Sequence of the gene encoding the major outer membrane protein of the mouse pneumonitis biovar of Chlamydia trachomatis

Gene. 106 (1991) 137-138 C 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

137

0378-l 119/91/$03.50

06035

Sequence of the gene encoding the major outer membrane protein of the mouse pneumonitis biovar of Chlamydia trachomatis (Recombinant

DNA;

Gram-

bacteria;

variable

domain;

constant

domain;

vaccine;

cervicitis;

infertility;

arthritis)

Thomas J. Fielder, Sukumar Pal, Ellena M. Peterson and Luis M. de la Maza Department of Pathology, University of California Irvine, Irvine, CA 92717 (U.S.A.) Received by J.A. Engler: 24 April 1991 Accepted: 21 May 1991 Received at publishers: 2 July 1991

SUMMARY

The gene encoding the major outer membrane protein of the Chlumydiu truchomatis mouse pneumonitis biovar was sequenced and the amino acid sequence deduced. The primary structure of this protein is similar to that of the lymphogranuloma venereum and trachoma biovars in that it consists of four variable domains interspersed with live constant domains. This protein may be an ideal candidate for a vaccine in chlamydia-infected mouse experimental models.

C. truchomutis are Grambacteria that cause a variety of infections including urethritis and epididymitis in males and cervicitis, salpingitis and infertility in females. Three C. truchomuris biovars have been described: LGV, trachoma and MoPn. The LGV biovar can be divided into three serovars, L 1, L2 and L3, and the trachoma into twelve serovars, A, B, Ba, C, D, E, F, G, H, I, J and K. Nigg (1942) isolated MoPn from the lungs of albino Swiss mice. Since then this microorganism has been reported to produce cervicitis, salpingitis, infertility, pneumonitis and arthritis in the mouse, thus providing very useful experimental models (Williams et al., 1981; Barron et al., 1984; Swenson and Schachter, 1984; Hough and Rank, 1988). The MOMP of C. truchomutis has been considered by several authors as the most likely candidate for a vaccine (Stephens et al., 1987; Su et al., 1990). In vivo and in vitro studies indicate that the MOMP is strongly antigenic and Correspondence to: Dr. L.M. de la Maza, Medical

Sciences

I,

University

of

Department

California

Irvine,

of Pathology, Irvine,

CA

92717-4800 (U.S.A.) Tel. (714)856-7450; Fax (714)725-2160. Abbreviations: constant venereum; membrane

aa, amino

domain(s);

acid(s);

kb, kilobase

bp, base pair(s);

C., Chlumydiu; CD,

or 1000 bp; LGV, lymphogranuloma

omplL3, gene encoding L3 MOMP; MOMP, major protein; MoPn, mouse pneumonitis; nt, nucleotide(s);

oligodeoxyribonucleotide; PCR, polymerase

ompZMoPn, gene encoding

chain reaction;

VD, variable

outer oligo,

the MoPn MOMP;

domain(s).

can elicit neutralizing antibodies and a T-cell response (Su et al., 1990). Ramsey et al. (1989) characterized the humoral immune response to a chlamydial genital infection with C. truchomutis MoPn. Here, immunoblot analysis showed a strong antibody response in plasma and high IgA titers. Thus, the MoPn MOMP appears to be an ideal candidate to test as a vaccine for its ability to protect mice experimentally infected with C. truchomutis. The nt sequence of omplMoPn was determined following amplification of the DNA of the Nigg II strain (American Type Culture Collection, Rockville, MD) using the oligos 5’-CTACAGGACATCTTGTCTGGC and 5’-TTAGAAGCGGAATTGTGCATT. The PCR conditions were as previously described, except that the MgCl, concentration was increased to 5.0 mM (Peterson et al., 1990). The amplilied DNA was purified in a l”/, low-melting-temperature agarose gel, extracted with phenol-chloroform and precipitated with alcohol. Both strands were sequenced following the method described by Casanova et al. (1990). Based on a comparison of the DNA and aa sequences of the L2, B and C serovars of the C. truchomutis MOMP, Stephens et al. (1987) determined that this protein has four VD interspersed with live CD. The nt sequence of omplMoPn (Fig. 1) indicates that this protein has a similar overall structure in the mouse, trachoma and LGV biovars. Compared with the C. truchomutis omplL3 sequence (Fielder et al., 1991) the omplMoPn has 10 less aa, 3

158 161

Phe

Gly

Arg

*ix

Asp Lys

G1” mlr

581 AGA GCA GCT TTC Tcx 601 c T c 195 *rg Ala Ala Phe Trp b” 201

. . . . . . wlr Gl” ser

Ah Am

“al Pile

GAG TGC GGA XT cc* RTG G G1” cys ay cys Pro Ala

. . . Ala AS” mr

cm

RAG CCT A&A GGA TAC GTT GGA CAA GAG TTT G GCG A G T I,ys Pro Lys ay Tyr “al Gly Gl” Gl” Pile Ala

823 841 275 281

TCT TAC AGA CTG AAT ATG TTC ACT CCT TRC I\TT TA ser ryr Arg I,eu As” Met Ph.2 mr Pro Tyr Ile

1051 lcml 351 361

CTT AAC ATT A G T Pro Le” AS” Ile

*w

ws

Ile “al

Pro

*sn “al

mr

of ompZMoPn and omplL3 EMBL/GeneBank

AAA GCT a.4 cc A lys Ala Gly Thr

. . . .. . .. . GCT TCC GGC . . .. . . . Ala ser my

TGC GGT CTT GCA RTT GGA ACA AC.4 ATT GTA GAT GCT GRT Am A GA G T AC cys my Leu Ala Ik my mr Thr Ile “al Asp *ia Asp I,ys Ile “al

in one of the serovars.

Asp Leu

GGA GTT AAG TGG TCT *GA A my “al I,y* n-p sei *rg

TGG AAC cc* ACG ATC XT GGA TCT GGT *lx GAC GTT G TANI A GT TG c CT* GC Trp As” Pro *tlr Ile ser my ser ay Ile Asp “al ser “al h” Ala

Fig. 1. The nt sequence absent

Asp Lys

ser Ah

Leu

ser

an

Ala

“al

“ai

a”

RCA GTT AGC GCT ACR GAT ACT .&AA GM AAGCTG lx G mm “al ser Ala nlr *sp mr l.ys Asp au Ala GlY GCA AGC TTT GAT GCC GAC ACT AK G T T Ala ser Pile Asp *la Asp mr Ile “al

. . . GAT ACA Am AGC A ACG . . . Asp Thr Lys ser Gl” *sn G1”

AX ACG GAT ACA CGGT Ile Thr ASP Thr Ie” Ala

and deduced accession

aa sequences

This work was supported by grant Al-26807 from the National Institute of Allergy and Infectious Diseases.

GTT CTC TGT T Le” cys

Ala Thr

Phe

Ala

Trp

ser

“al

my

RAT

GCG

GUI

@.A

TX

ACT

ATT

AAC

Gl”

T Phe ThT

Ii.?

AS”

G

*sn Ala *sp

“al

ATC Al.3

Pd.3

T

ser

GCT TCC RTC GAT TRC CRT GAG TGG CAR GCA AGC TTG GCT TTG CTTC T R CCT ser Ile Asp Tyr HIS G1” rrp Gl” Ala ser Leu ida I,eu

CGC ATT GCG CAG CCT AAG CTT GAG ACC TCT AK TCT ATG CTGAAGAG Iie Ala Gl” Pro Lys. I,..?” au mr Sex Ile Ala Glli Ala “ai

TTA Ax+ ATG ACC GGTGC T k” r,ys Met mr Psp “al

*rg

TTA CPA ATT GTT WC TTG CAG CTC AK AG c TG Ia” Gl” Il.2 “al ser Le” an I&” AS”

A/G

RTG AAA KC

I.ys

i-let

*GA

PAA TCT

*rg

Lys

T

ser

l.y.5

ser

net

of the MOMP

of C. rrachomatis

conditions

for directly

with Sequenase.

MoPn and L3. Dots represent

nt and aa

the major

trachomaris serovar

Peterson,

antigen.

P.: Optimal

pneumonia

and systemic

J. and de la Maza, L.M.: The for

Plasmid 23 (1990) 144-148. Rank, R.G.: Humoral immune

response to chlamydial genital infection of mice with the agent of mouse pneumonitis. Infect. Immun. 57 (1989) 2441-2446. Stephens,

R.S.,

brane

Sanchez-Pescador,

M.S.: Diversity protein

Su, H., Morrison,

chlamydial Barron, A.L., Rank, R.G. and Moses, E.B.: Immune response in mice infected in the genital tract with mouse pneumonitis agent (Chfamydia trachomafis biovar). Infect. Immun. 44 (1984) 82-85.

mice by

in Chlamydia trachomatis is not essential

the growth of this microorganism. Ramsey, K.H., Newhall, W.J. and

Transm.

R., Wagar,

E.A., Inouye,

of Chlamydia trachomatis major

genes. J. Bacterial. R.P., Watkins,

169 (1987) 3879-3885.

N.G. and Caldwell,

of T helper

C. and

outer mem-

cell epitopes

H.D.: Identification of the major

outer

protein of Chlamydia trachomatis. J. Exp. Med. 172 (1990)

203-212. Swenson, C.E.

REFERENCES

in C57B1/6

95 (1942) 49-50.

B.A., Schachter,

present

sequence

of Chlamydiu

130 (1988) 163-172.

virus which produces

E.M., Markoff,

protein

of arthritis

Am. J. Pathol.

in mice. Science

7.5 kb plasmid

PCR products

101 (1991) 159-160.

R.G.: Induction

Nigg, C.: An unidentified infection

outer membrane

L3. Gene

A.J. and Rank,

chlamydial

double-stranded

Acids Res. 18 (1990) 4028.

E.M. and de la Maza, L.M.: Nucleotide

of DNA encoding Hough,

sequencing

Nucleic

Fielder, T.J., Peterson,

membrane

C. and Kourilsky,

mr

Asp

Ala

and characterization

C., Jaulin,

Ttx

No. M64171

Urdea,

J.L., Pannetier,

Tyr

TAT GCR GTT ACT GTT GAG ACA ccc TTG ATC GAT GAA AGA GCA GCT CAC GTA ART GCR CA& TTC CGC TTC TAA G c A T Tyr Ala “al Thr “al G1” mr *rg Ie” Ile Asp G1” *rg *la Ala HIS “al AS” Ala Gl” Pk *rg Phe End

missing from the VDl and VD2 and 4 from the VD4. In addition, there are 240 nt changes in the omplMoPn compared to the omplL3 resulting in 63 aa substitutions. Of these aa substitutions 10 occur in the VDl, 12 in the VD2, 5 in the VD3 and 16 in the VD4. It is interesting to note that these changes result in approx. a 53-63 y0 aa replacement in the VDl, 2 and 4, while in the VD3 this represents only 36% aa substitutions. Based on these data it appears that the VD3 is better conserved than the other VD between different biovars of C. trachomatis, perhaps indicating less evolutionary pressure for this region, supporting the finding that no antigenic determinants have so far been mapped to this domain (Su et al., 1990).

Casanova,

Leu

AS”

ACT TTA GGA GCT KC TTC CAR TAT GCT CAP. TCT AAG cc* AAh GTA GAG @.A TTA MC G T A T RGGT mr k” GiY Ah ser Phe Gl” Tyr Al.3 Gh ser 5y.5 PI0 LYS “al Gl” mu Is%” AS”

703 721 235 241

943 ACT 961 315 TM 321

Ala

and

Schachter,

infection

J.: Infertility

of the upper genital

as a consequence

tract

of

in female mice. Sex.

Dis. 11 (1984) 64-67.

Williams, D.M., Schachter, J., Drutz, D.J. and Sumaya, C.V.: Pneumonia due to Chlamydia trachomatis in the immunocompromised (nude) mouse. J. Infect. Dis. 143 (1981) 238-241.