Isolation of an (O,H,Vi)-Free Immunoprotective Antigenic Fraction with Mannose ReceptorLike Activity from Salmonella typhi Author(s): Hlne Jouin,

Anne-Marie Staub and Joseph E. Alouf Source: The Journal of Infectious Diseases, Vol. 143, No. 1 (Jan., 1981), pp. 106-113 Published by: Oxford University Press Stable URL: http://www.jstor.org/stable/30081764 . Accessed: 16/01/2014 02:49
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THE JOURNAL OF INFECTIOUS DISEASES * VOL. 143, NO. 1 * JANUARY 1981 c 1981 by The University of Chicago. 0022-1899/81/4301-0018$00.81

Isolation of an (O,H,Vi)-Free ImmunoprotectiveAntigenic Fraction with Mannose Receptor-Like Activity from Salmonella typhi
H61bneJouin, Anne-Marie Staub, and Joseph E. Alouf From the Unitddes Antighnes InstitutPasteur, Bactdriens, and the CentreNationalde la Recherche Scientifique, Paris, France

An antigenicfractionof Salmonella typhistrainTY6S, obtainedfroma bacterial pellet in a 5% -20% sucrosedensitygrawith 1.5 MNaClandultracentrifugation by washings dient, inducedserumantibodiesin rabbitsthat protectedchickenembryoschallenged with lethal doses of the virulentmicroorganism. The antigenicpreparation was free of Vi, O, and H antigens.It containedabout 85%proteinand Rd1P'lipopolysaccharide. of 45,000, 32,000,and20,000daltons, The proteinmoietycomprised threepolypeptides In addition,the purifiedpreparation exhibited the properties of a bacterial respectively. adhesinwith receptor-like of activityfor mannosylresidues,as shownby agglutination targetcells containingthese residuesat their surfaces. Salmonella typhi is the causative agent of typhoid in humans. Evaluation of the effectiveness of typhoid vaccines or of the immunoprotective effects of antisera from convalescent patients or immunized humans or animals cannot be undertaken on mice or other laboratory animals because they are not susceptible to infection by S. typhi. In contrast, chicken embryos are susceptible to infection with human strains of this species. Thus, they are a useful biological tool for the study of the protective properties of immune sera to S. typhi [1, 2]. The protective effect of these sera was not related to their content of antibodies (agglutinins, hemagglutinins, or precipitins) to O, H, and Vi antigens [1]. This finding is in agreement with studies that have reported that antibodies to O, H, and Vi antigens are of little or questionable importance in reflecting the state of protective immunity to typhoid fever [3]. We postulated in a previous communication [2] that the immunoprotective properties of the antisera could be due to a still unidentified antigen(s) present on the surface of S. typhi organisms. We report here the isolation and some properties of a Vi-free immunoprotective preparation from S. typhi strain TY6S (O-H+Vi+),which lacks O antigen. The chicken embryo challenge was undertaken with strain TY2 (O*H+Vi+), isolated from a typhic patient.
Receivedfor publication May29, 1980,and in revisedform August 15, 1980. Pleaseaddressrequestsfor reprints to Dr. JosephE. Alouf, Unite des AntighnesBacttriens,InstitutPasteur,28, rue du 75724Paris Cedex 15, France. Docteur-Roux,

We also describe receptor-like properties of the isolated fraction related to adherence of S. typhi to mannose residues on yeast cells or on erythrocytes coated with mannans. Materialsand Methods Bacterial strains. The virulent TY2 strain of S. typhi possessing O, H, and Vi antigens and the nonvirulent O- strain TY6S possessing H and Vi antigens were used (provided by Dr. L. Le Minor, the World Health Organization International Salmonella Center, Institut Pasteur, Paris). The strains were maintained lyophilized. Preparation of the Vi-freefraction. Fractionation of S. typhi strain TY6S was carried out by the method of Forsberg et al. [4] for Pseudomonas aeruginosa. Bacterial cells in the early logarithmic phase of growth were harvested from the medium by centrifugation at 16,300 g for 20 min at 4 C and were suspended in 0.5 M NaCl to a concentration of about 5.0 x 1010cells/ml. All subsequent operations were carried out at 4 C (figure 1). The cell suspension was centrifuged immediately at 16,300 g for 30 min, and the opalescent supernatant was removed by aspiration and retained. The cells were suspended again in the initial volume of 0.5 M NaC1 and centrifuged, and the supernatant was separated and retained. This procedure was repeated once. The three supernatants were pooled, centrifuged at 35,000 g for 30 min to eliminate any remaining cells, and filtered (pore size, 0.22 tm; Millipore Corp., Bedford, Mass.). The filtrate (fraction I) contained large amounts
106

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Immunoprotective S. typhi Fraction

107

TY6S strain

cells

(logarithmic centrifugation

phase) 16,300xg, 20 min

mg
1.
1. 1.2

3 times

Cells + 0.5 M NaC1 centrifugation 16,300xg,

Supernate 30 min

(discarded)

1.1!

Cells (discarded) Pooled supernates 35,000xg, 20 min

oc. o.
0.7

proteins o---o ,c .... Vi

centrifugation

o.
O*

Pellet (discarded)

filtered Supernate Millipore through 0.22 I) (Fraction

0. 0.1 42

A-------4 Q3\

o3
o bn t mu1

/"
boto
4

"
r

ultracentrifugation 2 h 105,000xg,

tbenubr

to

Pellet

Supernate (discarded) 0.5 M NaC1 washing (twice)

Pellet (Fraction II)

Supernates (discarded) ultracentrifugation sucrose gradient in 5-20 (250,000xg, l 18 h)

pattern of fraction II Figure 2. Ultracentrifugation (see figure1) of Salmonella typhistrainTY6S.The samwas layeredon a 5%-20% sucrosedensity ple (100 Ml) at 250,000g for 18 hr at 4 C. gradientand centrifuged The total volume of the fractionscollectedwas 500 l.
Vi = Vi antigen.

Bottom (Fraction P)

Top (Fraction Vi)

Figure 1. Flow diagramof the purificationof a cellsurface antigenicextractfrom Salmonellatyphi strain TY6S. Millipore = filter from MilliporeCorp., Bedford, Mass. of proteins and Vi antigen in addition to lipids and carbohydrates. This material was ultracentrifuged at 105,000 g for 2 hr. The pellet was washed twice with 0.5 M NaC1, and the supernatant was discarded. The pellet (fraction II), dissolved in 0.5 M sucrose density NaC1, was applied to a 5%o-20%o gradient and centrifuged at 4 C at 250,000 g for 18 hr (figure 2). A protein-rich fraction completely free from Vi antigen (fraction P) was found in the bottom of the gradient; Vi antigen was in the top. Immunization. Male and female outbred Bouscat white rabbits, weighing 2-3 kg, were obtained from an animal colony at the Institut Pasteur, Garches, France. Rabbits having natural antibodies to Vi antigen were not used. Groups of six (or 12) rabbits were immunized by four injections each containing about 0.1 mg of antigen. On day 0, the first injection, consisting of 1 ml of antigen and 2 ml of complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.), was given sc in the back. On day 22 an injection was

given im in the leg. The remaining injections were given on days 24 and 27, iv in the marginal ear vein. The rabbits were bled seven days after the last injection, and the sera were stored at - 180 C. No antibodies to O or H antigens were detectable in the sera before or at the end of immunization. Culture conditions. S. typhi strain TY6S was incubated at 37 C for 12 hr in the following medium, containing, per liter: KH2PO4, 13.60 g; KC1, 0.50 g; (NH4)2SO4, 0.75 g; MgSO4, 0.05 g; nicotinic acid, 0.01 g; ammonium ferric citrate, 0.02 g; CaC12,0.02 g; glucose, 3.00 g; tryptophan, 1 mM;and the other 19 L-amino acids, 50 1M. The pH was adjusted to 7.5. Cells cultured under these conditions did not contain H antigen detectable by agglutination with standard antisera to H antigen (Institut Pasteur Production, Paris, France). S. typhi strain TY2 was first incubated in the following liquid medium, containing, per liter: Liebig meat extract (Brooke Bond Co., Agnitres, France), 5 g; peptone (Difco), 10 g; and NaCl, 2.5 g. The pH was adjusted to 7.5. When the culture was developed, it was transferred into the same medium containing 13 g of agar/liter. Chicken embryo test. Fertile eggs from white Leghorn chickens (France-Ponte, Arpajon, France) were incubated with automatic turning, controlled temperature (37.3-37.8 C), controlled humidity (50%-55%0), and air circulation. Eleven-day-old

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Jouin, Staub, andAlouf

living chicken embryos were selected for inoculation. The undiluted rabbit sera (10 Al)were added to 190 of 0.15 M NaCl containing 20 to 2.0 x al 104 living S. typhi TY2 organisms. Small windows were cut in the shell with a drill. The serum-bacteria mixtures (10 eggs per mixture) were injected with a tuberculin syringe onto the chorioallantoic membrane, and the windows were sealed with adhesive tape. The eggs were then placed horizontally in the incubator with the window up. Viability was assessed by candling after a three-day incubation. The surviving embryos were counted. Control procedures consisted of the injection of 200 Cl of 0.15 M NaCI. The LD50 of S. typhi was checked by injection of the serum-free, diluted bacterial suspensions. The LDso always corresponded to the inoculation of one bacterium. The results were expressed as the number of LDsoneutralized by 10 jl of antiserum. Normal rabbit sera never protected against more than 20 LD5s0. This value was the lower limit for the assessment of the protective titer of immune sera. Assay of Vi antigen. This homopolysaccharide was assayed as described elsewhere [5] by

HAI of sheep erythrocytes sensitized with purified antigen by antiserum to Vi antigen. Chemical assays. Concentrations of proteins were determined by the method of Lowry et al. [6] with use of bovine serum albumin as the standard. Concentrations of sugars were determined by the phenol-H2SO4 method with use of glucose as a standard [7]. Lipids were extracted by the methanol-chloroform method [8]. Heptose concentrations were determined as described by Osborn [9]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were electrophoresed according to the method of Laemmli [10]. The molecular weights were determined by the method of Weber and Osborn [11]. Yeast cell aggregate test. Saccharomyces cerevisiae cells harvested from a standard medium were suspended in phosphate-buffered saline to a concentration of 107 cells/ml as described by Ofek et al. [12]. S. typhi strain TY6S cells (2.5 x 109/ml) were incubated with the yeast cell suspension at room temperature (about 24 C) for 30 min. Aggregation was then observed microscopically. Mannose-sensitized erythrocyte agglutination

20000

2O00

Vi

(U (U
U,

., r (3

LA

( 200

20

o N

N
oc

0c LA c-

2C ........

11
0 2

Rabbi numberI
12c 4

Rabbit number

Rabbit number

immunization of rabbitswith fractionsI (left), P Figure 3. Serumantibodytitersbefore ( - -- ) and after (-) (middle),and Vi (right)of Salmonella typhistrainTY6S(see figure1). The resultsare significantfor titersexceeding neutralization of 20 LDso f S. typhiin chickenembryos.

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S. typhiFraction Immunoprotective

109

Table 1. Chemicalcompositionof fractionP of Salmonellatyphi strainTY6S.

Component Dry weight,total Proteins Lipids Neutralsugarst Phosphorus Weight(mg) 7.35 6.40 0.60 0.80 0.17 Percentage weight* 100 87 8 10 2.3

NOTE. For procedures, see figure 1. * Becauseof the limitations of most analytical methods,the calculated totalwithrespect to dryweightdoes not equal100%. t The amountof heptosepresentin the neutralsugarswas about0.3 mg.

test. Sheep red blood cells (2.5%0suspension in phosphate-buffered saline) were sensitized with yeast mannans by the benzoquinone method of Ternynck and Avrameas [13]. The agglutination of these cells by S. typhi cells was carried out similarly to that of yeast cells. Results Immunoprotective properties of fraction P. Rabbits were immunized with fractions I, P, and Vi. The first two preparations (figure 3) induced the appearance of a protective activity of various titers in immune sera, depending on the individual animal. In contrast, fraction Vi did not elicit such

an activity. Chicken embryos were not protected when they were challenged with Salmonella typhimurium instead of S. typhi, a difference indicating the specificity for this species. Fraction P adsorbed on aluminum hydroxide as adjuvant elicited protective activity measured in rabbit sera. However, the titer was twofold lower than that obtained with complete Freund's adjuvant. Physical and chemical characteristics of fraction P. The chemical composition of purified fraction P is shown in table 1. A high protein content (85%) was found, with low amounts of lipids (8%) and neutral carbohydrates (10%). Heptoses represented about 40% of the total neutral carbohydrates. Ribose, deoxyribose, and Vi antigen (D-N-acetylgalactosaminuronic acid, partly O-acetylated) were not detected. SDS-PAGE of fraction P (figure 4) disclosed a major protein band with two faint components of lower molecular weights. The migration position of the three bands was compared with those of the reference proteins of different molecular weights; the values were 45,000, 32,000, and 20,000, respectively. Whether the two faint bands correspond to degradation products of the major component is not clear. This possibility is suggested by the observation of a single band exhibited by some preparations in SDS-PAGE.

7~472~

sodium Figure 4. Results of gel dodecyl sulfate-polyacrylamide of fractionP (see disc electrophoresis figure 1) of Salmonellatyphi strain TY6S: (left) fractions and (right) relative mobility, as distance in cm from the cathode.The standardmol wt markerproteins are: (1) bovine serum albumin, 67,000; (2) ovalbumin, 45,000; (3) chymotrypsinogen, 25,000; and (4) myoglobin, 17,800.Threecomponents(a, b, and c) of fractionP are seen.

o 67.0o
x 450-

32 b.0 250 S2o0.0_ 0 178 3

06

0.7

0.8 0.9

1.0

1.1

Mobility (cm)

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110

Jouin,Staub,andAlouf

Figure 5. Resultsof gel diffusionimmunochemical analysisof fractionP (see figure 1) of SalmonellatyphistrainTY6S.Left: The upperwell contains rabbitantiserum to fractionP, and the bottomwell containsfraction P. Right:The contentsof the wellsarethe same, but the gel containsheptose (100 mg/ml).

Immunochemical analysis of fraction P. Gelimmunodiffusion of fraction P against homologous rabbit antisera disclosed a main precipitation zone and another faint band (figure 5, left). The same immunochemical analysis was undertaken in gels in which glucose, galactose, N-acetylglucosamine, and heptose, which are components of the core moiety of S. typhi lipopolysaccharides, were incorporated. Immunoprecipitation was inhibited in the presence of heptose (figure 5, right), an inhibition suggesting that fraction P contains lipopolysaccharide with heptose as the immunodominant group. As shown in table 1 this sugar was in fraction P. This finding was confirmed by testing the antisera with the three different lipopolysaccharides from Salmonella R forms with terminal heptose (figure 6) [14], provided by Dr. O. Liideritz, Max-Planck Institut fir Immunbiologie, Freiburg, Federal Republic of Germany. Only Rd1P* polysaccharide was shown to react (in the ring test) with the rabbit antiserum to fraction P. This fraction very likely contained lipopolysaccharide of Rd1P' type. Rabbits were then immunized with a rough muR chemotype
GIcNAc Gal Hep P Ra GIc Gal -Ge --Hep -P-P-EtN Hep --4 KDO -

tant of Salmonella minnesota, which has the Rd1P' lipopolysaccharide. The antisera obtained did not protect chicken embryos from challenge with S. typhi strain TY2. This result indicated that the lipopolysaccharide present in fraction P was not a significant immunoprotective moiety of this fraction. Fraction P was free of H antigen as demonstrated by immunologic tests. Toxicity of fraction P. Groups of five Swiss mice (weighing 20 g) were inoculated with fivefold dilutions of fraction P in pyrogen-free saline. Each animal received 0.5 ml ip. Mice inoculated with pyrogen-free saline served as controls. The mice were observed for five days after inoculation. The LDso was calculated by the method of Reed and Muench [15] as 2.5 yg. This toxicity was very likely due to the lipopolysaccharide present in fraction P (about 20%7on the basis of heptose content). Aggregation of yeast cells by fraction P. Yeast cells are aggregated by S. typhi cells (figure 7, left) under the conditions described in Materials and Methods. A similar aggregation as shown by the formation of large clumps was observed by in-

01 igosaccharide

KDO-

P-

EtN

KDO-

[LIPID A)

O-P KDO P - P--EtN Rd,P+ Hep

- EN

-4

Hep

-*

KDO --

KDO -4 KDO -

Figure 6. Structures of lipopolysaccharides of four core-defectiveR mutantsof Salmonella[14].

[LIPID A] P - EtN

RdP-

Hep --

Hep

DO ---0

-4 KOO KDO -

[LIPID A] P - FtN

Rd

Hep

-41

( KDO

0 -KDO

LIPID A]

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S. typhiFraction Immunoprotective

111

c.4

in the presence of Salmonella of yeastcells:left, clumping typhistrainTY6S;middle,clumpFigure 7. Aggregation of 5 mg of of clumping in the presence of 5 mg of fractionP/ml (see figure1);andright,inhibition ing in the presence fractionP/ml with mannose(5 mg/ml) (x 400). cubation of fraction P at a concentration of 5 mg/ml with yeast cells (107/ml) (figure 7, middle). Clumping by both S. typhi cells and fraction P was inhibited in the presence of D-mannose, mannans, or methyl a-D-mannopyranoside (5 mg/ml) in the incubation medium (figure 7, right). In contrast, no inhibition was found with galactose, glucose, N-acetylgalactosamine, or N-acetylglucosamine at the same concentration. A similar agglutination of D-mannose-sensitized erythrocytes by fraction P and S. typhi cells was also observed. D-mannose inhibited agglutination. These findings indicate that fraction P possesses a specific receptor-like activity for mannose residue. Serum absorption by erythrocytes. Absorption was performed with 50 plof serum incubated at 4 C for 10 min with 0.5 ml of erythrocyte suspension. The protective properties of rabbit sera immunized with fraction P were suppressed after absorption with mannose-sensitized erythrocytes as well as with unsensitized erythrocytes. Discussion The immunity of humans against typhoid fever resulting from vaccines prepared from bacterial cells of S. typhi appears to be humoral as well as cellmediated [3, 16, 17]. However, although S. typhi was discovered 100 years ago and immunization against the disease has been performed for over 65 years, our knowledge of the antigen(s) involved in immunoprotection is still very limited, as most studies on salmonella antigens have been predominantly directed toward serologic classification [14, 18]. Several lines of evidence suggest that the classical and widely studied cell surface antigens O, H, and Vi are of little importance in reflecting the state of immunity to the disease [1, 3]. This finding led us to postulate that another cell-surface antigen(s) still to be identified must be involved in immunoprotection [2]. The chicken embryo, which is known as an excellent model for the study of infection and immunity to microorganisms pathogenic for humans [19-21], proved a valuable experimental tool for the assessment of the immunoprotective properties of antisera to S. typhi [5]. This model was used in the present study. We report here the isolation and purification of a protein-rich, immunogenic preparation (fraction P) distinct from the O, H, and Vi antigens of S. typhi. Sera from rabbits immunized with this preparation, which was fractionated from the super-

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112

Jouin,Staub,andAlouf

strains of S. typhi obtained by natant of O-H cell washing with 0.5 M NaCl (figure 1), were shown specifically to protect chicken embryos human strain challenged with a virulent (Oi') of S. typhi. In contrast, no protection was afforded by the same sera upon infection of the embryo with S. typhimurium under the same conditions. Fraction P mainly comprised proteins (about with smaller amounts of lipids and carbohy85%7) drates, including Rd1P' type lipopolysaccharide. The protein moiety disclosed slight heterogeneity by SDS-PAGE (apparent mol wt, 20,000-45,000). The protective properties of the rabbit antisera to fraction P did not appear to involve any contribution of the antibodies to the lipopolysaccharide present in this fraction. Like intact S. typhi cells as shown here, and like several human strains of Escherichia coli [22], fraction P aggregated yeast cells, the surfaces of which are known to be covered with mannans [23]. It also agglutinated erythrocytes coated with mannans. Aggregation or agglutination was prevented by prior incubation of either fraction P or S. typhi cells with D-mannose or methyl a-D-mannopyranoside but not with other sugars tested at the same concentration. It has been reported that certain strains of E. coli and S. typhi produce adherence factors (adhesins) at their surface that mediate specific attachment of the organisms to mannose or mannoselike receptors on the surface of human epithelial cells, macrophages, and polymorphonuclear cells [12, 22, 24-26] as well as of various animal cells [27, 28]. The binding of the bacterial strains on target cells is specifically inhibited by D-mannose and methyl a-D-mannopyranoside [24, 28]. A mannose-sensitive adhesin of E. coli has been shown to be associated with type 1 pili [29]. This adhesin is protein in nature. Recently, Eshdat et al. [30] isolated from E. coli extracts, by differential centrifugation and gel filtration, a protein that appears to be involved in the mannose-specific adherence of the organism to eukaryotic cells. This protein (mol wt, 36,000) was different from that of type 1 pili and appeared to be a surface component of the cell. The mannose-specific lectin-like properties of fraction P reported in the present study indicate that this purified preparation contains S. typhi adhesin. It is likely but not certain that this cell-bind-

ing factor is a component of the protein moiety of fraction P (relative mol wt, 20,000-45,000). The mild treatment of S. typhi cells used to obtain fraction P and the presence of some lipopolysaccharide in this fraction suggest that the protein(s) present in fraction P originates from the outer membrane. Several outer membrane proteins have been shown to be virulence factors in the mouse infected with S. typhimurium [31]. As adherence to endothelial and mucosal surface is considered a prerequisite for successful invasion of host tissues by many bacterial pathogens [32], S. typhi adhesin may be an important virulence factor. The immunoprotective properties of the immune rabbit sera to fraction P might be due to the neutralization of adhesins at the surface of virulent S. typhi cells, which would prevent adherence onto chicken embryo cells. Thus, it was of interest to determine whether the protective antibodies in antisera to fraction P were to the mannose-like receptor. We did indeed find disappearance of the immunoprotective properties after serum absorption with mannose-sensitized erythrocytes. However, this finding was not conclusive because a similar result was obtained with unsensitized erythrocytes. This result may be due to either nonspecific adsorption of the antibodies or combination with mannose residues of membrane glycoproteins of control erythrocytes [33]. This problem must be investigated further before the answer is known.

References 1. Joyeux, Y., Jouin, H., Leluc, B., Staub, A.-M. Recherches sur l'antigene X responsable de la production des anticorps anti-Salmonella typhi, protecteurs pour l'embryon de poulet. C. R. Acad. Sci. [D] (Paris) 279:1507-1510, 1974. 2. Joyeux, Y., Jouin, H., Leluc, B., Staub, A.-M. Recherche sur l'antigtne X responsable de la production des anticorps anti-Salmonella typhi, protecteurs pour l'embryon de poulet. Ann. Immunol. (Paris) 128:221-223, 1977. 3. Warren, J. W., Hornick, R. B. Immunization against typhoid fever. Annu. Rev. Med. 30:457-472, 1979. 4. Forsberg, C. W., Costerton, J. W., MacLeod, R. A. Separation and localization of cell wall layers of a gramnegative bacterium. J. Bacteriol. 104:1338-1353, 1970. 5. Eiguer, T., Staub, A.-M. Etudes immunologiques sur l'antighne Vi. I. Propri6t6s immunologiques d'une pr6paration d'antighne Vi: effet de l'ac6tolyse et du traitement par le formol. Annales de l'Institut Pasteur 110:707-726, 1966.

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6. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275, 1951. 7. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. Colorimetric method for determination of sugars and related substances Anal. Chem. 28:350-356, 1956. 8. Folch, J., Lees, M., Stanley, G. H. S. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509, 1957. 9. Osborn, M. J. Studies on the gram-negative cell wall. I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A. 50:499-506, 1963. 10. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685, 1970. 11. Weber, K., Osborn, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412, 1969. 12. Ofek, I., Mirelman, D., Sharon, N. Adherence of Escherichia coli to human mucosal cells mediated by mannose receptors. Nature 265:623-625, 1977. 13. Ternynck, T., Avrameas, S. A new method using p-benzoquinone for coupling antigens and antibodies to marker substances. Ann. Immunol. (Paris) 127:197-208, 1976. 14. Jann, K., Westphal, O. Microbial polysaccharides. In M. Sela [ed.]. The antigens. Vol. 3. Academic Press, New York, 1975, p. 1-125. 15. Reed, L. J., Muench, H. A simple method of estimating fifty per cent endpoints. American Journal of Hygiene 27:493-497, 1938. 16. Collins, F. M. Vaccines and cell-mediated immunity. Bacteriol. Rev. 38:371-402, 1974. 17. Collins, F. M. Cellular antimicrobial immunity. CRC Crit. Rev. Microbiol. 7:27-78, 1979. 18. Roantree, R. J. Salmonella O antigens and virulence. Annu. Rev. Microbiol. 21:443-466, 1977. 19. Buddingh, G. J. The chick embryo for the study of infection and immunity [editorial]. J. Infect. Dis. 121:660663, 1970. 20. Tieffenberg, J., Vogel, L., Kretschmer, R. R., Padnos, D., Gotoff, S. P. Chicken embryo model for type III group B beta-hemolytic streptococcal septicemia. Infec. Immun. 19:481-485, 1978. 21. Powell, C. J., Jr., Finkelstein, R. A. Virulence of Escherichia coli strains for chick embryos. J. Bacteriol. 91:1410-1417, 1966.

22. Ofek, I., Beachey, E. H. Mannose binding and epithelial cell adherence of Escherichia coli. Infec. Immun. 22:247-254, 1978. 23. Ballou, C. E. Some aspects of the structure, immunochemistry, and genetic control of yeast mannans. Adv. Enzymol. 40:239-270, 1974. 24. Bar-Shavit, Z., Ofek, I., Goldman, R., Mirelman, D., Sharon, N. Mannose residues on phagocytes as receptors for the attachment of Escherichia coli and Salmonella typhi. Biochem. Biophys. Res. Commun. 78:455-460, 1977. 25. Mangan, D. F., Snyder, I. S. Mannose-sensitive interaction of Escherichia coli with human peripheral leukocytes in vitro. Infec. Immun. 26:520-527, 1979. 26. Thorne, G. M., Deneke, C. F., Gorbach, S. L. Hemagglutination and adhesiveness of toxigenic Escherichia coli isolated from humans. Infec. Immun. 23:690-699, 1979. 27. Cheney, C. P., Boedeker, E. C., Formal, S. B. Quantitation of the adherence of an enteropathogenic Escherichia coli to isolated rabbit intestinal brush borders. Infec. Immun. 26:736-743, 1979. 28. Aronson, M., Medalia, O., Schori, L., Mirelman, D., Sharon, N., Ofek, I. Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence with methyl a-D-mannopyranoside. J. Infect. Dis. 139:329-332, 1979. 29. Salit, I. E., Gotschlich, E. C. Type 1 Escherichia coli pili: characterization of binding to monkey kidney cells. J. Exp. Med. 146:1182-1194, 1977. 30. Eshdat, Y., Ofek, I., Yashouv-Gan, Y., Sharon, N., Mirelman, D. Isolation of a mannose specific lectin from Escherichi coli, and the role in the adherence of the bacteria to epithelial cells. Biochem. Biophys. Res. Commun. 85:1551-1559, 1978. 31. Valtonen, M. V., Nurminen, M., Johansson, V., Mikeli, P. H. Mouse virulence of Salmonella typhimurium mutants deficient in two major outer membrane proteins. Infec. Immun. 18:454-458, 1977. 32. Gibbons, R. J. Adherence of bacteria to host tissue. In D. Schlessinger [ed.]. Microbiology- 1977. American Society for Microbiology, Washington, D.C., 1977, p. 396-406. 33. Sharon, N. Complex carbohydrates: their chemistry, biosynthesis and functions. Addison-Wesley, Reading, Mass., 1975, p. 235-256.

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