WO1999015690A1

WO1999015690A1 - Novel serratia marcescens strain, prodigiosin and the use of the same as an immunosuppressive

Info

Publication number: WO1999015690A1
Application number: PCT/KR1998/000287
Authority: WO
Inventors: Hwan Mook Kim; Young Kook Kim; Sang Bae Han; Sung Rak Yoo
Original assignee: Korea Institute of Science and Technology KIST
Current assignee: Korea Institute of Science and Technology KIST
Priority date: 1997-09-20
Filing date: 1998-09-19
Publication date: 1999-04-01
Anticipated expiration: 2000-03-20
Also published as: DE69834128T2; US20050069560A1; KR100252197B1; EP1017840A1; US7083969B2; US6645962B1; EP1017840B1; KR19990025976A; DE69834128D1; AU9188998A

Abstract

There are disclosed a novel microorganism Serratia marcescens strain and a prodigiosin isolated from the microorganism. The prodigiosin is useful as an immunosuppressive in various fields, including the treatment of the diseases requiring immunosuppression and the basic research for the diseases, the transplantation of the organs or tissues, and the immune cells.

Description

NOVEL Serra tia marcescens STRAIN, PRODIGIOSIN AND

THE USE OF THE SAME AS AN IMMUNOSUPPRESSIVE

TECHNICAL FIELD

The present invention relates to a novel Serratia

marcescens strain, a prodigiosin, and the use of the

prodigiosin in immunosuppression fields. More

particularly, the present invention relates to a novel

Serra tia marcescens strain which can produce the

prodigiosin, and the use of the prodigiosin as an

immunosuppressive .

BACKGROUND ART

Over the recent few years, active study and research

have been and continued to be directed to the development

of immunosuppressives, which are useful for the study on

immunocytes and immune responses and for the treatment of

the diseases requiring immunosuppression. For instance,

immunosuppressives are utilized in researching almost all

of immune responses, including cytokine production, T-cell activation, antibody production, cell death, DNA synthesis,

lmmunocyte differentiation, mtracellular signal

transduction, etc. The immunosuppressives are also used

to treat the diseases attributable to exaggerated immune

responses, such as hypersensitive immune response and

allergies. In addition, they are needed to suppress

excess immune responses upon transplantation of organs,

such as the kidney, the liver, the pancreas, marrow, the

heart, skin, the lung, etc.

Prevailing immunosuppressives include, for example,

cyclosporm A, cyclophosphamide, rapamycm, FK-506, etc.

Many immunosuppressives which show similar or different

suppressing behaviors are now under research.

The microorganisms belonging to genus Streptomyces or

Serra tia produce red substances of pyrrolylpyromethene

structures, examples of which include prodigiosin,

metacycloprodigiocm, prodigiosene, methoxyprodigiosm, and

prodigiosin 25-C. They are now known to be of

antibacterial and antimalarial activity and, particularly,

prodigiosin 25-C shows an lmmunosuppress g effect. DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a

novel strain Serra tia marcescens which produce a

prodigiosin.

It is another object of the present invention to

provide a prodigiosin as an immunosuppressive.

BEST MODES FOR CARRYING OUT THE INVENTION

The detailed description of the present invention

will follow isolation of a desired microorganism strain;

mycological characterization of the strain; extraction of

prodigiosin with organic solvent; purification of

prodigiosin through silica gel column and thin layer

chromatography; structure analysis through nuclear magnetic

resonance; utility of the prodigiosin as an

immunosuppressive .

Germ-free test animals, mice BDF1 and B6C3F1,

obtained from Genetic Resources Center, Korean Research

Institute of Bioscience and Biotechnology in the Korean

Institute of Science and Technology, were used for the assay of the immunosuppressive activity of prodigiosin.

The data from the ex vivo experiments concerning the

immunosuppressive effect of prodigiosin show that as much

as 300 nM of prodigiosin has a cytotoxic effect, but no

effects at less than 100 nM. At such concentrations as

show no cytotoxic effects, prodigiosin cannot suppress the

immune response of B lymphocytes. Prodigiosin had no

influence on the antibody production and proliferation of

B lymphocytes, but has a potential suppressive effect on

the proliferation and activity of T lymphocytes. This

selective immunosuppression for T lymphocytes is not

ascribed to the selective cytotoxicity for T lymphocytes.

The same immunosuppression results as m the ex vivo

experiments were obtained m m vivo experiments. When T

lymphocyte activity was measured by use of a graft versus

host reaction and a T cell-dependent antibody producing

reaction, the prodigiosin suppressed the immune response,

but exerted no toxicity on animals. Therefore, the

immunosuppressive activity of the prodigiosin is thought

to be attributed to the selective suppression for T lymphocyte activity.

Prodigiosin 25-C, an immunosuppressive analogous to,

but different from prodigiosin m structure and molecular

weight, is known to suppress the proliferation of T

lymphocytes, but not the proliferation of B lymphocytes.

Of T lymphocytes, CD8 T lymphocytes are suppressed, but

CD4 T lymphocytes are not. In contrast, the prodigiosin

of the present invention has an immunosuppressive activity

on CD8 T lymphocytes and CD4 T lymphocytes, both. This

immunosuppressive activity is similar to those of other

preexisting immunosuppressives. Like commercially

available immunosuppressives, such as Cyclosporm A,

Cyclophosphamide, FK-506 and Rapamycm, the prodigiosin of

the invention selectively suppress the immune response of

T lymphocytes.

The reaction systems used m the present invention

are illustrative of the application of prodigiosin for a

basic research of immunology, but not limitative of the

use of prodigiosin. The immunosuppressives m current use

are needed m various fields. First of all, the treatment of the diseases requiring immunosuppression and the basic

research therefor require them. Immunosuppressive drugs

are useful to remove the immune response which follows the

transplantation of organs or tissues. Another application

field of immunosuppressives is a basic research related to

immune cells. In this field are included studies on

cytokmes, activation and differentiation of immune cells,

and mtracellular signal transduction . Cyclosporm A,

Cyclophosphamide, FK-506 and Ripamycm are available for

this field. Because the prodigiosin of the present

invention has an activity similar to that of the above

immunosuppressives, it can be used as a curing agent and a

standard in such various fields.

The prodigiosin of the present invention was found to

have the following chemical formula with a molecular

weight of 323 as measured by NMR.

A better understanding of the present invention may

be obtained in light of the following examples which are

set forth to illustrate, but are not to be construed to

limit the present invention.

EXAMPLE I : Culturing of a Serra tia marcescens strain and

Isolation of Prodigiosin

Soil samples were taken from a silt area in Mokpo,

Korea. A bacterial group belonging to Serra tia spp. was

isolated from the samples and named Serra tia marcescens B-

1231. It was deposited in Korean Collection for Type

Cultures, Korean Research Institute of Bioscience and

Biotechnology on Sep. 19, 1997 and received a Deposition

No. KCTC-0386BP. In order to obtain an immunosuppressive,

the Serratia marcescens B-1231 was cultured at 28 °C for

62 hours in a 1L Erlenmeyer flask containing a basic

medium which consisted of soluble starch 1%, phamamedia

0.5%, glucose 0.2%, ammonium sulfate 0.1%, potassium

phosphate 0.1%, MgS0₄«7H0 0.05%, calcium chloride 0.1% and

NaCl 0.3%, at pH 7.0. An equal amount of ethyl acetate was added to the culture and they were sufficiently mixed

for 30 mm to give an organic layer. As the organic layer

was concentrated under a reduced pressure, a red substance

was obtained. This was separated by silica-gel column

chromatography using as a mobile phase a mixture of

chloroform and methanol . Following this, silica gel thin

layer chromatography was carried out to purify the object

material .

EXAMPLE II : m vi tro Experiment for Cytotoxicity Effect

of Prodigiosin on Lymphocytes

Immune cells were separated from the spleens of the

germ-free animals and cultured in vi tro . The cultures

were treated with the prodigiosin at various amounts from

3 nM to 30,000 nM and the viability of the cells were

measured from the first day to the third day after the

treatment. Based on the initial viability of the immune

cells, the viabilities of the test groups were calculated.

The results are given as shown in Table 1, below. As

apparent from the data, the viability of the treated immune cells is significantly decreased at a concentration

not less than 300 nM when being compared with that of an

untreated control. So, subsequent experiments for

immunoactivity were carried out at not more than 100 nM in

order to exclude the cytotoxicity and to measure only the

immunosuppressive effect of the prodigiosin.

TABLE 1

Effect of Prodigiosin on the Viability of Immune Cells

EXAMPLE III : in vi tro Experiment for the Effect of

Prodigiosin on Immune Cell Proliferation

Three standard substances which induce lymphocytes to

proliferate were employed to measure the effect of the

prodigiosin on proliferation of lymphocytes. 5 μg/ml of

lipopolysaccharide were used to induce B lymphocyte to

proliferate, 5 μg/ml of Concanavalin A for T lymphocyte

and 5 μg/ml of Pokeweed mitogen for B and T lymphocytes,

both. Prodigiosin was added, together with the

proliferation-inducing substance. Three days after the

addition, the proliferation effect was monitored by

measuring the amount of DNA synthesized. In order to

exclude the cytotoxicity of prodigiosin, it was used at a

concentration of not more than 100 nM. The effect of

prodigiosin on the proliferation of lymphocyte is shown in

Table 2, below. In Table 2, the proliferation percentages

mean the proliferated amounts of prodigiosin-treated

lymphocytes relative to that of an non-treated group. As

shown, the suppression percentage effected by prodigiosin

in amounts of 30-100 nM reaches up to 96-98 % for the T lymphocyte induced by concanavalm A while the

proliferation of B lymphocyte induced by lipopolysaccharide

and the proliferation of B/T lymphocytes induced by

pokeweed mitogen are suppressed to the extent of 13-19%

and 45-83%, respectively. Consequently, the data

demonstrate that the prodigiosin of the present invention

has a potential immunosuppressive activity which is

exerted selectively on T lymphocytes.

TABLE 2

Effect of Prodigiosin on the Proliferation of Immune Cells

EXAMPLE IV : m vi tro Experiment for the Effect of

Prodigiosin on the Immune Response The influence of prodigiosin on the functions of

lymphocytes was measured using three reaction systems.

First, the ability of B lymphocyte to proliferate in

response to lipopolysaccharide stimulus was assessed. For

this, on the third day after stimulation with

lipopolysaccharide, the antibody production of the B

lymphocyte was measured. When B lymphocytes are

stimulated with lipopolysaccharide, they can produce

antibodies without the aid of T lymphocyte. Second, a

mixed lymphocyte reaction was induced in order to assess

the effect on T-cell response. The reaction needs no aids

from the B lymphocyte. On the third day after two types

of heteroimmune cells, which are different from each other

in histocompatibility antigen, were mixed to stimulate the

activity of T lymphocytes, the T-cell response was

assessed. Third, the T-cell dependent antibody producing

reaction was utilized to assess the effect of prodigiosin

on the simultaneous immune response of both of the B and T

lymphocytes. This reaction requires the functions of B

and T lymphocytes, simultaneously. On the fifth day after immunization of the lymphocytes with the red blood cells

of sheep, their antibody production ability was assessed.

The effects of prodigiosin on the immune response of

lymphocytes are shown in Table 3, below. As apparent from

Table 3, the immune response in which T lymphocytes are

involved is significantly suppressed whereas the B cell

response is not at all throughout the concentration range.

In Table 3, the values are relative to the immune response

of the lymphocytes untreated with prodigiosin.

Taken together, the data of Examples III and IV

demonstrate that the prodigiosin potentially suppresses the

proliferation and immune response of T lymphocytes,

selectively.

TABLE 3

Effect of Prodigiosin on the Immune Response of Immune

Cells

EXAMPLE V : Selective Cytotoxicity of Prodigiosin for B,

CD4 T and CD8 T Lymphocytes

Whether the selective immunosuppression of prodigiosin

for T cells is attributed to the selective cytotoxicity

for T cells or not was assayed by measuring the proportion

of the cells. On the third day after treatment of the

immune cells with prodigiosin, the number of the cells was

counted. Because T lymphocytes consist of CD4 T cell

(helper T cell) and CD8 T cell (cytotoxic T cell), the

proportion of T and B lymphocytes was calculated in this Example. The results are shown in Table 4, below. The

data of Table 4 show that the prodigiosin has no selective

cytotoxicity. Thus, the selective immunosuppression for T

lymphocytes is proved to be attributed to the suppression

of immune response, but not of cytotoxicity. This result,

together with the result of Example II, also demonstrates

that the prodigiosin is not toxic within an effective

experimental concentration range.

TABLE 4

Cytotoxicity of Prodigiosin on Lymphocytes

EXAMPLE VI : in vivo Experiment for the Effect of

Prodigiosin on T Lymphocyte A graft versus host reaction was utilized for the m

vivo assay of prodigiosm's immunosuppression. The graft

versus host reaction enables an assessment of the immune

response of T lymphocytes. On the sixth day after

transplantation of the T lymphocytes of BDF1 mice

different in histocompatibility antigen, the lymphatic

nodes were measured for weight, thereby assessing the

immune response of T lymphocyte to the grafted

heteroantigens . The prodigiosin was peπtoneally injected

at a dose of 30-100 mg per kg of body weight for five days

while cyclophosphamide, as a positive control, was

peπtoneally injected at a dose of 100 mg/kg for five

days. The body weights of the injected mice were measured

to compare the toxicity of prodigiosin with that of

cyclophosphamide. The results are given in Table 5,

providing testimony that the prodigiosin potentially

suppress the immune response of T lymphocytes, like the

positive control, cyclophosphamide. As for the body

weight, it was not changed m the mice injected with

prodigiosin at an effective concentration. This demonstrates that the prodigiosin suppresses the immune

response of T lymphocyte without exerting toxicity in

vivo . In contrast, a loss of body weight occurred in the

mice injected with cyclophosphamide at an effective

concentration, showing the toxicity of the chemical.

TABLE 5

Effect of Prodigiosin on T Lymphocyte

EXAMPLE VII : Effect of Prodigiosin on T Lymphocytes in

vivo (T-Cell Dependent Immune Response)

A T cell-dependent immune response reaction was used

to assess the influence of prodigiosin on T lymphocytes in

vivo . Test animals were immunized with sheep red blood cells by peritoneal injection. 4 days after the

immunization, the number of the antibody producing cells

was counted. Prodigiosin was peritoneally injected

everyday. Based on the number of the antigen-producing

cells in the non-treated animals, the influence of

prodigiosin on T lymphocytes in vivo was assessed as

percentage. Also, the weight ratio of the spleen to the

body was measured to assay the toxicity of prodigiosin to

the animals. Cyclophosphamide was used as a positive

control.

The results are given in Table 6, below. As apparent

from the data of Table 6, the number of the antibody-

producing cells was significantly reduced by the treatment

of prodigiosin, which is comparable to the positive

control, cyclophosphamide, in the immunosuppression.

Taking account of the weight ratio of the spleen to the

body, the prodigiosin showed no toxicity at its effective

concentrations while cyclophosphamide was very toxic at

its effective concentration. TABLE 6

Effect of Prodigiosin on T cell -Dependent Immune Response

INDUSTRIAL APPLICABILITY

As apparent from the data of the Examples, the

prodigiosin of the present invention has a potentially

suppressive effect on the immune response of T

lymphocytes, in vivo and in vi tro, both. What is better,

the prodigiosin shows no toxicity at its effective

concentration ranges. Therefore, the prodigiosin of the

present invention can be used as an immunosuppressive or a

standard substance in various fields, including the

treatment of the diseases requiring immunosuppression and

the basic research for the diseases, the transplantation

of organs or tissues, and the immune cells 19/1

=0» --S ⁵UΛP S2 '^OCΞDt.3

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ϊssυed pursuant to Rule 7 J

TO- Him . Kwan Moo

- 33- 130 i Hanbit -Apt Chm- ocg. Yusong-ku . Taejon 305-333, ⁽ rT)"blic of Korea

Claims

-20- CLAIMS

1. A novel microorganism Serra tia marcescens B1231

which produces prodigiosin (KCTC 0386BP) .

2. Use of prodigiosin as an immunosuppresive,