ELSEVIER FEMS Microbiology Letters 141 (1996) 25529

Murine splenocyte proliferation by porin of Shigella dysenteriae type 1 and inhibition of bacterial invasion of HeLa cell

by anti-porin antibody

Sudipta Roy, Tapas Biswas *

Division of Immunology and Vaccine Development, National Institute of Cholera and Enteric Diseases, Beliaghata. Calcutta 700 010.

West Bengal, India

Received 12 February 1996; revised version received 25 March 1996; accepted 12 April 1996

Abstract

The purified porin of ShigeZlu dysenteriae type 1 showed strong mitogenic activity for murine splenocytes. Preincubation of

S. dysenteriae type 1 with anti-pork antibody reduced the bacterial plaque formation in HeLa cell monolayers by 45%. The two immunobiological activities indicate that porin might be important in the induction of protective immunity against shigellosis.

Keywords: Porin; ShigelZa dysenteriae; Splenocyte proliferation; HeLa cell invasion

1. Introduction

Shigella dysenteriae type 1, a Gram-negative en- teric bacterium, causes bacillary dysentery in humans by invading epithelial cells of the colon [l]. The in-

vasion of the epithelial cells elicits severe inflamma-

tory reaction, characterized by abscesses and ulcera- tions that destroy the epithelial lining of the colon [2]. These processes constitute the pathology of the early stages of shigellosis. Lipopolysaccharide (LPS),

a glycolipid unique to the outer membrane of Gram- negative bacteria, plays a major role in triggering immune cell activation leading to the release of host-derived proinflammatory cytokines and further amplification of inflammatory response [3]. Porins,

*Corresponding author. Tel.: +91 (33) 350 4478; Fax: +91 (33) 350 5066.

the channel-forming proteins of the outer membrane of Gram-negative bacteria [4], have also been found to be associated with immunobiologic activity [S] pointing to their role as modulators of pathogenesis

of infectious diseases. The study of these surface molecules appear to be of interest for the possible

role they might play in the pathology of the disease. Previously, we purified the major outer membrane protein of S. dysenteriae type 1 and showed it was porin [6]. Immunoelectron microscopy demonstrated the porin of S. dysenteriae type 1 to be surface ex- posed which makes it a potentially significant mole-

cule for pathogenicity. The OmpC, an outer mem- brane protein with pore-forming ability, has been shown to be involved in invasion of epithelial cells by S. JEexneri [7]. As porins of several bacteria have been studied for lymphocyte proliferation [5,8] and release of cytokines [9], we evaluated the role of pu-

0378-1097 /96/ $12.00 0 1996 Federation of European Microbiological Societies. All rights reserved

PIISO378-1097(96)00199-l

S. Roy. i? EiswslFEA4S Microbiology Letters 141 (1996) 25-29

Fig. 1. Proliferative response of murine splenocytes with purified

porin from S. dysenteriae type 1, or purified LPS from S. &en-

teriae type 1 in the presence or absence of polymyxin B. Results

are reported as geometric means f standard errors of values taken

from three separate experiments. Splenocyte stimulation index

was calculated by dividing the optical density (at 570 nm) values

of mitogen-containing wells by that of controls in which no mi-

togen (porin or LPS) was added.

rified porin of S. dysenteriue type 1 for proliferation

of murine splenocytes. The process of cellular invasion and multiplication

of Shigella spp. can be studied by in vitro assays using mammalian cell monolayers, such as HeLa.

Infected cell may eventually be killed by intracellular

Shigella, developing an area of dead or dying host cells or a plaque [lo]. As we found the porin to be exposed on the cell surface of S. dysenteriae type 1,

we studied whether murine anti-porin antibody blocked HeLa cell penetration by the bacteria. This

blockade of bacterial entry through HeLa cells by anti-porin antibody suggested the importance of porin in the pathogenicity of Shigella.

2. Materials and methods

2.1. Bacteria and HeLa cells

S. dysenteriae type 1 (N-120) was isolated from diarrhoeal patients admitted to the paediatric and

general wards of the Infectious Diseases Hospital, Calcutta. Identification of the strain was performed by standard microbiological methods and serotyping confirmed by agglutination with species- and type- specific antisera (Wellcome Diagnostics, Dartford, UK). Virulence of the isolated bacteria was checked routinely by their ability to invade the cornea1 epithelium of guinea-pig causing keratoconjunctivitis

[lll.

HeLa cells for monolayer culture was obtained from the National Facility for Animal Tissue and

Cell Culture, Kothrud, Pune, India, and stored in liquid nitrogen before use.

2.2. Animal and anti-porin antibody

BALB/c mice, originally obtained from Jackson

Laboratories (Bar Harbor, ME), were bred and reared in the animal care facility of the National

Institute of Cholera and Enteric Diseases, Calcutta,

India. BALB/c mice were used for generation of anti- porin antibody [6] or killed for obtaining spleno-

cytes. Anti-LPS activity in murine anti-porin anti- body at l/10 dilution could not be detected by en- zyme-linked immunosorbent assay (ELISA).

2.3. Preparation and estimution of lipopolysaccharide

The LPS was extracted from acetone-dried cells of

S. dysenteriue type 1 [12] to be used as controls in proliferation assays of murine splenocytes. The total carbohydrate content of the LPS was estimated by

the phenol-sulfuric acid method [13]. Trace amounts of LPS at the picogram level in porin of S. dysente-

riae type 1 were identified by Limulus amebocyte lysate assay [14] with endotoxin free water and E. coli 055:B5 LPS (E-Toxate Kit no. 210-Al, Sigma

Chemical Co., St. Louis, MO).

2.4. Murine splenocyte culture and proliferation assay

Spleen was removed aseptically from 6-week old

female BALB/c mice and transferred to culture me- dium. The spleen was gently teased with a syringe, passed through 105 ym nylon mesh and a single cell suspension of splenocyte was obtained. Cells were

devoid of erythrocytes and viable cells were counted by the trypan-blue exclusion method [15]. Cells were then seeded in 96-well round-bottomed tissue culture plates (Corning Glass Works, Corning, NY) at 1 X lo5 cells/well per 200 ~1 of RPM1 1640 supple- mented with penicillin and streptomycin (100 U/l) and 10% fetal bovine serum (FBS; Gibco BRL Life Technologies Inc., Grand Island, NY). To the splenocytes LPS of S. dysenteriae type 1, LPS with 2 pg/well of polymyxin B (Sigma Chemical Co., St. Louis, MO), porin or porin with 2 pg/well of poly-

S. Roy, T BiswaslFEMS Microbiology Letters 141 (1996) 25-29 21

B

Fig. 2. Formation of plaques on confluent monolayers of HeLa

cells infected with S. dysenteriae type 1 and bacteria preincubated

with anti-porin antibody. (a) 1 x lo5 bacteria were preincubated

with preimmune mouse sera and added to HeLa cells for plaque

formation. (b) 1 X IO5 bacteria were preincubated with a 1:25 di-

lution of anti-porin antibody and added to HeLa cells for plaque

formation.

myxin B was added and cells were grown for 72 h

and subjected to (3-(4,5-dimethylthiazol-2-yl)-2,5-di-

phenyl tetrazolium bromide (MTT) assay [16]. 20 pl

of a 5 mg/ml stock solution of MTT (Sigma Chemi- cal Co., St. Louis, MO) was added in a total volume of 200 ~1. 96-well plates were incubated at 37°C for 4 h for formation of formazan. Acid-isopropanol (100

~1 of 0.04 N HCl in isopropanol) was added to the wells and mixed thoroughly to dissolve the dark-blue

crystals. After a few minutes the 96-well plates were measured at 570 nm in a Titertek Multiskan ELISA reader (Flow Lab, Helsinki, Finland) against a blank 96-well plate.

2.5. Assay for bacterial penetration of HeLa cells

2 X lo6 HeLa cells in minimum essential medium (MEM) containing 10% horse serum (Gibco BRL Life Technologies Inc., Grand Island, NY) and pe- nicillin and streptomycin were seeded in six-well flat-

bottomed tissue culture plates (Corning Glass Works, Corning, NY). The cells were left to grow in a humidified incubator at 37°C in 5% CO2 until confluent monolayers were formed. The confluent HeLa cell monolayers were washed several times with MEM without serum and antibiotics and then infected with various dilutions of S. dysenteriae type 1. Prior to infection virulent S. dysenteriae type 1

were grown in trypticase soy broth (Difco Labora-

tories, Detroit, MI) at 37°C for 5 h, centrifuged, suspended in antibiotic free MEM. HeLa cell mono-

layers were infected with similar dilutions of the bac- teria preincubated for 1 h with either anti-porin anti-

body at 1125 dilution in MEM without antibiotics

and horse serum or preimmune mouse sera. HeLa

cells were allowed to grow further for 90 min at

37°C. The media from the culture plates were aspi- rated and washed with MEM containing 10% horse

serum and antibiotics. Infected HeLa cells were then

grown in MEM, 10% horse serum, penicillin, strep- tomycin and 20 pg/ml of gentamycin finally for 48 h,

at 37’C in 5% COz. After 48 h cells were stained

with 1% crystal violet (Sigma Chemical CO., St. Louis, MO) in 45% methyl alcohol for 2 min, washed with water and dried for visualization of

plaques.

3. Results

3.1. LPS content of porin of S. dysenteriae type 1

The LPS content of the purified porin of S. dy-

senteriae type 1 could not be detected biochemically. All possible traces of LPS were identified by Limulus

amebocyte lysate assay and found to be in the order

of 5 pg per 10 pg of porin.

3.2. Porin-induced prollyeration of murine splenocytes

Incubation of murine splenocyte with varying con-

centrations of porin profoundly stimulated prolifera-

tion of the cells (Fig. 1). The proliferative response of splenocytes to porin of S. dysenteriae type 1 in- creased in a dose-dependent manner between

1 X low4 and 1 pg of protein. Proliferation of spleno- cytes reached a plateau at 1 pg of protein or above. To exclude the possibility that the observed stimula- tion of murine splenocytes by porin was contributed by traces of LPS, polymyxin B was added over wide

range of porin concentrations, prior to stimulation. Addition of polymyxin B had no significant effect on splenocyte proliferation by porin. LPS of S. dysen-

teriae type 1 stimulated cell proliferation and addi- tion of polymyxin B completely removed the stimu- latory effect of LPS. These data indicated that the

28 S. Roy, T. BiswaslFEMS Microbiology Letters 141 (1996) 25-29

porin-mediated stimulation of murine splenocyte was

specific for protein rather than LPS.

3.3. Depletion of bacterial plague jbrmation by

anti-porin antibody

Fig. 2 shows the decrease in plaque forming ability

due to preincubation of S. dysenteriue type 1 with

anti-porin antibody. Compared to the control sets in which the bacteria were directly applied to the lawn

of HeLa cell monolayers or preincubated with pre-

immune mouse sera (Fig. 2a), the bacteria preincu- bated with the anti-porin antibody showed an about

45% drop in plaque formation (Fig. 2b). A similar

result was obtained on varying the S. dysenteriae

type 1 number compared to a parallel set of controls.

4. Discussion

The factors that elicit a protective immune re- sponse in shigellosis are still an important question,

as this infection remains a major health problem in many countries. One method of studying this prob- lem is to determine which immunogens, particularly

those located on the bacterial surface, have an im-

munomodulatory role and are also capable of elicit- ing protection. This should lead to the development of an effective vaccine urgently required against Shi-

gellu spp. as the clinical management of the disease has become difficult with the emergence of drug-re-

sistant strains of the bacteria [17]. Therefore, it is important to study bacterial components that have

vaccine potential. Since porins, the most abundant outer membrane protein (OMP), showed promise as potential vaccine [18,19], proliferate splenocytes [5] and also release cytokines not inhibited by poly- myxin B [9], we studied the immunomodulatory role

of S. dysenteriae type 1 porin [6]. We found that porin of S. dysenteriae type 1 was capable of induc- ing profound proliferation of murine splenocytes. The proliferation of cells of the immune system is an important immunomodulatory role played by porin. Further, porins as protein antigens may have the advantages of inducing antibodies of higher affinities and be free of endotoxic effects associated with LPS [3] and are capable of eliciting the cellular immune response necessary to control intracellular

bacteria. Immunization with porins of Salmonella

typhi and Pseudomonas aeruginosa led to protection against the bacteria in experimental animals [18,19].

Moreover, the surface-exposed pot-in of S. dysen-

teriae type 1 [6] has importance in pathogenesis as

preincubation of the bacteria with anti-porin anti-

bodies inhibited the penetration of HeLa cells, the

model system that mimics the process of cellular in- vasion and multiplication of Shigella spp in human,

its only natural host. The presence of trace amounts of LPS (LPS:protein = 1:2X lo6 by weight) in S. dy-

senteriue type 1 porin might elicit an immune re-

sponse to LPS as well, as found earlier with bacterial

outer membrane proteins prepared by elution from polyacrylamide gels [20]. However, the anti-LPS antibody, even if elicited, was not detectable by ELI- SA, indicating that inhibition of plaque formation by

murine anti-porin antibody was mediated specifically

by antibody to proteins. Since the anti-porin anti-

body interfered with a critical step in pathogenesis, it is likely to be an important immunogen with pos- sible relevance in protection. Further study will be of interest to evaluate the in vivo role of S. dysenteriae

type 1 porin in the pathogenesis of shigellosis and the induction of protective immunity in the host.

Acknowledgments

We thank Dr. S.K. Bhattacharya, Director for

support. We also thank Mr. Shyamal Kr. Das for

secretarial assistance.

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