Comp. lmmun. Microbiol. inject. Dis. Vol. 8, No. 1, pp. 9 16, 1985 0147-9571/'85 $3.00 + 0.00 Printed in Great Britain Pergamon Press Ltd

R O L E O F O U T E R M E M B R A N E P R O T E I N S I N

I M M U N I T Y A G A I N S T M U R I N E S A L M O N E L L O S I S - - 1 .

A N T I B O D Y R E S P O N S E TO C R U D E O U T E R M E M B R A N E

P R O T E I N S O F SALMONELLA T Y P H I M U R I U M

M. NATARAJAN, V. UDHAYAKUMAR, K. KRISHNARAJU a n d

VR. MUTHUKKARUPPAN*

Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, India

Almtract--The humoral immune response to crude outer membrane proteins (comp) of S. typhimurium in mice has been characterized. Maximal and quicker antibody response was observed when 50/tg of comp was injected intraperitoneally. The comp of smooth C5 strain of S. typhimurium evoked antibody response to both lipopolysaccharide (LPS) and proteins. Absorption of these sera with LPS coated erythrocytes eliminated the antibodies to LPS completely, while the antibody level to protein was left unaltered. The comp from rough mutant (lacking O-specific chain of LPS) of S. typhimurium elicited antibodies to proteins but not to LPS. These results indicate the concomitant production of antibodies to Salmonella outer membrane proteins also. The significance of such antibodies in protection and diagnosis has been discussed.

Key words: Salmonella typhimurium, outer membrane proteins, lipopolysaccharide, anti-protein antibodies, anti-lipopolysaccharide antibodies

R6sum6----La r6ponse immunitaire humorale aux extraits prot6i'ques bruts de la membrane externe (comp) de S. typhimurium a 6t6 caract6ris6e chez la souris. La r6ponse anticorps est maximale et la plus rapide quand 50/lg de comp est inject6 par vole intrap6riton6ale. Le comp de la souche C5 de S. typhimurium induit une r6ponse anticorps contre le lipopolysaccharide (LPS) et les prot6i'nes. L'absorption de ces s6rums avec des 6rythrocytes sensibilis6s au LPS 61imine com- pl&ement les anticorps contre le LPS tandis que le taux d'anticorps antiprot6i'nes reste inchangb. Le comp de mutant rugueux de S. typhimurium induit des anticorps contre les prot6ines mais non contre le LPS. La signification de ces anticorps dans la protection et le diagnostic est discut6e.

Mots-clefs: Salmonella typhimurium, prot6i'nes de la membrane externe, lipopolysaccbaride, anticorps antiprot6i'nes, anticorps antilipopolysaccharide

INTRODUCTION

The exposed cell wall components of Gram-negative bacteria have direct interactions with host cells, antibodies and phages [10, 21,20]. An immune response evoked against the surface components of Gram-negative bacteria would be able to recognize the invading pathogens and confer protection [6]. Hence the search for possible vaccines for Gram- negative bacterial infections need to be directed to a greater extent to the surface components.

The outer membrane of Salmonella typhimurium is constituted by lipopolysaccharide (LPS), proteins, phospholipids, and entero bacterial common antigen. Of these LPS and proteins have gained attention because of the immune response evoked by them.

*To whom correspondence should be addressed. Q

l0 M. NATARAJAN et al.

The O-specific chains of LPS are exposed on the surface of the outer membrane [14]. They are virulent factors which help the invading bacteria to survive in the host by resisting phagocytosis and the action of serum complement [4]. They are highly immunogenic and are capable of inducing specific antibodies [2]. However, LPS was found to be least effective in providing protection when compared with other nonviable vaccines [1, 2].

Major outer membrane proteins of S. typhimurium are porin, OmpA and lipoprotein. These have been shown to stimulate antibody response in rabbits [6]. Importance of protein antigens [13, 18] and protein-LPS complex [19] in inducing protective immunity have also been suggested in mice.

However, these sutidies have been restricted to the use of crude preparation of proteins and the nature of these protective antigen(s) was not identified. Later Kuusi et al., [7] using porin, a protein purified from the outer membrane of S. typhimurium demonstrated that passive transfer of antiporin serum could protect the mice from a lethal challenge with virulent form of S. typhimurium. However, little attempt has been made to characterize the immune response to outer membrane proteins and this information would be valuable in developing a suitable vaccine against typhoid. In the present study we have attempted to characterize the antibody response to outer membrane proteins. Its significance in protective immunity and diagnosis has been discussed.

MATERIALS AND METHODS

Animals

Balb/c mice aged 6 weeks belonging to both sexes bred in our colony were used. They were fed with commercial feed (Hindustan Lever, Bombay) and watered ad libitum.

Bacterial strains

The virulent C5 strain of S. typhimurium was kindly supplied by Dr C. Hormaeche, U.K. The rough mutant, Rb2 strain of S. typhimurium (deficient in O-specific chain of LPS) was obtained from Dr P. H. Makela, Finland.

Bacteria[ preparation

S. typhimurium, grown overnight at 37°C in tryptic soy broth, was inoculated into a Roux flask containing nutrient agar and incubated at 37°C for 18 h. The cells were harvested using Tris-HCl buffer (pH 7.8).

Crude outer membrane proteins (comp)

Comp from either C5 or Rb2 strain of S. typhimurium was prepared following the method of Kuusi et al. [8]. Lysozyme-EDTA treated envelopes [15] were extracted twice

o/ with 2/0, (wt/vol) Triton X-100 in the presence of 0.01 M MgCI2. The pellet obtained after centrifugation at 7000 rpm (designated Triton X-100-1ysozyme-EDTA-treated envelopes) was suspended in 0.2%. Triton X-100 and electrodialysed against distilled water for 1 h. Then the pH of this suspension was raised to 8.0 using 1 M Tris-HC1 (pH 8.8). After 10 min of incubation at 37°C this suspension was spun at 7000 rpm for 15 rain and the supernatant was collected. The supernatant contained the crude outer membrane proteins. The total protein content of the comp preparation was estimated following the method of Lowry et al., [12]. The LPS content was determined by the method of Janda and Work [5].

Antibody response to outer membrane proteins of Salmonella 11

Immunization

Groups of mice received 5, 25, 50, 100 or 500/~gs of comp by intraperitoneal (i.p.), intramuscular (i.m.) or subcutaneous (s.c.) route. Control animals received the saline similarly.

Collection of sera

Control and immunized mice were bled by retroorbital puncture and the blood was allowed to clot for 1 hour at room temperature (28°C) and kept overnight at 10°C. Serum was separated at 2000 rpm for 10 min and decomplemented at 56°C for 30 min. The sera were stored at - 7 0 ° C in aliquots.

Antigen coated sheep erythrocytes

To 0.1 ml of packed sheep erythrocytes (SRBC) 2.5 ml of 2.5~o glutaraldehyde solution in phosphate buffer (pH 7.2) was added. With gentle stirring 1 mg o f c o m p was added and the mixture was incubated at 25°C for 1 h. Then the cells were washed in saline thrice and finally suspended in phosphate buffer. These cells were used at 1~o concentration for the assay.

The saline extract containing LPS was prepared from C5 strain of S. typhimurium [16]. This LPS was coated onto SRBC by following the method of Padmanaban and Mittal [17].

Passive haemagglutination assay (PHA)

Two fold serial dilution of the sera were made in microtiter plates. To this an equal volume of 1~o protein or LPS coated cells were added. This mixture was incubated at 37°C for 1 h and the titre was read after overnight incubation at 4°C. The reciprocal of the highest dilution which gave clear agglutination was taken as the antibody titre.

Statistics

Statistical analyses were done according to Student's t-test [3].

RESULTS

Influence of dose of Immunization on the antibody response

In order to determine the optimal dose of antigen concentration required for eliciting maximal antibody response, varying doses of comp ranging from 5-500/~g were used for immunization. Sera were collected from the immunized animals at an interval of 15 days and the antibody response to comp and LPS were detected using comp or LPS coated sheep erythrocytes.

A dose dependent effect on antibody response to comp could be observed from Fig. 1. The data indicated that as low as 50 ~g of comp was optimal to induce a maximal level of antibody response. Since the preparation of comp contained trace amounts of LPS (100/~g LPS/1 mg protein) the anti-LPS antibody level was also estimated. The same sera were titrated for antibodies directed against comp and LPS. The kinetics of the anti-LPS antibody response was found to be similar to the anti-protein antibody response (data not shown). As low as 50 ~g of comp was sufficient to evoke a maximum (Log2 titre of 4.5) anti-LPS response.

12 M. NATARAJAN et al.

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Fig. 1. Effect of various doses of comp on the antibody response in mice. Groups of mice were immunized with designated antigen doses. Individual sera were collected at two week intervals and titrated by PHA method using comp coated erythrocytes. [] [] 5#g; O O 25#g;

• • 501~g; • • 1001lg; /~ A 500/tg.

In normal mouse serum, no anti-LPS or anti-comp antibodies could be detected. When the normal mouse sera or immune sera were tested with uncoated erythrocytes, no agglutination was observed.

Influence of route of immunization on the humoral immune response

To study the influence of the route of immunization on the humoral immune response to comp, the standard dose of 50/~g of antigen in saline was injected through i.p., i.m. or s.c. route. At a time interval of two weeks, the antibody levels for comp and LPS were assayed using SRBC coated with comp or LPS.

Though there was no significant difference in the levels of antibody produced with the various routes of immunization, a difference in the kinetics was noticed among these groups (Fig. 2). Intraperitoneal route of immunization evoked a quicker onset of the anti-comp response and the peak level was reached by 4th week. But in the i.m. and s.c. routes, the peak occurred 6 weeks after immunization.

Demonstration of antibodies to LPS and protein

The interesting similarity observed in the kinetics of the LPS and comp response for a single injection of comp warranted the confirmation of the fact that these two responses were directed towards two different antigens. The study was further required because of the possibility of detecting the same antibodies by two different methods, as the antigens used in these assays were crude preparation of outer membrane proteins with LPS contamination.

Mice were immunized with 50 #g comp i.p. and the high titre sera were pooled. The sera were absorbed twice, using LPS coated sheep erythrocytes at 37°C. The unabsorbed and

Antibody response to outer membrane proteins of Salmonella 13

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Fig. 2. Influence of different routes of immunization on the anti-comp antibody response. Groups of mice received 50/~g of comp in different routes. Sera were collected at two week intervals and the individual sera were titrated with comp coated erythrocytes. (P value i.m. vs i .p .-->0.4; i.m.

t,s s .c .-->0.1; i.p. t,s s .c.-->0.5). O O i.p., ~ A s.c., • - • i.m.

absorbed sera were tested for the presence of antibodies to LPS and proteins. The observations are shown in Table 1.

Absorption with LPS eliminated anti-LPS antibody completely while there was no significant change in the level of antibody to comp. This indicated that these sera contained both protein specific and LPS specific antibodies and that the contaminating LPS in comp induced a significant anti-LPS antibody response.

Antibody response to comp of Rb2 strain of S. typhimurium

In order to find the influence of O-specific chain of LPS on antibody response to proteins and to further confirm that comp from smooth C5 strain of S. typhimurium elicited both protein and LPS specific antibodies, the comp from rough Rb2 mutant strain was used for immunization. Mice were injected i.p. with 50/~ g of Rb2 comp. The serum antibody response was assayed using LPS or comp coated sheep erythrocytes.

The results (Fig. 3) showed that Rb2 comp could elicit antibody response to comp but not to LPS. This is explainable since this antigen does not contain O-specific chain of LPS. The kinetics of the antibody response to comp of Rb2 strain was similar to that of C5

Table 1. Demonstration of anti-comp antibodies after absorption with LPS coated erythrocytes

PHA assay Before absorption After absorption* with (Log 2 titre) (Log 2 titre)

LPS coated cells 6.3 + 0.87t 0 Comp coated cells 4.6 _+ 0.33 3.7 _+ 0.33

*Sera were absorbed twice with equal volume of LPS coated erythro- cytes at 37C for l h.

tMean values of three different pooled sera.

14 M. NATARAJAN et al.

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Fig. 3. Antibody response to Rb2-comp in mice. Mice were injected with 50/.~g of Rb2-comp subcutaneously. Individual sera were assayed using Rb2-comp coated erythrocytes [~ ] or LPS

coated erythrocytes [O].

strain. This showed that the antibody response to LPS in C5 comp immunized mice was due to the low amount of LPS in this preparation. Further, in the absence of LPS, Rb2 comp could elicit a high level of antibody response to outer membrane proteins.

DISCUSSION

It is evident from previous observations that other than LPS, protein antigens are also important for protection in Salmonellosis [6, 7, 13, 18]. However, no major attempt had been made to identify and characterize the nature of protective antigen(s). In many of these studies [13, 18] though the antigenic preparations were shown to contain protein antigens their identities were obscure. The comp preparation we have used in this study was shown to contain the outer membrane proteins of S. typhimurium in the non-denatured form [8]. It is desirable to understand the role of these protein antigens isolated under non- denaturing conditions in protective immunity to Salmonellosis. Hence in the present study we have characterized the antibody response to outer membrane proteins of Salmonella.

Anti-protein antibodies were produced in mice immunized with comp. Interestingly, the same sera were also found to have anti-LPS antibodies. Presence of the anti-LPS antibodies in this sera could be accounted for the presence of low amount of LPS in the comp preparation. In general there was a similarity in the kinetics of both anti-LPS and anti-comp antibody responses. There were two observations which clearly indicated that the protein and LPS specific antibodies detected in these studies were different from each other. Firstly, when the CS-comp immune sera were absorbed with LPS coated cells, only anti-LPS titre was eliminated while there was no change in the anti-protein antibody titre (Table 1). Secondly, sera from mice immunized with Rb2 comp (O-chain deficient Rb2) had only anti-protein antibodies and the O-specific LPS antibodies were absent (Fig. 3). This observation confirmed the earlier findings of Kuusi et al. [6] who had shown that

Antibody response to outer membrane proteins of Salmonella 15

immunization with different preparations of outer membrane proteins in mice and rabbits resulted in the production of both protein specific and LPS specific antibodies. Similarly vaccination in calves with live and heatkilled vaccines of S. typhimurium induced both anti-LPS and anti-protein antibodies [11].

The antibody response persisted for a longer period to both the antigens. A clear dose dependent effect on the antibody response was also noticed (Fig. 1). Route of immu- nization did not have any major influence on the antibody response except for the delay in the peak response obtained in the i.m. and s.c. routes (Fig. 2).

The present study indicated that the outer membrane proteins were immunogenic. An interesting correlation was, presence of anti-protein antibodies in addition to anti-LPS antibodies in the immune sera of mice infected with S. typhimurium (Rajeshwari, unpublished). Tsang et al. [22] reported using radioimmunoassay, that typhoid patients had both protein specific and LPS specific antibodies in their sera. These observations implied that detection of antiprotein antibodies might serve as a useful diagnostic test for typhoid particularly in endemic areas where the Widal test had been found to be unsatisfactory [9]. In view of this the possibility of using outer membrane proteins prepared under non-denaturing conditions, for diagnostic test could be studied in detail.

Kuusi et al. [6] have reported that rabbit antisera raised against outer membrane proteins had a highly significant protective capacity against intraperitoneal Salmonella infection in mice. In a subsequent study Kuusi et al. [7] demonstrated that antisera raised against porin, a major outer membrane protein could protect the mice even after the removal of anti-LPS antibodies by specific absorption. These observations suggested the importance of protein antigens in protective immunity. Similarly our observations in this study showed that Rb2 comp which lacks O-specific chain of LPS could evoke anti-protein antibody response in mice (Fig. 3). These mice were also protected when challenged with 50 LDs0 of S. typhimurium (unpublished). This finding indicated that protein antigens in the absence of O-specific chain of LPS could induce protective immunity. We are further characterizing the significance of anti-protein antibodies in protective immunity.

Acknowledgements--We thank Professor P. H. Makela and Dr M. Nurminen, Finland, for providing rough mutant strain of S. typhimurium. The financial assistance from C.S.I.R. in the form of JRF is gratefully acknowledged by V.U.

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