosyl-like receptors (probably glycolipids) onthe surface tegument of the adult schistosome.

MATERIALS AND METHODSBacteria. Three strains of S. typhimurium were employed:

LT2 (supplied by M. Levinthal, Purdue University), whichhas no known mutations and properties similar to those ofwild-type strains, S7471N type FIRN, which is genotypicallynonpiliate and therefore cannot spontaneously give rise tofim+ (pili-producing) mutants (34), and S7471 4F, a fim+lysogenic transductant of S7471N. S. typhimurium S7471Nand S7471 4F were supplied by B. A. D. Stocker, StanfordUniversity. Stock cultures of these bacterial strains werestored in nutrient agar stabs (Difco Laboratories). Bacteriawere cultured overnight under static conditions in Luriabroth.

Bacteria-parasite association. Hamsters with patent S.mansoni infections were sacrificed, and the adult wormswere removed from the blood vessels, rinsed two times inminimal essential medium in a base of Earle salts, enrichedwith L-glutamine (GIBCO) (MEM), and then incubated for 1h at 37°C in tissue culture medium (TCM; consisting ofMEMand 10% heat-inactivated fetal calf serum) with a suspensionof approximately 106 bacteria per ml. After incubation, TCMwas decanted and replaced with 5 ml of sterile 0.2 M sodiumphosphate-buffered saline (PBS; pH 7.2). To remove anyloosely adhering bacteria, the flukes were washed 20 times;each wash consisted of 5 ml of sterile PBS, followed byvortexing for 5 s. The male and female schistosomes (one perplate) were then streaked on selective solid medium, Mac-Conkey agar, or brilliant Green agar, incubated at 37°C, andexamined for the presence of bacterial colonies after 24 and48 h.To determine whether the bacteria were associating in the

gut and/or on the surface of the tegument of Schistosoma

274

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PATHOGEN-PARASITE INTERACTIONS 275

sp., adult flukes were ligatured (24) by tying a thread justbehind the acetabulum to close off the only opening of thedigestive system. After the ligatured flukes were incubatedwith the bacteria, each fluke was removed, a cut was madebehind the ligature, and the posterior piece of the worm waswashed 20 times in sterile PBS, plated, and scored as

described above.Results were only considered in those experiments in

which as a control a sample (0.1 ml) of wash 20 was culturenegative, indicating that all loosely adhering bacteria hadbeen removed.

Use of antipili antibodies. S. typhimurium LT2 cells weregrown for 48 h at 37°C in Luria broth under static conditions.The cells were harvested by centrifugation at 10,000 x g for20 min in a refrigerated (4°C) Sorval centrifuge. The bacteriawere then washed in 10 mM Tris-hydrochloride buffer (pH7.6) and resuspended in the same buffer. The bacteria weredepiliated by shearing forces in a Virtus homogenizer at11,500 rpm (32) for 5 min.The suspension was then centrifuged at 10,000 x g for 20

min at 4°C. The supernatant containing pili was decanted andcentrifuged at 10,000 x g for 30 min at 4°C to remove any

remaining bacteria. The solution was then dialyzed against0.1 M acetate buffer (pH 3.9) overnight, and the aggregatedpili were removed by centrifugation at 2,000 x g for 20 min.The pellet was resuspended in 10 mM Tris-hydrochloridebuffer (pH 7.6), and then a filtered, saturated ammoniumsulfate solution was added dropwise to this stirred solutionto a final concentration of 10% (39). The suspension was

allowed to stand overnight at 4°C. Pili aggregates were thencollected by centrifugation at 4,000 x g for 15 min, resus-pended in 10 mM Tris-hydrochloride buffer, and dialyzedagainst Tris buffer with several changes for 2 days. Sodiumdeoxycholate (Sigma Chemical Co.) was added to the pilisuspension at a concentration of 0.5% (wt/vol), and thesolution was dialyzed against deoxycholate buffer (0.5%deoxycholate in 10 mM Tris-hydrochloride buffer [pH 7.6])for 2 days. Deoxycholate disaggregates pili-pili and pili-vesicle aggregates (21). The solution was then centrifuged at10,000 x g for 30 min, and the supernatant fluid was

collected and dialyzed for 2 days against 10 mM Tris-hydrochloride buffer with several changes of the buffer toremove the deoxycholate. The pili preparation was dialyzedagainst 0.1 M acetate buffer (pH 3.9) overnight, and theaggregated pili were collected by centrifugation at 4,000 x g

for 20 min. The pellet was resuspended in 10 mM Tris-hydrochloride buffer, and a saturated ammonium sulfatesolution was added dropwise (while stirring) to a finalconcentration of 10%. Several cycles of this precipitationyielded a pure preparation as determined by electron micros-copy (negative stain) and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis.

Protein was estimated by the method of Lowry (25), withbovine serum albumin as a standard.

Rabbits were injected intramuscularly with 750,ug of piliprotein emulsified in Freud complete adjuvant (GIBCO).The intramuscular injections were repeated at weeks 2, 3,and 5. During weeks 6 and 7, 250,ug of pili suspended in 10mM Tris-hydrochloride buffer (pH 7.5) was administeredintravenously. Three days after the last injection the rabbitswere bled. The serum was separated and tested for aggluti-nation against whole organisms and purified pili using astandard slide agglutination test.For antibody purification, the serum was dialyzed over-

night at4°C against Tris-hydrochloride buffer (0.02 M Tris,0.28 M NaCl) (pH 8.0) containing 0.2% sodium azide. The

serum was then passed through a DEAE-Affi blue gel (Bio-Rad Laboratories) column, and the immunoglobulin G (IgG)fraction was collected in the void volume. The IgG fractionwas then concentrated by lyophilization and reconstituted in6 ml, and the antibody protein was estimated (25) and thenstored at -70°C. To remove any possibility of interference ofantibodies to nonpili surface components, the IgG fractionwas absorbed three times with S. typhimurium S7471N (fim).The absorbed serum was then shown to agglutinate fim' S.typhimurium and purified pili but not fim S. typhimuriumcells.To avoid bacterial agglutination by intact IgG that might

bias the results, IgG was digested with papain (Sigma) toproduce monovalent Fab fragments (36).

Antipili antibodies were incubated with S. typhimuriumLT2 for 20 min at 37°C. The solution was centrifuged, thesupernatant was removed, and the bacteria were incubatedwith ligatured worms in TCM for 1 h at 37°C. The wormswere then washed and plated, and the number of bacteria permale fluke was determined.Use of carbohydrates. The sugars tested for their effect on

the association of S. typhimurium and S. mansoni were: D-fructose, D-galactose (Fisher Scientific Co.), D-glucose(MallineKradt), D-mannose (Matheson, Coleman and BellChemical Co.), D-fucose, L-fucose, a-methyl-D-mannoside,L-mannose, and N-acetyl-D-galactosamine (Sigma).

Bacteria were incubated for 30 min at 37°C in TCMcontaining a certain carbohydrate concentration before theaddition of ligatured worms. The procedure then followedthe bacteria-parasite association protocol, with the numberof salmonellae per male worm determined.To determine whether the bacteria-parasite interaction

was competitively reversible, bacteria and adult schisto-somes were incubated for 30 min at 37°C in TCM, afterwhich 50 mg of carbohydrate per ml was added and incuba-tion was continued for another 30 min. Alternatively, bacte-ria and worms were incubated for 1 h at 37°C in TCM. Halfof the worms were washed in PBS the remaining wormswashed in PBS containing 50mgof D-mannose per ml.

Use of lectins. Concanavalin A (ConA), Dolichous biflorusagglutinin (DBA), soy bean agglutinin, Lens culinaris agglu-tinin (LCA-A), pea agglutinin, wheat germ agglutinin, andUlex europeus agglutinin, all obtained from Sigma, wereused to study the specificity of the Salmonella-Schistosomasurface interaction.

In the experiments employing lectins, fixed ligatured maleworms were used. The worms were fixed overnight in 2%glutaraldehyde in PBS. After fixing, the worms were washedthree times in PBS and incubated with 50,ug of lectin per mlin 4 ml of PBS for 30 min at 37°C, after which bacteria wereadded and the incubation was continued for another hour.The procedure then followed the bacteria-parasite associa-tion protocol, with the number of salmonellae per maleworm determined.Enzyme treatment of adult schistosomes. Ligatured worms

were incubated with 1 U of pronase E, protease type XIV(Sigma) for 15 min in 3 ml ofMEM at 37°C or with lipase typeVII (Sigma) at a concentration of 150 U/3 liters of MEM for30 min at 37°C. Four glycosidases, a-glycosidase (frombakers yeast), P-glucosidase (from almonds),a-mannosidase(from jack beans), and neuraminidase (from Clostridiumperfringens), were incubated with fixed (PBS) and live(MEM) ligatured worms for various periods of times and atdifferent concentrations. All the enzymes employed wereobtained from Sigma and were tested for activity before usein any experiments (1).

VOL. 44, 1984

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

276 MELHEM AND LOVERDE

TABLE 1. Role of pili in the association of S. typhimurium FIRNmutants with S. mansoni

No. of schistosomes positiveS. typhimurium Ligatured No. of for bacteria/no.

strain worms replicates tested (%)Male Female

S7471 OF (fim') - 4 47/47 (100) 27/36 (75.0)+ 4 41/41 (100) 15/32 (46.9)

S7471N (fim) - 5 54/60 (90.0) 6/22 (27.2)+ 3 0/27 (0) 0/25 (0)

Control experiments. Control experiments were performedsimultaneously with treatment experiments, and the result ofeach treatment was compared with that of its correspondingcontrol. This was done because there were significant varia-tions in the number of bacteria per worm in similar experi-ments when done on different days but not on the same day.However, the relative number of bacteria per worm (treat-ment compared to control) was similar. This variation amongdays may be due to changes in bacterial culture as well as

differences in the worms (obtained from different hamsters).Data analysis. In experiments using pili mutants a worm

was considered culture positive if it had one or more bacteriaassociated with it. The result is reported as the total numberof positive worms (either males or females) over the totalnumber of worms (males or females, respectively) in allsimilar experiments.

In experiments using antipili antibodies, carbohydrates,lectins, and enzymes the average number of bacteria permale worm (X ± S, where X is the mean and S is thestandard deviation) of each individual treatment was com-

pared to the average number of bacteria per worm of itscorresponding control.To compare the average number of bacteria per worm

between treatment and control experiments, the two-samplet test was used; however, when the t test was not valid, i.e.,when the ratio of the variance of the two samples comparedwas significantly different (a = 0.05) by the F test, the Mann-Whitney U test was performed. In both tests, P < 0.01 was

the level of significance used (28).Moreover, in those experiments in which there was signifi-

cant change in the average number of bacteria per worm dueto a specific treatment, the percent change was calculated as

[1 - (x of treatment/x of control)] x 100 (where x is themean).

Also, the number of worms which were culture positive(i.e., had bacteria associated with them) was expressed as a

fraction of the total number of worms in the experiment.

RESULTS

Interaction of S. typhimurium FIRN phenotypes with schis-tosomes. S. typhimurium S7471N, which is incapable ofproducing pili, showed no interaction with ligatured male or

female schistosome flukes as compared to the pilus-produc-ing transductant (S74741 XF) of S. typhimurium, whichshowed 100% association with ligatured male worms and46% with ligatured female schistosomes (Table 1). All groupsof nonligatured worms had positive cultures, even if thebacteria were nonpiliated.

Effect of antipili antibodies. When Fab fragments fromantipili antibodies were incubated with piliated S. typhimur-ium and then added to ligatured schistosome worms, therewas a significant reduction (72 to 90%) in the number ofbacteria associating with schistosome worms as comparedwith controls (Table 2). Two sets of controls were employed:one in which no sera was added and one in which the IgGfraction had been absorbed with piliated bacteria to deter-mine whether any nonspecific factors in the serum were

inhibiting the Salmonella-Schistosoma interaction. Therewas no significant difference between the two controls.

Effect of carbohydrates. Of the nine different carbohy-drates used, D-mannose and cx-methyl-D-mannoside were themost potent inhibitors of the Salmonella-Schistosoma inter-action (Table 3). They caused an ca. 85% reduction at 5mg/ml to almost 100% reduction at 25 mg/ml compared withtheir corresponding controls. D-Fructose reduced bacterialattachment to the tegument of S. mansoni by about 85% atall the sugar concentrations employed. N-Acetyl-D-galactos-amine followed with a reduction of about 40% at the highersugar concentrations employed. D-Glucose reduced the bac-terial attachment by about 50% in half of the experiments atthe higher sugar concentrations (Table 3). The other foursugars tested (L-mannose, L-fucose, D-fucose, and galac-tose) showed no significant reduction even at concentrationsof 40 mg/ml when compared with controls (data not shown).The results of experiments performed to determine wheth-

er the bacterial attachment to S mansoni is reversibleshowed that the addition of 50 mg of either D-glucose or a-

TABLE 2. Effect of Fab fragments of antipili antibodies on the association of S. typhimurium LT2a with the surface tegument S. mansoni

Avg no. of bacteria per male wormbAntipilli antibodies Control expt

TreatmentS. typhimurium S7471N- S. typhimuriumG Untreated controlabsorbed IgG fragments fragments (no sera added)

Expt 1. S. typhimurium LT2 (2 x 107 cells) 1.7 ± 1.6 (9/11) [77.4] 7.6 ± 2.4 (9/9) No dataincubated with 3 mg of IgG fragments for 2.1 ± 1.1 (8/9) [72.4]20 min at 37°C

Expt 2 S. typhimurium LT2 (2 x 107 cells) 2.4 ± 2.1 (5/7) [80.0] 12.1 ± 4.4 (6/6) 13.1 ± 4.7incubated with 5 mg of IgG fragments for 1.7 ± 1.3 (7/9) [85.3]20 min at 37°C

Expt 3. S. typhimurium LT2 (2 x 107 cells) 1.1 ± 1.2 (6/10) [89.3] 10.3 ± 2.2 (10/10) 11.8 ± 1.8 (6/6)incubated with 5 mg of IgG fragments for20 min at 37°Ca The bacterial concentration used was 4 x 106 bacteria per ml.b X ± s. The numbers in parentheses represent the number of male worms with associated bacteria/total number of worms. The numbers in

brackets represent the percent reduction (significant reduction, P < 0.01) in the number of bacteria per worm (compared with S. typhimurim-absorbed IgG fragments). There was no significant difference (P < 0.01) between the control experiments (LT2-absorbed sera and no sera).

INFECT. IMMUN.

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PATHOGEN-PARASITE INTERACTIONS 277

TABLE 3. Effect of various concentrations of carbohydrates on the Salmonella-Schistosoma surface interactionaAvg no. of bacteria per male worma

CarbohydrateControlc 5 mg/ml Control 25 mg/ml Control 40 mg/ml

D-Mannose 12.2 ± 8.7 (10/10) 2.8 ± 3.0 (7/10) 12.2 ± 8.7 (10/10) 0.3 ± 0.8 (2/11) 12.2 ± 8.7 (10/10) 0 (O0)[77.0] [97.0] [100]

a-Methyl D-mano- 14.9 ± 6.1 (14/14) 1.4 ± 1.4 (9/14) 9.1 ± 2.3 (9/9) 1.0 ± 0.8 (6/9) 15.1 ± 3.8 (9/9) 0 (0/9)side [90.4] [89.0] [100]

D-Fructose 15.1 ± 3.8 (9/9) 2.4 ± 2.4 (7/9) 11.4 ± 3.3 (6/6) 1.3 ± 1.8 (4/8) 11.4 ± 3.3 (6/6) 1.3 ± 1.8 (4/9)[83.8] [87.8] [88.3]

N-Acetyl-D-galac- 13.3 ± 3.4 (7/7) 29.5 ± 11.3 (6/6) 18.6 ± 5.1 (9/9) 10.1 ± 3.3 (9/9) 21.4 ± 6.2 (8/8) 13.5 ± 4.4 (8/8)tosamnine [45.5] [36.8]

D-Glucose 19.3 ± 6.4 (17/17) 31.1 ± 6.4 (15/15) 14.9 ± 6.1 (14/14) 7.3 ± 3.4 (14/14) 7.3 ± 6.4 (7/8) 5.3 ± 2.8 (8/8)[51.0]

a Representative data. Each experiment was repeated four times.b X ± s. The numbers in parentheses represent the number of male worms with associated bacteria/total number of worms in the

experiment. The numbers in brackets represent the percent reduction (significant reduction, P < 0.01) in the number of bacteria per worm.c Control experiments were similar to treatment experiments, but no carbohydrate was added.

methyl-D-mannoside per ml to TCM containing Salmonellaand Schistosoma sp. that had been incubated for 30 minreduced the bacterial association 75 and 95%, respectively,compared with controls (Table 4). In other experiments, inwhich the schistosome worms, after 1 h of incubation withbacteria, were washed with PBS containing 50 mg of D-mannose per ml, a 99% reduction in bacterial associationcompared with controls was seen.

Effect of lectins. Glutaraldehyde-fixed ligatured wormswere used in these experiments because the worms wereincubated in PBS containing the various lectins and PBS hasan adverse effect on the schistosome tegument. The wormswere incubated in PBS instead ofTCM because it was fearedthat the lectins might interact with components of the TCMand bias the results. It was found that three times as manybacteria would bind to glutaraldehyde-fixed worms com-pared with live worms (unpublished data).To determine the nature of the worm surface receptors for

bacterial pili, various lectins were incubated with fixed,ligatured adult schistosomes before their incubation withbacteria. First it was determined for ConA that at concentra-tions of 50 p.g/ml, all of the putative receptors were saturated(data not shown); thus, all the experiments employing lectinsused concentrations of 50 ,ug/ml. ConA, which binds to D-mannose and D-glucose, reduced the bacterial-parasite sur-face interaction by 80 to 90%. DBA, which binds to N-acetyl-D-galactosamine, reduced the association by 35 to50% (representative data in Table 5). The other lectins hadno consistent significant effect on the Salmonella-Schisto-soma interaction.

Effect of enzymes. The ability of various enzymes torelease surface tegument moieties important in the associa-

tion of bacterial pili to the surface tegument was tested byincubating the worms with the enzymes before their incuba-tion with the bacteria.

(i) Lipase. To determine whether lipids (glycoplipids) areinvolved in the bacteria-parasite association, S. mansoniligatured worms were treated with a lipase. This treatmentsignificantly reduced (50%) the association of S. typhimur-ium with the surface of S. mansoni (Table 6).

(ii) Protease. To determine whether proteins (glycopro-teins) are involved in the bacterial association with thetegument of S. mansoni, ligatured worms were treated with 1U of pronase E for 15 min before incubation with thebacteria. This treatment resulted in about twofold increase inthe number of bacteria associating per worm (Table 6).Higher protease concentrations and longer incubation peri-ods resulted in the digestion of the worm tegument.

(iii) Glycosidases. a-Glucosidase, ,B-glucosidase, a-manno-sidase, and neuraminidase were employed to determinewhether a-glucosides, 3-glucosides, a-mannosides, and sial-ic acid, respectively, were involved in the association of S.typhimurium to the tegument of S. mansoni. In most of theexperiments, employing live or fixed worms, the glycosi-dases showed no consistent effect on the bacterial-parasitesurface interactions (data not shown). There were someexceptions, however. a-Mannosidase, in one of four experi-ments, reduced the association by 44%, P-glucosidase re-duced the association by 55% in one of seven expenments.

DISCUSSIONIn nature an association between chronic salmonellosis

and schistosomiasis has been established. LoVerde et al.(24) have demonstrated, using piliated S. typhimurium, that

TABLE 4. Displacement of S. typhimurium LT2' associated with the surface tegument of S. mansoni by D-glucose and a-methyl-D-mannoside

Avg. no. of bacteria per male worm"Expt Control' 50 mg of D-glucose per ml 50 mg of a-methyl

D-mannoside per ml

1 45.9 ± 10.4 (10/10) 11.8 ± 4.0 (11/11) [74.2] 0.6 0.9 (5/11) [98.6]2 42.5 ± 13.0 (10/10) 9.2 ± 2.5 (7/7) [78.1] 0.2 ± 0.4 (3/12) [99.4]3 30.5 ± 9.4 (11/11) 5.7 ± 1.9 (9/9) [81.1] 0.4 ± 0.5 (3/7) [98.5]a The bacterial concentraion was 3 x 106 bacteria per ml.b s+ S. The numbers in parentheses represent the number of male worms with associated bacteria/total number of male worms in the

experiment. The numbers in brackets represent the percent reduction (significant reduction, P < 0.01) in the number of bacteria per worm.Control experiments are similar to treatment experiments but no carbohydrate was added.

VOL. 44, 1984

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

t

0 -N

ClA 00*) - ie"- -

+I

s- +al +1 'I IT

- 1-o N C

o 0r

o 0 NN

^ N

0 \ O 0 Cl

Z r '- C Cl

- Ns

_ :4

r,£F7 ~Cl- - zi

ONONON

:4 :4~~0 tl

C O~~~~~~~~~~4CZ Z £~~~4

40

00

:4, :4 +l

o o_ _ _0

4,. .0 4.

o3 0 00XZ l Cl 40

-

+1

~C3

+1

o0cll

+l

INFECT. IMMUN.

vIIt"N- Cz

00

--00

+1 O-r-

clicl

ON

+ 1 V>,tl

C,I

-4

r-

+1

C,I

O'N

ON "

" o0

+1 +1

-

+l +l1t

-

+l

'-4 00

N~~~~~~~0

- oo~~~~~~~~~~I

+1 + o+I

fol- ~ ~ so

ND--

+l 6+l +

+l+1o*,4 o

C:Cl_i

I

z:4

0z

-0

0

z

r-:4

:-4

0-0rze

).;

4)o '~

.0;

0

O .E.0 -

E6oVD

00E

0. -C4)4)

C V0:4-

.0 o

.0 n

Q04

* -.

:4 Q4) 04. _0

Q.4 .0

._ ._

0 E

.0.0.g 0.._=_00 ~

._0_

=

0 0Q4;eQ_k..

4: EWn0 S4.)4

4. 0: D DC.'E. 0C 3

4_* ).Q -.3_C0.0Q-o<iEQ*,X0

HQ Q 5Q-s *t S

0

these appendages are important in the attachment of S.typhimurliiiin to S. miansoni. The use of the FIRN pheno-types of S. tvphiminrium provide unequivocal evidence thatpili are the appendages used for the attachment of Saloenotiel-la sp. to the surface tegument of Schistosomna sp.These results also show that salmonellae are able to

colonize the surface tegument as well as the digestive tract,but nonpiliated bacteria are only able to colonize the diges-tive tract (Table 1). The association of Sailmoniella sp. withthe digestive tract of S. inianisonii and its role in dualinfections is unclear. Authors have reported previously thatcolonization of the schistosome intestine by gram-positiveand -negative bacteria is detrimental to the flukes (3. 35). Inunpublished studies we have found that various gram-nega-tive bacteria colonize the surface tegument and digestivetract of S. inansonli. However, we have not observed apathogenic effect of these bacteria on the schistosomeworms.The importance of pili in the surface interaction between

Salmonella and Schistosomna sp. was further demonstratedby the use of antipili antibodies. Monovalent fragment (Fab)of IgG antipili antibodies was used in these experimentsbecause whole IgG will agglutinate the bacteria and thus biasthe results. Interaction of S. typhimuirillm with antipiliantibodies before their incubation with ligatured adult schis-tosomes reduced the association by about 70 to 90%(/ (Table2). This implies that the pili were blocked by the antibodies(no bacterial agglutination was seen) and thus preventedfrom associating with the surface receptors on the schisto-some tegument. This has been shown in other systems. inwhich antipili antibodies (raised against pure pili or againstpiliated bacteria and absorbed with nonpiliated bacteria)blocked the hemagglutinating ability and adherence of pili tocell surfaces (6, 17, 18. 41).Our evidence concerning the role of pili in bacterial

adherence agrees with other studies that have demonstratedthat pili are the ligands responsible for binding of certainbacterial species to cell surfaces. There are several differenttypes of pili; of interest to this study are type I pili which arecharacterized by the mannose-sensitive haemagglutinating,adhesive, and pellicle-forming properties they confer on thebacteria. Type I pili are found on various strains of Esche-richia (oli, Shigellai flexnieri, Klebsiella (ier-ogeties, andSalmonella sp. (10-14, 17, 19).

Since type I pili are found in the Enterobactriacea innature, and since these pili are adhesive and the hemaggluti-nation properties are mannose-sensitive, we tested the abili-ty of these ligands to recognize and bind to mannosereceptors on the schistosome surface. Table 3 shows thatbacterial incubation with D-mannose and ox-methyl-D-man-noside reduced their association with S. mnaiisonii. However,L-mannose had no significant effect on the interaction, evenat a high concentration, suggesting that the interaction of S.typhimuriuim LT2 and the surface tegument of S. incanisonii ismediated by pili recognition and association with D-manno-side residues on the surface of the worm. Moreover, thisinteraction seems highly specific, since the D-configurationof the mannoside is necessary to bind to the pili receptorsite. Thus, the addition of mannoside to the bacteria willblock the pili receptor sites and reduce the association withthe surface tegument of S. inanisonti.The results mentioned above are in agreement with sever-

al studies available on the hemagglutination properties oftype I pili and their adhesion to cell surfaces (11, 20, 33, 47).Ofek et al. (33) demonstrated that the adherence is reversibleand probably does not involve any irreversible alterations of

278 MELHEM AND LOVERDE

00-

c

:4

C4

a..

0

0

0

+1

C).2

CZC)

-C6;,

0

0j

0

E

:4

3

4)

.0

.0

H

0*_

0

4)0

.44)

-c

._

0

4)

4)

00

:4

_.

4._

H

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PATHOGEN-PARASITE INTERACTIONS 279

TABLE 6. Effect of enzyme on the association of S. typhimuriurm LT2" with the surface tegument of S. inansoniAvg no. of salmonellae/male schistosomeh

TreatmentTreated worms Untreated controls"

100 U of lipase per 3 ml of 4.08 ± 5.88 (7/12) [82.82]" 23.75 ± 9.03 (8/8)MEM at 37°C for 30 min 2.67 ± 3.85 (7/12) [88.76]d

12.00 ± 3.43 (9/9) [49.47]"

1 U of pronase E per 3 ml 46.13 + 18.68 (8/8) [87.22]e 24.64 ± 5.03 (14/14)of MEM at 37°C for 15 min 50.86 ± 15.57 (8/8) [99.79]1

60.10 ± 17.22 (10/10) [143.911]a The bacterial concentration was 3 x 106 bacteria per ml.b Numbers in parentheses represent the number of male worms with associated bacteria/total number of male worms in the experiment.Control experiments were similar to treatment experiments but no enzyme was added.

d Experiments showing significant reduction (P < 0.01) in the number of bacteria per worm have percent reduction in brackets.Experiments showing significant increase (P < 0.01) in the number of bacteria per worm have percent reduction in brackets.

either cell type. We also demonstrated in this study that theability of pili to bind to worm surface receptors is reversible.To further investigate the specificity of the interaction

between S. typhimurium pili and the surface tegument of S.mansoni, several lectins were employed because of theirbinding specificity to carbohydrates (receptors) on the schis-tosome surface. ConA reduced the bacterial association byabout 80%. This result was expected because D-mannoseand x-methyl-D-mannoside almost inhibited the bacterial-parasite interaction completely. Moreover, Simpson andSmithers (43) have demonstrated that ConA binds stronglyto the surface tegument of S. mansoni, indicating thatmannose and/or glucose are exposed on the surface of theadult male schistosome. LCA-A also binds to mannose andglucose; however, it had no effect on the bacterial associa-tion to the schistosome tegument at the concentration used(50 ,ug/ml). This is probably due to the lower specificity ofLCA-A for mannose and/or glucose. Salit and Gotschlich(40) have shown that LCA-A is a less potent inhibitor of themannose binding ability of E. coli to Vero cells. Anotherlectin which reduced the association of S. typhimurium andS. mansoni was DBA, which reduced the bacterial associa-tion by about 40%. This result was expected because N-acetyl-D-galactosamine (the sugar to which DBA binds)reduced significantly bacterial association to S. mansoni.Moreover, Simpson and Smithers (43) found that N-acetyl-D-galactosamine is exposed on the schistosome surface.The results reported here show that the carbohydrates

involved in the adherence of S. typhimurium to S. mansoniare most likely glycolipids, since lipase reduced bacterialadherence to the surface of S. mansoni. Interestingly, theresults from other laboratories have shown that glycosphin-golipids extracted from urinary tract epithelial cells inhibitthe adherence of E. coli to uroepithelial cells (22, 23). Daviset al. (8) have provided evidence that rat bladder epithelialcell receptors for mannose-sensitive adherence of E. colistrains are glycolipids. The employment of pronase E in-creased the association of S. typhimurium to the surface ofS. mansoni, suggesting that the proteins on the surface of theworm may mask some of the receptors for the bacterialassociation. Similar results were obtained by Salit andGotschlich (39), who showed that treatment of erythrocyteswith a protease mixture enhanced agglutination. However,the results of experiments using enzyme treatments on livingschistosomes should be interpreted with some caution, asthe schistosome tegument is very sensitive to culture condi-tions and easily disrupted (see, e.g., references 7 and 42).To further study the importance of carbohydrates in the

surface interaction of S. typhimutrium LT2 and S. mansoni,several glycosidases were employed to cleave specific sug-ars from the surface of living and fixed worms. The results ofthese experiments, with occasional exceptions, showed thatthe glycosidases used had no significant effect on the bacte-ria-parasite interactions whether live or fixed worms wereused. At first these results seemed contradictory to theresults presented above, in which carbohydrates, especiallyD-mannose, were shown to be important in the association ofthe bacteria to cell surfaces; however, even though theenzymes were active, the carbohydrates on the surface ofthe worms might not have been accessible to the enzymesand thus were not cleaved. Salit and Gotschlich (40), study-ing the effect of the treatment of Vero cells with mannosi-dase on the association of purified pili to these cells, alsoobtained negative results, suggesting that the bulk of themannose molecules are subterminal or otherwise shieldedfrom cleavage.The results of the experiments clearly show that the

association of S. typhimurium LT2 and S. mansoni isspecific and that it is mediated by bacterial pili that recognizeand associate with mannose-like receptors (glycolipids) onthe surface tegument of S. mansoni.

It is known that schistosome parasites can exist in thehuman host for long periods of time. We suggest that thisproperty of Schistosoma sp., along with the adhesive prop-erty of piliated Salmonella sp., enables schistosomes toprovide a long-term focus for bacterial multiplication in theportal-mesenteric flow, thus, in part, accounting for theprotracted course of typhoid fever in patients dually infectedwith Salmonella and Schistosoma sp.

ACKNOWLEDGMENTS

This work was supported by a David Ross Fellowship from thePurdue Research Foundation and a Biomedical Research SupportGrant from the School of Medicine, State University of New York atBuffalo.We thank Daniel Weeks for technical assistance, Gene I Higashi,

University of Michigan, for valuable discussions, and B. A. D.Stocker, Stanford University, for supplying the FIRN strains of S.typhimurium.

LITERATURE CITED

1. Agrawal, K. M. L., and 0. P. Bahl. 1972. a-Galactosidase, ,B-galactosidase, f-glucosidase, p-N-acetyl glucosaminidase, anda-mannosidase from pinto beans (Phaseolus vulgaris). Methods

VOL. 44, 1984

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

280 MELHEM AND LOVERDE

Enzymol. 28:720-721.2. Aitken, M. M., P. W. Jones, G. A. Hall, D. L. Hughes, and K. A.

Collins. 1978. Effects of experimental Salmonella dublin infec-tion in cattle given Fasciola hepatica thirteen weeks previously.J. Comp. Pathol. 88:75-84.

3. Basch, P. F. 1971. Infection of schistosomes with gram-positivecocci. Nature (London) 233:492-493.

4. Bottjer, K. P., D. C. Hirsh, and G. F. Slonka. 1978. Nematospir-oides dubius as a vector for Salmonella typhimurium. Am. J.Vet. Res. 39:151-153.

5. Bouillard, C., M. Miegeville, and C. Verneil. 1982. Etudes invivo et in vitro des relations entre Schistosoma mansoni etcertaines souches D'enterobacteries. Bull. Soc. Pathol. Exot.75:426-433.

6. Brinton, C. C., Jr. 1967. Contributions of pili to the specificityof the bacterial surface, and a unitary hypothesis of conjugalinfectious heredity, p. 37-70. In B. D. Davis and L. Warren(ed.), The specificity of cell surfaces. Prentice-Hall, Inc., Engle-wood Cliffs, N.J.

7. Carlisle, S., L. S. Weisberg, and A. G. Bentley. 1983. Schisto-soma mansoni: morphologic changes induced by maintenancein vitro. J. Parasitol. 69:319-334.

8. Davis, C. P., A. E. Avots-Avotins, and R. C. Fader. 1981.Evidence for a bladder cell glycolipid receptor for Escherichiacoli and the effect of neurominic acid and colominic acid onadherence. Infect. Immun. 34:920-929.

9. Dijkstra, R. G. 1973. Does the control of liver fluke decrease theoccurrence of salmonellosis in cattle? Vet. Rec. 93:467-468.

10. Duguid, J. P. 1959. Fimbriae and adhesive properties in Klebsi-ella strains. J. Gen. Microbiol. 21:271-286.

11. Duguid, J. P., E. S. Aronson, and I. Campbell. 1966. Fimbriaeand adhesive properties in Salmonella. J. Pathol. Bacteriol.92:107-138.

12. Duguid, J. P., S. Clegg, and M. I. Wilson. 1979. The fimbrial andnon fimbrial haemagglutinins of Escherichia coli. J. Med. Mi-crobiol. 12:213-227.

13. Duguid, J. P., M. R. Darekar, and D. W. F. Wheather. 1976.Fimbriae and infectivity in Salmonella typhimurium. J. Med.Microbiol. 9:459-473.

14. Duguid, J. P., and R. R. Gillies. 1957. Fimbriae and adhesiveproperties in dysentery bacilli. J. Pathol. Bacteriol. 74:397-411.

15. Hathout, S. E. D., Y. A. El-Ghaffar, and A. Y. Awny. 1967.Salmonellosis complicating schistosomiasis in Egypt. Am. J.Trop. Med. Hyg. 16:462-472.

16. Hathout, S. E. D., Y. A. El-Ghaffar, A. Y. Awny, and K. Hassan.1966. Relation between urinary schistosomiasis and chronicenteric urinary carrier state among Egyptians. Am. J. Trop.Med. Hyg. 15:156-161.

17. Isaacson, R. E., P. C. Fusco, C. C. Brinton, and H. W. Moon.1978. In vitro adherence of Escherichia coli to porcine smallintestinal epithelial cells: pili as adhesive factors. Infect. Im-mun. 21:392-397.

18. Korhonen, T. K., S. Eden, and C. Svanborg-Eden. 1980. Bindingof purified Escherichia coli pili to human urinary tract epithelialcells. FEMS Microbiol. Lett. 7:237-240.

19. Korhonen, T. K., H. Leffler, and C. Svanborg-Eden. 1981.Binding specificity of piliated strains of Escherichia coli andSalmonella typhimurium to epithelial cells, Saccharomycescerevisiae, and erythrocytes. Infect. Immun. 32:796-804.

20. Korhonen, T. K., K. Lountama, H. Ranta, and N. Kousi. 1981.Characterization of type I pili of Salmonella typhimurium LT2.J. Bacteriol. 144:800-805.

21. Korhonen, T. K., E.-L. Nurmiaho, H. Ranta, and C. Svanborg-Eden. 1980. New method for isolation of immunologically pure

pili from Escherichia coli. Infect. Immun. 27:569-575.22. Leffler, H., and C. Svanborg-Eden. 1980. Chemical identifica-

tion of a glycosphingolipid receptor for Escherichia coli attach-ing to human urinary tract epithelial cells and agglutinatinghuman erythrocytes. FEMS Microbiol. Lett. 8:127-134.

23. Leffler, H., and C. Svanborg-Eden. 1981. Glycolipid receptorsfor uropathogenic Escherichia coli on human erythrocytes anduroepithelial cells. Infect. Immun. 34:920-929.

24. LoVerde, P. T., C. Amento, and G. I. Higashi. 1980. Parasite-

parasite interaction of Salmonella typhimurium and Schisto-soma. J. Infect. Dis. 41:177-185.

25. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

26. Marinho, R. P., and J. P. Neves. 1974. Salmonelose septicemica,prolongada. Tratamento da esquistossomese mansoni intercor-rente como hycanthone. Rev. Inst. Med. Trop. Sao Paulo16:70-76.

27. Mikhail, I. A., G. I. Higashi, N. S. Mansour, D. C. Edman, andS. H. Elwan. 1981. Salmonella paratyphi in hamsters concur-rently infected with Schistosoma mansoni. Am. J. Trop. Med.Hyg. 30:385-393.

28. Miller, I., and J. E. Freund. 1977. Probability and statistics forengineers. Prentice-Hall, Inc., Englewood Cliffs, N.J.

29. Neva, F. A. 1949. Urinary enteric carriers in Egypt. Incidence in76 cases and observations on the urinary carriers' state. Am. J.Trop. Med. Hyg. 29:909-919.

30. Neves, J., P. Marinho, N. R. L. Martins, L. Dalu, P. K.DeAraujo, and A. Lucciola. 1969. Prolonged septicaemic salmo-nellosis: treatment of intercurrent schistosomiasis with nirida-zole. Trans. R. Soc. Trop. Med. Hyg. 63:79-84.

31. Neves, J., and N. R. Martins. 1967. Long duration of septicae-mic salmonellosis: 35 cases with 12 implicated species ofSalmonella. Trans. R. Soc. Trop. Med. Hyg. 61:541-552.

32. Novotny, C., J. Charnahan, and C. C. Brinton. 1969. Mechani-cal removal of F pili, type I pili and flagella from HFr and RTFdonor cells and kinetics of their reappearance. J. Bacteriol.98:1294-1306.

33. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherence ofEscherichia coli to human mucosal cells mediated by mannosereceptors. Nature (London) 265:623-625.

34. Old, D. C., I. Corneil, L. F. Gibson, A. D. Thomson, and J. P.Dugiud. 1968. Fimbriation, pellicle formation and the amount ofgrowth of salmonellae in broth. J. Gen. Microbiol. 51:1-16.

35. Ottens, H., and G. Dickerson. 1972. Studies on the effects ofbacteria on experimental schistosome infections in animals.Tran. R. Soc. Trop. Med. Hyg. 66:85-107.

36. Porter, R. R. 1959. The hydrolysis of rabbit y-globulin andantibodies with crystalline papain. Biochem. J. 73:119-126.

37. Rocha, H., J. W. Kirk, and C. D. Hearey. 1971. ProlongedSalmonella bacteremia in patients with Schistosoma mansoniinfection. Arch. Intern. Med. 128:254-257.

38. Rocha, H., M. M. G. Oliveira, V. S. Oliveira, and A. Prata. 1971.Algumas caracteristicas de infeccao por Salmonella typhi emcamundongos com esquistossomose experimental: multipicacaeda bacteria nos Schistosoma mansoni. Rev. Inst. Med. Trop.Sao Paulo 13:399-404.

39. Salit, I. E., and E. C. Gotschlich. 1977. Hemagglutination bypurified type I Escherichia coli pili. J. Exp. Med. 146:1169-1181.

40. Salit, I. E., and E. C. Gotschlich. 1977. Type I Escherichia colipili: characterization of binding to monkey kidney cells. J. Exp.Med. 146:1182-1194.

41. Silverblatt, F. J., and L. S. Cohen. 1979. Antipili antibodyaffords protection against experimental ascending pyelonephri-tis. J. Clin. Invest. 64:333-336.

42. Simpson, A. J. G., and D. J. McLaren. 1982. Schistosomamansoni: tegumental damage as a consequence of lectin bind-ing. Exp. Parasitol. 53:105-116.

43. Simpson, A. J. G., and S. P. Smithers. 1980. Characterization ofthe exposed carbohydrates on the surface membrane of adultSchistosoma mansoni by analysis of lectin binding. Parasitology81:1-15.

44. Spaldonova, R., 0. Tomasovicova, Z. Koppel, and D. Duvel.1969. Trichinella spiralis as the carrier of Salmonella typhimur-ium. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.1 Orig. Reihe A 211:47-52.

45. Tai, T. Y., C. Y. Hsu, H. C. Chang, and Y. K. Liu. 1958.Typhoid and paratyphoid fevers occurring in cases of schistoso-miasis. Chin. Med. J. 76:426-435.

46. Teixeira, R. 1960. Typhoid fever of protracted course. Rev.Inst. Med. Trop. Sao Paulo 2:65-70.

INFECT. IMMUN.

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PATHOGEN-PARASITE INTERACTIONS 281

47. van der Bosch, J. F., U. Verboom-Solmer, P. Postma, J. DeGraff,and D. M. Maclaren. 1980. Mannose-sensitive and mannose-resistant adherence to human uroepithelial cells and urinaryvirulence of Escherichia coli. Infect. Immun. 29:226-233.

48. Walker-Smith, J. A., B. McMillan, A. W. Middleton, S. Robert-son, and A. Hoperoft. 1969. Strongyloidiases causing small-

bowel obstruction in an aboriginal infant. Med. J. Aust. 2:1263-1265.

49. Young, S. W., G. Higashi, R. Kamel, A. Zein El Abdin, and I. A.Mikhail. 1973. Interaction of salmonellae and schistosomes inhost-parasite relations. Trans. R. Soc. Trop. Med. Hyg. 67:797-802.

VOL. 44, 1984

on Decem

ber 30, 2020 by guesthttp://iai.asm

.org/D

ownloaded from