Isolation of principal outer membrane protein and antigen of Chlamydia trachomatis

Procedures are presented for isolating the major outer membrane protein of Chlamydia trachomatis. The isolated protein is a species specific antigen which comprises about 60% of the C. trachomatis cell outer membrane structure. The protein has a molecular weight ranging from about 38,000 to 44,000 daltons, with a mean molecular weight of about 39,500 daltons. The protein antigen is purified from C. trachomatis cells by first extracting the cell contents with a mild anionic detergent, preferably sarcosyl, to leave a residue of intact outer cell membranes. These outer cell membranes are then extracted with a strong anionic detergent, preferably sodium dodecyl sulfate, which solubilizes the 39,500 dalton antigen. The antigen is then purified by hydroxlapatite chromatography. The antigen is species specific for Chlamydia trachomatis and may be utilized in assaying Chlamydial infection in mammals.

TECHNICAL FIELD 
This invention relates generally to the isolation of cell protein of 
microorganisms which exhibit antigenic properties and more particularly to 
the isolation of the principal outer membrane protein of Chlamydia 
trachomatis, which protein exhibits antigenic properties common to all the 
Chlamydia trachomatis serotypes. 
BACKGROUND OF THE INVENTION 
Chlamydia trachomatis is one of the two microorganism species of the genus 
Chlamydiaceae, order Chlamydiales. The other species is Chlamydia 
psittaci. Chlamydia trachomatis in its some 15 various strains, are the 
etiologic agents for a number of human ocular and genital diseases 
including trachoma, inclusion, conjunctivitis, lymphogranuloma venereum, 
"nonspecific" or non-gonococcal urethritis and proctitis. C. trachomatis 
infection is pervasive throughout the general population. It has been 
estimated, for instance, that C. trachomatis is accountable for several 
million cases per year of nongonococcal urethritis. 
Since C. trachomatis mediated disease is widespread, a reliable, simple and 
inexpensive test for the organism's presence is highly desirable and of 
great importance so that proper treatment may be undertaken. The only 
serological test in current use is the microimmunofluoresence test. This 
test however requires that the strains of C. trachomatis be used as 
serological test antigen. In addition, the facilities for conducting this 
test are available in only a limited number of laboratories throughout the 
world. The test is very laborious, time consuming and difficult to 
perform. 
Recently, U.S. Pat. No. 4,118,469, noted the preparation of an antigen of 
C. trachomatis useful in serological testing for lymphogranuloma venereum 
and nongonoccocal urethritis. Such antigen was purified from C. 
trachomatis organisms by immunoadsorption chromatography using the 
monospecific antiserum as a specific ligand covalently bound in an agarose 
gel column. This antigen had a molecular weight of about 160,000 daltons, 
and in counter-immunoelectrophoresis testing was capable of detecting 
antibodies from the sera of lymphogranuloma venereum patients. However, 
when utilized in a similar test with sera of non-gonoccocal urethritis 
patients, this antigen failed to detect antibodies. It was successful, 
however, in detecting antibodies in two dimensional immunoelectrophoresis 
testing. 
In any event, however, there is still great medical interest in the 
isolation of species specific antigens of C. trachomatis which are capable 
of the detection of C. trachomatis infection, preferably by commonly 
practiced antigen-antibody assay methods. 
BRIEF SUMMARY OF THE INVENTION 
The present invention presents a species specific antigen which comprises 
the principal outer membrane protein of Chlamydia trachomatis. Such 
protein comprise about 60% of the total associated outer membrane protein 
of C. trachomatis, and have a size or subunit molecular weight of between 
38,000 and 44,000 daltons, with a mean molecular weight of 39,500 daltons. 
Hereinafter for ease in reference, this principal outer membrane protein 
group will be referred to as MP 39.5 signifying "major outer membrane 
protein having a mean subunit molecular weight of 39,500 daltons". 
When tested against C. trachomatis antibodies derived from all the 
serotypes thereof, MP 39.5 reacts with species specificity. Thus MP 39.5 
is a C. trachomatis species specific antigen. MP 39.5 is a unique protein 
common to all C. trachomatis serotypes, and as an antigen provides a basis 
for the identification of all the C. trachomatis serotypes. 
MP 39.5 is isolated from C. trachomatis elementary bodies, i.e., the intact 
microorganism cells, by first growing suitable strains of the organism and 
collecting the elementary bodies free from the growth medium. The purified 
elementary bodies are treated by means hereinafter described to isolate 
the outer cell membranes. These outer cell membranes are selectively 
separated from the cell cytoplasm membrane and protoplasm. The isolated 
outer cell membranes are then further treated by a method hereinafter 
described to yield essentially pure MP 39.5. 
The MP39.5 recovered from the outer membranes is then available for either 
(1) direct reaction with C. trachomatis antibodies generated in the serum 
of C. trachomatis infected hosts; or (2) to be injected into laboratory 
animals to produce antiserum against MP39.5. Thus the recovered MP39.5 may 
be utilized in immunodiagnostic assay procedures for C. trachomatis. 
It is therefore an object of the invention to provide the principal outer 
membrane protein (MP39.5) of Chlamydia trachomatis. 
It is another object of the invention to provide a C. trachomatis species 
specific antigen. 
It is yet another object of the invention to provide a method for isolating 
C. trachomatis principal outer membrane protein. 
It is still another object of the invention to provide an antigen suitable 
for assaying chlamydial infection. 
Other objects and advantages of the invention will be apparent from a 
review of the following description and the claims appended hereto.

DETAILED DESCRIPTION OF THE INVENTION 
MP39.5 is the principal outer membrane protein of C. trachomatis and it is 
species specific antigen to all C. trachomatis serotypes. 
isolation of MP39.5 from C. trachomatis elementary bodies is accomplished 
by an essentially two step extraction procedure. In the first step, 
purified elementary bodies are contacted with an aqueous solution of a 
mild anionic sarcosine detergent, preferably sodium N-lauroyl sarcosine 
(commonly referred to as sarcosyl). The sarcosyl selectively dissolves out 
the elementary body cytoplasm including the protein, nucleic acids and 
other molecular structures associated therewith, leaving the elementary 
body outer membrane as an insoluble residue. 
Electron microscopic studies indicate that the sarcosyl treatment leaves an 
insoluble residue which consists of uniform particles of single intact 
double-track unit membranes of a size and morphology characteristic of 
native chlamydial elementary body outer membrane. 
In the second process step, the residual elementary body membranes are 
lysed with a strong anionic detergent, preferably sodium dodecyl sulfate, 
which solubilizes the principal outer membrane protein, MP39.5. The MP39.5 
is then recovered from the detergent solution, and purified to yield the 
MP39.5 antigen. 
The purified MP39.5 protein, when tested against antibody derived from 
known C. trachomatis serotypes demonstrates that MP39.5 is a species 
specific antigen of C. trachomatis organisms. 
The entire and detailed isolation procedure and characterization of MP39.5 
as a C. trachomatis antigen may be best understood from a review of the 
following detailed procedures and tests: 
Growth and purification of C. trachomatis organisms.--The following C. 
trachomatis strains were used: L2/434/Bu(L2), E/UW-5/Cx(E) and 
C/TW-3/OT(C). Chlamydiae were grown in HeLa 229 cells as described 
previously in the publication in October of 1975 in the Journal of 
Immunology v. 15, pgs 963-968 by Caldwell et.al., entitled "Antigenic 
Analysis of Chlamydiae by Two-Dimensional Immunoelectrophoresis." Such 
disclosure is incorporated herein by reference. The L2 strain was also 
grown in suspension cultures of L-929 cells. L-cell-propagated L2 
organisms were used for the isolation and purification of the 39,500 
dalton protein. 
Chlamydiae were harvested from HeLa cell monolayers grown in 150 cm.sup.2 
polystyrene culture flasks (Corning Glass Works, Corning, N.Y.) with 90% 
of the cells containing inclusions at 48 hours postinoculation. Medium was 
poured off and cells were removed with 4 mm glass beads and 10 ml of cold 
Hanks' balanced salt solution. These cell suspensions were pooled and the 
cells ruptured by sonication (Braunsonic Model 1510). This suspension was 
centrifuged at 500.times.g for 15 min at 4.degree. C. The supernatants 
were layered over 8 ml of a 35% Renographin solution (v/v) (diatrizoate 
meglumine and diatrizate sodium, 76% for injection, Squibb and Sons, N.Y.) 
in 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid containing 
0.15 M NaCl. They were then centrifuged at 43,000.times.g for 1 hour at 
4.degree. C. in an SW 27 rotor (Beckman Instruments Inc., Fullerton, Ca). 
The pellets were resuspended in 0.01 M sodium phosphate (pH 7.2) 
containing 0.25 M sucrose and 5 mM L-glutamic acid (SPG). pooled and 
layered over discontinuous Renographin gradients (13 ml of 40%, 8 ml of 
44% and 5 ml of 52% Renographin, v/v). These gradients were centrifuged at 
43,000.times.g for 1 hour at 4.degree. C. in an SW 27 rotor. 
The Chlamydiae elementary body bands, located at the 44/52% Renographin 
interface, were collected, diluted with three volumes of SPG and then 
centrifuged at 30,000.times.g for 30 min. The elementary body pellets were 
washed in SPG to remove residual Renographin. The purified elementary 
bodies were resuspended in SPG and stored at -80.degree. C. The purity of 
the elementary body preparations was determined by electron microscopy and 
Macchiavello stained smears. 
Isolation of chlamydial outer membrane complexes (COMC) by sarcosyl 
extraction of intact elementary bodies.--C. trachomatis L2 elementary 
bodies as collected above were suspended (approximately 5 mg protein/ml) 
in 5 ml of phosphate buffer solution (PSB) comprising 0.01 M sodium 
phosphate, and 0.15 M Nacl, pH 8.0, also containing 2% sarcosyl and 1.5 mM 
ethylenediaminetetracetic acid (EDTA). This suspension was incubated at 
37.degree. C. for 1 hour and then centrifuged at 100,000.times.g for 1 
hour. The insoluble pellet was resuspended in the same sarcosyl buffer and 
centrifuged as before. The pellet was washed twice in PBS to remove excess 
detergent and then resuspended in 0.02 M sodium phosphate, pH 8.0, 
containing 19 mM MgCl.sub.2 and 25 g/ml deoxyribonuclease (Worthinton 
Biochemical Corp., Freehold, N.J.) and ribonuclease (Millipore Corp., 
Freehold, N.J.). This suspension was then incubated for 2 hours at 
37.degree. C., centrifuged and the insoluble pellet washed twice with PBS 
to remove any remaining nucleases. This sarcosyl insoluble material 
consisted of chlamydial outer membrane complexes (COMC). 
Purification of the 39,500 dalton outer membrane protein.--Isolated COMC 
prepared from 25-30 mg L2 EB protein in the procedures as noted above, was 
suspended in 5 ml of 2% sodium dodecyl sulfate buffer and incubated at 
37.degree. C. for 1 hour. This suspension was centrifuged at 
100,000.times.g for 1 hour and the soluble supernatant fraction collected. 
This sodium dodecyl sulfate extract, enriched in the 39.500 dalton protein, 
was then dialyzed against 200 volumes of 0.01 M sodium phosphate, pH 6.4, 
containing 1 mM dithiothreitol (DTT) and 0.1% sodium dodecyl sulfate 
(column equilibration buffer) for 24 hours with several changes of 
dialysate. This extract was fractionated by hydroxylapatite chromatograpy 
in the presence of sodium dodecyl sulfate using the technique disclosed by 
Moss and Rosenblum in J. Bio. Chem., 1972, V. 247, pgs. 5194-5198. 
Briefly, the dialyzed extract (8-10 ml) was supplied to a pre-equilibrated 
hydroxylapatite column (0.9.times.30 cm). The column was washed with 100 
ml of equilibration buffer and eluted with a 150 ml linear gradient of 0.1 
M to 0.6 M sodium phosphate, pH 6.4, containing 1 mM DTT and 0.1% sodium 
dodecyl sulfate. The column eluate was collected in 40 drop fractions at a 
flow rate of 5-6 ml per hour and spectrophotometrically monitored at 280 
nm absorbence. Those fractions showing positive absorbence were analyzed 
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 
The polyacrylamide gels were stained with Coomassie blue for protein and 
Stains-All to detect nucleic acid and glycolipid moieties. The phosphate 
molarity of every tenth column fraction was determined by measuring total 
phosphorous and converting to phosphate molarity by using a standard curve 
prepared wiht known sodium phosphate standards. Those fractions that 
contained only the MP39.5 were pooled and concentrated to a 1-2 ml volume 
by vacuum dialysis against 0.05 mM Tris-HCl, pH 8.5, containing 0.15 M 
NaCl and 0.1% sodium dodecyl sulfate. These concentrated preparations were 
used for performing analytical assays to test for protein purity and as a 
source of immunogen for the preparation of antisera in laboratory animals. 
It will be understood that the detergent extraction temperatures and times 
noted in the procedures above may be varied from those as stated. It is 
perfectly feasible to extract at higher temperatures e.g., 45.degree. C., 
60.degree. C., 80.degree. C., or even 100.degree. C. Higher extraction 
temperatures may be accompanied by shorter extraction times. For instance, 
extraction at 100.degree. C. for 10 minutes, is sufficient to solubilize 
essentially all the elementary body components which are soluble in the 
particular detergent. Generally speaking, however, if time is not a 
problem, it is desirable to extract at the lower temperatures, e.g., 
37.degree. C., in order to avoid any chance of denaturing the desired 
proteins. 
The molecular weights of the purified COMC proteins were determined by 
polyacrylamide gel electrophoresis. Specifically, the Chlamydial proteins 
were electrophoresed on 12.5% acrylamide slab gels in the discontinuous 
Tris (hydroxymethyl) aminomethane-glycine (Trisglycine) system described 
by Laemmli in Nature (London) v. 227, pgs. 680-685 (1970). The ratio of 
acrylamide to N, N'-methylenebisacrylamide was 30:0.8 in both the 12.5% 
separating gel and 5% stacking gel. Before electrophoresis samples were 
mixed with an equal volume of solubilizing solution (0.1 M Tris HCl, pH 
6.8), containing 2.5% sodium dodecyl sulfate (BDH Chemicals Ltd.), 5% 
2-mercaptoethanol, 20% glycerol and 0.0001% bromophenol blue and boiled 
for 10 min. Electrophoresis in Tris-glycine buffer (ph 8.6) containing 
0.1% sodium dodecyl sulfate was carried out at a constant current of 25 
mA. Gels were stained in 0.25% Coomassie brilliant blue R-250 in 7% acetic 
acid and 30% methanol. The protein standards used for estimating 
chlamydial protein molecular weights were: phosphorylase b (94,000), 
bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase 
(30,000), soy bean trypsin inhibitor (20,100) and .alpha.-lactalbumin 
(14,400) (Pharmacia, Inc., Piscataway, N.J.). 
In one experimental study approximately 1.4 mg of purified MP39.5 protein 
was recovered after concentration, after following the procedures set 
forth above. Although the amount recovered was small compared to the 
recovery of outer membrane proteins from more readily cultivatable 
microorganisms, the yield was quite exceptional considering that only 
25-30 mg. of elementary body protein was used as the starting material. 
Preparation of Antisera--Swiss Webster mice strain 1CR (Charles River Co. 
Baltimore, MD) were immunized subcutaneously on day 0 with 30 .mu.g of 
purified Mp39.5 emulsified with Freund's incomplete adjuvant. 
Immunizations were repeated with the same amount of purified protein 
administered subcutaneously without adjuvant on days 16 and 27. Mice were 
bled by cardiac puncture 5 days after each immunization (days 21 and 32, 
respectively). The reactivity and specificity of the pooled sera collected 
from each bleeding was evaluated by indirect immunofluorescence. 
Table 1, below, presents the results of tests against elementary bodies of 
the various Chlamydiae serotypes (both trachomatis and psittaci) with the 
mouse generated antisera. 
TABLE I 
______________________________________ 
Indirect immunofluorescence of Chlamydia with mouse antiserum 
prepared against purified MP39.5 
Reciprocal antibody titer of 
mouse* anti-MP39.5 
Titer after 2nd 
Titer after 3rd 
Serotype or 
immunization 
immunization 
Organism strain (day 21) (day 32) 
______________________________________ 
C. trachomatis 
A -- -- 
B -- -- 
Ba 8 128 
C -- -- 
D 8 128 
E 8 64 
F -- -- 
G -- -- 
H -- -- 
I -- -- 
J -- -- 
K 8 128 
L1 8 128 
L2 64 512 
L3 8 128 
Mouse -- -- 
pneumonitis 
C. psittaci 
6BC -- -- 
Feline -- -- 
pneumonitis 
Guinea pig -- -- 
inclusion 
conjunctivitis 
______________________________________ 
*Highest dilution of antiserum (starting at 1:8) showing fluorescence. 
Serum antibody titers are lgG only, no fluorescence was observed with 
antilgM specific conjugate. 
In a procedure similar to that noted for the production of antiserum in 
mice, rabbits were inoculated with 300 .mu.g each of purified MP39.5 
protein. The protein was injected intramuscularly, and the rabbits were 
then bled after a suitable time was allowed for induction of the MP39.5 
antibodies. The pooled rabbit sera was then utilized for evaluation for 
reaction against all the various Chlamydeae elementary body serotypes. The 
results of these tests are set forth in Table 2, below. 
TABLE II 
______________________________________ 
Indirect fluorescent antibody staining of 
intact Chlamydeae with rabbit antiserum 
raised against the major outer membrane 
protein MP39.5 of the L2 C. trachomatis strain. 
Reciprocal Fluor- 
escent Antibody 
Organism Serotype Titer 
______________________________________ 
C. trachomatis 
A 64 
B 4096 
Ba 8192 
C 64 
D 512 
E 4096 
F 2048 
G 4096 
H 256 
I 64 
J 256 
K 4096 
L1 128 
L2 8092 
L3 4096 
C. psittaci 6BC &lt;8 
Mn &lt;8 
Feline pneumonitis 
&lt;8 
Guinea pig inclusion 
&lt;8 
conjunctivitis 
______________________________________ 
Fluorescence was determined by reacting elementary bodies of each Chamydia 
serotype with serial 2-fold dilutions of rabbit anti-MP39.5 (L2 
antiserum). Note that anti-MP39.5 reacts with every C trachomatis serotype 
but not with the C. psittaci strains. These results show that MP39.5 is a 
C. trachomatis species specific antigen. 
When MP39.5 protein prepared from other C. trachomatis serotypes, e.g. H, 
was utilized to generate antisera in laboratory animals, and the resultant 
antisera was reacted with elementary bodies of all the C. trachomatis 
serotypes, positive results similar to those set forth in Table II above 
were obtained. 
In any event, however, it is clear that the MP39.5 antigen has species 
specificity against all the C. trachomatis serotypes. 
As noted above monospecific antibodies against MP39.5 antigen can be 
generated by suitable inoculation procedures with laboratory animals such 
as mice and/or rabbits. The animal generated antibodies may be utilized in 
assays for Chlamydial infection in other mammals. These assays may be 
conducted in well known procedures for assaying the presence of bacterial 
antigen in the infected subject. Once a supply of monospecific antibodies 
has been secured from MP39.5 antigen inoculated laboratory animals, either 
direct or indirect assay procedures can be undertaken with specimens 
secured from mammals suspected of harboring Chlamydial infections. 
Assay techniques such as enzyme linked immunoabsorbent assays (ELISA) or 
radioimmune assays (RIA) are suitable for these purposes. 
In a direct assay procedure monospecific antibody against the MP39.5 
protein may be covalently or non-covalently attached to a solid phase 
support system. As is customary in these techniques the support system may 
be glass, plastics and the like. The solid phase support with attached 
monospecific antibody against MP39.5 may be incubated with a specimen 
previously secured from the individual suspected with having Chlamydial 
infection. Prior to incubation, the specimen is treated with a detergent 
such as sodium dodecyl sulfate or other anionic, nonionic or cationic 
detergent to extract the MP39.5 outer membrane antigen from any Chlamydial 
organisms which may be present therein. It is the extracted specimen which 
is incubated with the solid phase support. 
Monospecific antibody against MP39.5 antigen, which has been previously 
radiolabeled or conjugated with enzyme by known techniques, is then 
equilibrated against the support system. Any MP39.5 antigen present in the 
specimen and which had been bound to the antibody on the support system 
will in turn bind to the radiolabeled or enzyme conjugated antibody. 
If radiolabeled antibody is used, the amount of residual radioactivity in 
the sample may then determined. This value is compared to specimens that 
have been determined to be free of Chlamydial MP39.5 antigen. In the event 
enzyme conjugated antibody is used, a substitute specific for the enzyme 
is added to the solid support reaction mixture and the resultant color 
change is recorded spectrophotometrically. This color change is compared 
to samples known to be free of Chlamydial MP39.5 antigen. 
Thus the presence of MP39.5 antigen is mammalian specimens can be assayed 
directly. 
Alternatively, indirect assay procedures can be used. specifically, the 
Chlamydial (MP39.5) antigen secured as in the procedures set forth above, 
may be covalently or non-covalently bound to a suitable solid phase 
support system. A specimen from the individual suspected of having 
Chalmydial infection is treated with detergent, e.g., sodium dodecyl 
sulfate to extract the major outer membrane protein antigen from any C. 
trachomatis cells which may be present. 
The extract from the specimen may then be mixed with a known quantity of 
radiolabeled or enzyme conjugated antibody against the MP39.5 antigen, 
previously secured from a laboratory animal source. The specimen 
extract--antibody mixture may then be incubated with the solid support 
system and its bound MP39.5 antigen. 
The radioactivity of the solid support system is measured; or color 
development in the enzyme conjugated system is measured; and compared to 
specimens similarly treated as standards and which do not contain any 
Chlamydial antigen. 
The ability of the clinical sample suspected of containing C. trachomatis 
to inhibit the ability of the radiolabeled or enzyme conjugated antibodies 
to the MP39.5 antigen bound to the solid support thus reveals the 
presence, or absence, of the MP39.5 antigen in the clinical specimen. Any 
demonstrated inhibition indicates the presence of C. trachomatis 
infection. 
Other suitable assay method and variations will be apparent to those 
skilled in such assay techniques.