Vol. 58, No. 9

INFECTION AND IMMUNITY, Sept. 1990, p. 2760-2769 0019-9567/90/092760-10$02.00/0 Copyright © 1990, American Society for Microbiology

A Protective Protein Antigen of Rickettsia rickettsii Has Tandemly Repeated, Near-Identical Sequences BURT E. ANDERSON,'* GREGORY A. McDONALD, t DANA C. JONES,' AND RUSSELL L. REGNERY' Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 598402 Received 16 February 1990/Accepted 11 May 1990

The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type H), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever.

The intracellular bacterium Rickettsia rickettsii is the etiologic agent of Rocky Mountain spotted fever (RMSF), currently the rickettsial disease of greatest public health concern in the United States. Likewise, related spotted fever group rickettsiae are known to cause disease on five other continents. Although RMSF has been well recognized and studied for decades, the immune response against rickettsial infection is still not completely understood. Specifically, the role that individual rickettsial antigens play in eliciting the immune response during infection is not well defined. A thorough study of individual rickettsial components and the role each plays in conferring immunity will allow an assessment of the feasibility of producing new-generation vaccines. Additionally, such studies will provide basic knowledge regarding specific rickettsial proteins and their function in rickettsial pathogenicity and infection. Since previous vaccines for RMSF have been only marginally effective (11, 13, 35) and no vaccine for RMSF is currently available, recombinant DNA technology offers a possible avenue for producing new-generation RMSF vaccines. Two surface protein antigens of R. rickettsii have been identified as major protective antigens and possible candi-

(unpublished observations). An Escherichia coli clone, EM24(pGAM21), expressing a truncated version of the 190-kDa antigen has been reported by McDonald et al. (30). A subclone of EM24(pGAM21) expressing a 3.7-kilobase (kb) PstI fragment has been shown to protect both mice and guinea pigs from subsequent challenge with viable R. rickettsii (30, 31). To further characterize the 190-kDa antigen, we report the nucleotide sequence of pGAM21 and describe the corresponding deduced amino acid sequence. In addition, we describe the immediate 5' end of the gene cloned from the R strain of R. rickettsii; together with the sequence for pGAM21, this defines the entire reading frame for the 190-kDa-antigen gene. MATERIALS AND METHODS Plasmids and bacteria. R. rickettsii strain R was grown in Vero E-6 cells as described previously (7) and purified from host cell material by Renografin density gradient centrifugation (46). Rickettsiae for DNA extraction were inactivated with 0.1% Formalin and lysed with Sarkosyl and proteinase K (8). Lysis was followed by repeated phenol and chloroform extractions, and the DNA preparations were dialyzed against two changes of TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA). Rickettsiae for SDS-PAGE and immunoblot analyses were inactivated by gamma irradiation (106 rads) from a 'Co source and subsequently purified in the absence of trypsin. The construction of pGAM21 has been described previously (30). Since the nucleotide sequence revealed that pGAM21 lacked the 5'-terminal sequences of the 190-kDaantigen gene, an additional plasmid coding for the 5' terminus was obtained by direct cloning of R strain genomic DNA. Briefly, a 1.1-kb HindlIl fragment was found to contain sequences overlapping pGAM21 at the 5' end as determined by Southern blot analysis. A preparative gel was then used to electrophorese HindlIl-cleaved R strain R.

dates for use in subunit vaccines (1, 4, 30, 31). These proteins have apparent molecular masses of 120 and 155 kilodaltons (kDa), although the molecular mass estimation for both of these proteins appears to vary with the concentration of acrylamide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (2, 31). In our laboratory, these two proteins have apparent molecular masses of approximately 135 and 190 kDa as determined by SDS-PAGE and immunoblotting with 8% acrylamide gels * Corresponding author. t Present address: Department of Microbiology, School of Medicine, University of Missouri, Columbia, M653 Medical Science Building, Columbia, MO 65212.

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REPETITIVE PROTEIN ANTIGEN OF R. RICKETTSII

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rickettsii genomic DNA, and the 1.1-kb region was excised and purified from the agarose by using Gene-Clean (BiolOl, La Jolla, Calif.). The resulting DNA was ligated into plasmid vector pUC19 (50) and transformed into Escherichia coli DH5a [F' 4)80 d lacZAM15 (lacZYA-argF)U169 recAl endAl hsdRJ7 (rK- MK+) supE44 X thi-J gyrA relA]. Colonies were screened for the presence of the 1.1-kb insert by hybridization to an oligonucleotide probe corresponding to the 5' end of the pGAM21 nucleotide sequence. This new plasmid contained sequences upstream of pGAM21 (Fig. 1) and was termed pllOO. Sequencing was used to confirm that the overlapping region between pllOO and pGAM21 was the same. Phagemid constructs used for sequencing were transformed into E. coli XL1 Blue [recAl endAl gyrA96 thi hsdRJ7 (rj iK) supE44 relAl A- A(lac) {F' proAB lacIqZAM15, TnJO (TetD}]. The entire 190-kDa-antigen open reading frame was reconstructed in E. coli via a multistep cloning procedure. First, pllOO was unidirectionally deleted from the XhoI site for all sequences upstream of nucleotide 56 (Fig. 2) by using exonuclease III and S1 nuclease (Erase-a-base; Promega Biotech, Madison, Wis.). This results in a plasmid containing the 5' end of the 190-kDa-antigen gene deleted with respect to the presumed rickettsial promoter but not the putative ribosome-binding site. Subsequently, the 5.1-kb HindlIl fragment of pGAM21 was ligated to the single HindIll site remaining in the deleted p1100, and the junction was sequenced to confirm successful in-frame ligation. The 3.7-kb BamHI (vector derived)-XhoI fragment from the resulting plasmid was directionally ligated into expression vector pNH8A (15) (Stratagene Cloning Systems, La Jolla, Calif.). Finally, the 3.9-kb XhoI-XbaI fragment of pGAM21, containing the 3' half of the 190-kDa-antigen gene, was directionally ligated to the pNH8A-derived construct, and the ligation site was sequenced to confirm in-frame ligation. The resulting plasmid, pBA13 (Fig. 1), contains the entire promoterless gene downstream of the tandem lac and tac promoters, which face in the opposite direction from the 190-kDa-antigen gene. Subsequent to transformation into E.

coli D1210HP [hsdR hsdM supE44 ara-14 galK2 lacIq proA2 rspL20 xyl-5 mtl-i recAJ3 mcrB (Axis kil c1857)], int (encoded by lysogen)-mediated inversion of the promoters (to the active orientation) was accomplished by thermal induction (15). This should result in the 190-kDa-antigen gene being under the sole transcriptional control of the tandem lac and tac promoters of expression vector pNH8A. Chemicals and enzymes. Restriction endonucleases, ligase, and all other enzymes were purchased from New England BioLabs (Beverly, Mass.) unless otherwise noted. All isotopes were purchased from New England Nuclear Corp. (Boston, Mass.). Reagents for preparation of DNA sequencing gels were ultrapure quality from Bethesda Research Laboratories (Gaithersburg, Md.). Chemicals and salts were purchased from Sigma Chemical Co. (St. Louis, Mo.). DNA sequencing. DNA fragments to be sequenced were cloned into phagemid vector pTZ18R, pTZ19R, or pTZ19U (U.S. Biochemical Corp., Cleveland, Ohio), and the singlestranded DNA was isolated by packaging into M13 particles subsequent to infection with helper phage M13K07. The single-stranded template was sequenced by using the Sequenase T7 DNA polymerase-based kit (U.S. Biochemical Corp., Cleveland, Ohio) with the appropriate M13 sequencing primer (reverse or universal) or a primer internal to the rickettsial DNA insert derived from the sequence. Alternatively, the double-stranded plasmid sequencing method of Zhang et al. (52) was used to determine portions of the sequence. The nucleotide sequence for both strands of the entire open reading frame and flanking sequences was determined. The 3.8-kb PstI fragment of pGAM21 was subcloned into pTZ18R and termed pGM3-18R. Subsequent sequence analysis of pGM3-18R failed to yield clean and unambiguous sequence with internal primers but not with M13 primers, indicating the possibility of multiple internal priming sites. Consequently, a set of nested deletions for pGM3-18R were generated by using the Erase-a-base system. A second set of deletion plasmids for the 3.8-kb PstI fragment were generated by using phagemid pTZ19U to allow sequencing of the

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strand opposite that of pGM3-18R when the M13 universal primer was used. Southern blot analysis. DNAs from the R strain of R. rickettsii (20 ,ug) and pGAM21 (0.5 ,ug) were digested with restriction endonucleases, extracted with buffer-saturated phenol, and ethanol precipitated. The cleaved DNA was then electrophoresed on a 1.2% agarose-2.0% NuSieve GTG agarose gel and transferred to nitrocellulose by standard procedures (27). The 3.8-kb PstI fragment of pGAM21 was isolated, labeled with 32p via random primer labeling, and subsequently used for hybridization. After prehybridization for 4 h, hybridization was performed for 16 h at 67°C and then washing at the same temperature, with the final wash consisting of 0.1 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate) with 0.2% SDS. Immunoblot analysis. E. coli D1210HP containing pBA13 was grown at 30°C to mid-log phase and heat-pulsed at 42°C for 10 min. Additionally, the tandem lac and tac promoters were induced by addition of 1 mM IPTG (isopropylthiogalactopyranoside) at the same time as the heat-pulse. Samples were collected at various time points relative to induction and subjected to electrophoresis through an 8% acrylamide SDS-PAGE gel by the method of Laemmli (25). Subsequently, the resolved proteins were transferred to nitrocellulose, and the blot was developed by the method of Towbin et al. (43). Monoclonal antibody 14-19D4H8 (3, 4), specific to the 190-kDa antigen (G. A. McDonald, unpublished observations), was used as the primary antibody, and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Bio-Rad Laboratories, Richmond, Calif.). RESULTS Cloning the 5' terminus of the 190-kDa-antigen gene. Sequence analysis of pGAM21 revealed a single open reading frame starting at one edge of this clone and extending 6,455 nucleotides in the 3' direction (Fig. 1). To locate the 5' terminus of the 190-kDa-antigen gene and the corresponding flanking sequences, the oligonucleotide probe 5'-AAGGC TATAAATGTTGCGGGTACTACTCCCG-3' was used to identify a genomic fragment for subsequent cloning and sequencing. This oligonucleotide was located 9 bases downstream of the Sau3AI restriction site defining the 5' terminus for the pGAM21 insert and 86 bases upstream of the first HindIII site. A single 1.1-kb HindIIl fragment was identified by Southern blot analysis (data not shown), subsequently purified from an agarose gel, and cloned into pUC19, yielding pllOO. The nucleotide sequence for the 133-base overlap between pGAM21 and pllOO agreed perfectly, indicating that pllOO represents sequences that are upstream (on the R strain genome) of those found in pGAM21. Thus, pllOO should contain the 5' terminus of the 190-kDa-antigen gene. Nucleotide sequence analysis. The nucleotide sequence for the 190-kDa-antigen gene was provided to GenBank (accession no. M31227). Sequence analysis of pGAM21 indicated a single sizable open reading frame in only one of the six possible reading frames (Fig. 1). However, this open reading frame extended to the Sau3AI site at the end of the insert for pGAM21 (Fig. 2, nucleotide 361); as a result, pllOO was constructed to determine the sequence upstream of the open reading frame from pGAM21. Sequence analysis of both plasmids provided the continuous open reading frame for the 190-kDa antigen as diagrammed in Fig. 1. The presumed translational initiator codon (ATG) was located at nucleotide 70 (Fig. 2) and was preceded by a termination codon in the

INFECT. IMMUN.

same frame at nucleotide 28. This putative initiator codon was preceded 7 bases upstream by the sequence AAGGT, which has partial homology to the ribosome-binding site consensus sequence of E. coli (42) and is similar to those described for other genes from rickettsiae (7, 8, 49). We therefore defined the putative 190-kDa-antigen gene as a 6,747-nucleotide sequence in the R strain of R. rickettsii starting with the ATG triplet at nucleotide 70 and proceeding to the TAA terminator triplet at nucleotide 6817. The G+C content for the coding region itself was 39%. The deduced amino acid sequence for this open reading frame consisted of 2,249 amino acids. The open reading frame was used to search the GenBank data base, and no nucleotide sequences with significant homology to the 190-kDa-antigen gene were found. The region immediately upstream of the presumed initiator methionine codon consisted of sequences that were very A+T rich (approximately 20% G+C). This region is presumably involved in rickettsial RNA polymerase binding and transcription initiation. The sequence TAGACA, located at nucleotides 3 to 8 (Fig. 2), had homology with five of six bases from the E. coli -35 consensus promoter sequence (16). Likewise, the sequence TATAAC (nucleotides 26 to 31, Fig. 2) matched in five of six bases the E. coli consensus -10 promoter region. Similar promoter sequences are characteristic of sequenced rickettsial genes described to date (6, 8, 48, 49). Consequently, the putative transcription initiation site would be in the region of nucleotides 39 to 41. Recently, Policastro et al. used primer extension to identify the putative rickettsial transcription initiation site for the 190-kDaantigen gene as either nucleotide 38 or 39, as shown in Fig. 2 (American Society for Rickettsiology and Rickettsial Diseases, 8th Sesquiannual Meeting, Diamond Point, N.Y.). Immediately following the open reading frame was an inverted repeat, located at nucleotides 6840 to 6876 (Fig. 2), which was capable of forming a stable loop-and-stem structure with an estimated AG of -28.30 kcal/mol. This type of structure is characteristic of procaryotic transcription terminators. Therefore, the transcript for the 190-kDa antigen is likely to be monocistronic, with a predicted size of 6.4 kb. The 3,840-nucleotide fragment bounded by PstI sites at nucleotides 614 and 4424 (Fig. 2) was found to consist predominantly (>75%) of highly repetitive sequences. The repetitive units were tandemly arranged with no intervening nonrepetitive sequences and composed almost 43% of the coding region of the 190-kDa-antigen gene. There were 13 back-to-back repeating units, as diagrammed in Fig. 1, starting with the first complete repeating unit at nucleotide 703 (Fig. 2) and ending with the last complete unit at nucleotide 3564 (Fig. 2). There was a short region of homology to the repeating units immediately following nucleotide 3564; however, this was not a complete repeating unit. Each of the 13 repeating units could be divided into either a 225-nucleotide unit, termed type I, or a 216-nucleotide unit, termed type II, with units A, D, F, H, I, and J being type I (Fig. 1) and units B, C, E, G, K, L, and M being type II. The type I repetitive unit coded for 75 amino acids and was extremely well conserved with very little sequence variation among the six versions. Slightly greater sequence variation among the seven versions of the type II repeating units was evident; however, there was still a high degree of homology among each of the units. Additionally, there was considerable homology between the type I and type II units. Southern blot analysis. To confirm that the unusual highly repetitive sequences located within the 3.8-kb PstI fragment

REPETITIVE PROTEIN ANTIGEN OF R. RICKETTSII

VOL. 58, 1990

A B C D

E F G H

1353-

1078_ -

872-

603-

to~~~~~~~~ 310-

271/281234 194-

FIG. 3. Southern blot of the region containing the tandem

re-

peats. R. rickettsii R strain genomic DNA (lanes A to D) and pGAM21 DNA (lanes E to H) were doubly digested with PstI and a second enzyme. The second enzymes were as follows: lanes A and

E, BstEII; lanes B and F, DraI; lanes C and G, Hinfl; lanes D and H, NsiI. The blot was hybridized to a 32P-labeled 3.8-kb PstI fragment isolated from the pGM3-18R subclone of pGAM21 (see text). The positions of molecular size standards (+X174 phage DNA cleaved with HaeIII) are shown at the left (in base pairs). were arranged in the R. rickettsii R strain genome as they are within pGAM21, Southern blot analysis was performed. DNA was digested with PstI and each of four different enzymes known to cut within the repetitive region as determined by sequence analysis; the digested DNA was then electrophoresed and transferred to nitrocellulose. The resulting filter was probed with the 32P-labeled 3.8-kb PstI fragment isolated from a subclone (pGM3-18R) of pGAM21. The pattern of bands that hybridized to the probe appeared to be identical in the R strain genome (Fig. 3, lanes A to D) and in the cloned version of the gene (lanes E to H). There were some additional faint bands in the genomic digest that were not present in the cloned gene. This may be because 20 ,ug of genomic DNA was used compared with 0.5 ,ug of plasmid DNA to equalize the intensity of the signal on the autoradiograph; consequently, a higher background was observed. Alternatively, some degree of partial digestion of the genomic DNA may be present. In addition, there was excellent correlation between the sizes of the bands seen in the autoradiograph and those predicted from the nucleotide sequence. Thus, the unusual repetitive structure obtained for the 190-kDa-antigen gene nucleotide sequence is probably not the result of cloning artifacts or recombination occurring after the initial cloning of the gene. Analysis of the deduced amino acid sequence for the 190kDa antigen. The 6,747-nucleotide open reading frame assigned to the 190-kDa-antigen gene coded for a protein with 2,249 amino acids and a calculated molecular weight of 224,321. This represents a 15.3% error between the 190-kDa apparent molecular mass observed by SDS-PAGE and immunoblotting in our laboratory and the molecular mass calculated from the deduced amino acid sequence. An isoelectric point of 5.99 was calculated from the deduced

2765

amino acid sequence. The deduced protein could be divided into four domains, based on sequence and relative hydrophobicity, for the purpose of analysis. Domain I consisted of the amino-terminal portion of the deduced protein from residues 1 to 211 (Fig. 2). Domain II contained the repeating units starting with residue 212 and ending with residue 1180. Domain III contained the residues immediately following the repetitive region (1181 to 1780), whereas domain IV was the carboxy-terminal portion of the protein from residues 1781 to 2249. The immediate amino terminus (first 20 residues) of the deduced protein, contained within domain I, consisted of hydrophobic or uncharged amino acids, with four lysine residues at positions 7, 10, 11, and 18 (Fig. 2). This type of sequence is similar to signal peptide sequences described for bacterial proteins targeted for translocation across the cytoplasmic membrane (38). However, no consensus signal peptidase cleavage site (A-X-A) was found in the immediate amino terminus. This region was followed by a hydrophobic (Fig. 4, residues 19 to 42) potential membrane-spanning region predicted to form an a-helix. The remainder of domain I consisted of two hydrophilic regions (Fig. 4, residues 52 to 60 and 68 to 82) and a long region that was slightly hydrophobic (Fig. 4, residues 83 to 212). Domain II consisted entirely of the repetitive region of the deduced 190-kDa antigen. The first repeating unit started at residue 212 and was followed, back to back, by 12 additional repeating units. The entire repetitive portion of the deduced protein consisted of alternating hydrophobic and weakly hydrophilic regions, with an overall slightly hydrophobic composition (Fig. 4, residues 212 to 1180). The deduced amino acid sequence for the tandem repeats showed that each repeating unit fell into either a type I or type II unit (Fig. 5). The type I repeating unit was a 75-amino-acid peptide that was almost completely conserved among each of the six type I repeats (Fig. 5). The type II repeating unit was a 72-amino-acid peptide with slightly less conservation among each of the seven type II units than for the type I units. There was also considerable homology between the type I and type II units. Domain III was hydrophobic overall but contained several hydrophilic maxima that may represent surface-exposed regions (Fig. 4). No repetitive peptide sequences were found in domain III. Domain IV consisted predominantly of strongly hydrophilic regions that are probably external to the rickettsial cell. Additionally, domain IV contained 22 of the 30 tyrosine residues found in the 190-kDa protein that are the primary targets of lactoperoxidase-mediated surface labeling (28) demonstrated for this protein by Williams et al. (47). Transient expression of the 190-kDa antigen in E. coli. Several attempts to reconstruct (by subcloning) the entire 190-kDa-antigen gene and presumed control sequences in both pUC18 and pUC19 failed, suggesting that expression of the entire protein is detrimental to E. coli. Consequently, pBA13 was constructed as described in Materials and Methods and used for attempts to transiently express the 190-kDa antigen in E. coli. This plasmid contains the entire reading frame in the absence of the presumed rickettsial promoter under transcriptional control of the tandem lac and tac promoters upon both thermal and chemical induction. E. coli D1210HP(pBA13) grew at a rate approximately half that of the control strain, D1210HP(pNH8A), indicating the detrimental effect the 190-kDa antigen had on the cell. However, transient expression of the full-length product was obtained. An extremely faint band was seen (Fig. 6, lane E, top band) before induction that was reactive with a monoclonal anti-

INFECT. IMMUN.

ANDERSON ET AL.

2766

Inkx ..................

...

a.1, ,11,,1 4.~~~~~~~~~~~~~~7

FIG. 4. Hydrophobicity plot of the deduced amino acid sequence for the 190-kDa antigen of R. rickettsii. The algorithm of Kyte and Doolittle was used (24) with a window of six residues. Positive values indicate hydrophobic sequences, and negative numbers indicate hydrophilic regions.

body specific to the 190-kDa antigen. This band was not seen in the control (Fig. 6, lane F) strain D1210HP(pNH8A) and appeared to comigrate with antigen expressed in R. rickettsii (Fig. 6, lane G). Antigen production appeared to occur before induction, indicating that the repression of int-mediated promoter inversion is somewhat leaky. As the time TYPE I REPEATING UNITS A D

1 1

F

1

H

1..50

IGNTNALATV NVGAGTATLG GAVIKATTTK LTNAASVLTL

TNANAVLTGA

......................... S...I S.........

.....

51

D

51

F

51

H

51

51 51

50

I

S

50

..S...I S.............................

A

50

50

IDNTTGGDNV GVLNLNGALS QVTGD

..........

50

75

.......... ..........

.... N

75

. ......... ..........

.....

75

. ......... ..........

.....

75

......... V..........

.....

75

. ......... ..........

.....

75

TYPE II REPEATING UNITS

B

1

IGNTNSLATI SVGAGTATLG GAVIKATTTX LTDAASAVKF

c

1

V....A ...VN....LI.QVQ

E

1 ..........5..........0..................... .......... 50

G

TNPVVVTGAI

G.V..N.IN ...N....T.

50 50

K

1 ............................... I.N.V .50 1..50

L

1

M

1

B

51

c

51

E

51

G

51

...50

V.... A...V N... LLQVQ .G.V..N.IN

DNTGNANNGI VTFTGNSTVT GN ......... ..........

.

.. .........

K

51

.......... ..........

L

51

.......... .....

M

51

.....

72

D D

72

..

N ....T.

........

50

72

D

.......... . ..........

..........

elapsed after induction increased, the antigen produced was more severely degraded, indicating that the protein is highly susceptible to E. coli proteases. Plasmid DNA from the transcriptionally active (induced) form of pBA13 was isolated and transformed into E. coli Y1089 (pMC9-) (a lon protease-deficient strain cured of endogenous pMC9); however, there was no visible expression of the 190-kDa antigen (data not shown). Lack of expression may have resulted from rearrangement of pBA13 sequences following induction, since uncontrolled expression of the 190-kDa antigen is probably detrimental to E. coli.

72 72 72

72

FIG. 5. Alignment of the deduced amino acid sequence for each of the 13 tandemly arranged repetitive units. Each repeating unit is depicted as a single-letter symbol reflecting its position within the gene (Fig. 1). The units are divided into type I (75 amino acids) or type 11 (72 amino acids). Amino acid homology between each of the repeating units is indicated by a dot, and nonhomology is shown by substitution of the appropriate amino acid.

DISCUSSION Immunity to RMSF appears to involve both the humoral and cellular arms of the immune system (1, 20, 22, 23). Traditionally, two methods of assaying protective immunity to spotted fever group rickettsiae have been employed: the mouse toxin neutralization test (9), which detects the presence of antibodies capable of preventing immediate death from a lethal dose of viable R. rickettsii, and the guinea pig model, which assays for true protective immunity potentially involving both arms of the immune system. The latter model more closely resembles human RMSF infection. Recent work has shown that certain monoclonal antibodies directed to the 135-kDa antigen of R. rickettsii are protective in the mouse toxin neutralization assay (1, 4), suggesting that antibodies reactive with a specific domain(s) of this protein block a vital function of rickettsial pathogenesis or facilitate opsonization. The 190-kDa antigen, the subject of this report, is protective by both assay methods (1, 4, 31), indicating that this protein may be involved in the "toxic" reaction attributed to R. rickettsii pathogenesis as well as eliciting true immunity. Furthermore, McDonald et al. (30) demonstrated that this protective property of the 190-kDa protein is retained in a 3.7-kb PstI fragment subcloned into pUC8. Sequencing data described in this report indicate that this 3.7-kb (3.8 kb from the nucleotide sequence) PstI fragment codes for a long region of repetitive peptides (domain II) followed by a shorter nonrepetitive region. Thus, it is likely

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VOL. 58, 1990

-200 -93

-69

-46

-30

A

B

C

D

E

F

G

FIG. 6. Immunoblot of E. coli recombinant strain D1210HP (pBA13). Samples were solubilized at 100°C and resolved on a 8% acrylamide SDS-PAGE gel, transferred to nitrocellulose, and reacted with monoclonal antibody 14-19D4H8, specific to the 190-kDa antigen. Lanes A to E are D1210HP(pBA13) samples collected at different time points relative to induction: lane A, 120 min; lane B, 60 min; lane C, 30 min; lane D, 15 min; lane E, 0 min. Lane F is D1210HP(pNH8) collected after 60 min of induction, and lane G is solubilized R. rickettsii R strain. Molecular mass standards (in kilodaltons) are shown at right.

that the tandemly repeated peptides that compose most of the coding region of this fragment are involved in eliciting this protection. To our knowledge, the length and conservation of sequences in the repetitive portion of the protein are unique among bacterial antigens, encompassing almost 95 kDa of the molecular mass of the 224-kDa deduced protein. However, genes encoding repetitive peptides have been reported for a number of protozoan human parasites, including Trypanosoma cruzi (18), T. brucei (36, 37), Plasmodium falciparum (21), and Leishmania major (45). The function of some of the repetitive proteins of these human parasites has been shown to include attachment to cell surfaces or cellular invasion. For instance, recent reports indicate that antibodies raised to synthetic peptides corresponding to the repeats of the circumsporozoite protein of P. falciparum block invasion of hepatoma cells (29) by sporozoites in vitro. It is tempting to speculate on a similar role for the 190-kDa antigen in the process of RMSF pathogenesis; however, assignment of that role awaits further investigation. Expression of repetitive peptides within proteins from Plasmodium spp. and T. cruzi have also been implicated in diversion of the host immune response (5, 17). It has been hypothesized that cross-reactivities among and within proteins containing repeats may cause overstimulation of B cells, resulting in interference with the normal maturation of an effective humoral response and permitting long-term evasion of immune clearance of these organisms (5, 17, 51). Again, this phenomenon may be involved in rickettsial pathogenesis and warrants further study. Although the specific function that this unusual repetitive protein plays in rickettsial infection remains unclear, the conservation of the repetitive structure, coupled with previous results showing the protective properties of the 190-kDa antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to RMSF. Previous work by Williams et al. (47) has shown that

lactoperoxidase-mediated surface labeling was targeted to six or seven different antigenic proteins of R. rickettsii. One of these protein antigens was reported to have an apparent molecular mass of 186 kDa and is presumably the same antigen described in this report. The hydrophobicity plot,

2767

described in this report (Fig. 4), shows one long highly hydrophilic region (domain IV) that is a potential surfaceexposed portion of the 190-kDa antigen. Domain IV contains 22 of the 30 tyrosine residues found in the deduced amino acid sequence for the 190-kDa antigen. Tyrosine residues are the primary target of lactoperoxidase-mediated radioiodination (28); thus, there is strong evidence that domain IV is located external to the R. rickettsii cell and is an exposed region of the protein. Epitope mapping or additional surfacelabeling experiments are needed to determine the exact arrangement of the 190-kDa antigen on the surface of the rickettsiae. Recently, much attention has been paid to surface layer proteins of pathogenic bacteria, since these proteins come into close contact with host cells and tissue fluid (12, 39, 40) and therefore have important roles in interaction of organisms with host cells. Characteristically, surface layer (Slayer) proteins consist of a regular array of crystalline subunits or monomers that constitute the outermost layer of the cell envelope (39, 40). Previous investigators have shown that members of both the spotted fever and typhus groups of the genus Rickettsia have surface structures that are arranged in a square lattice with a regular spacing of 13 nm (33, 34) and resemble S-layer proteins. Typically, S-layer proteins have molecular masses ranging from 40 to 200 kDa, contain a high proportion of acidic and hydrophobic residues, and are deficient in cysteine; these characteristics are consistent with those of the deduced amino acid sequence obtained for the 190-kDa antigen. It is conceivable that the 190-kDa antigen is a crystalline surface layer protein and that the regular spacing of 13 nm observed by electron microscopy for R. akari corresponds to the possible surfaceexposed regions of the repeating units, assuming that the repetitive portion of the antigen is conserved between R. akari and R. rickettsii. Recently, the gene encoding the 120-kDa surface-exposed protein of R. rickettsii was sequenced and appears to share similarities with S-layer proteins as well (14). The exact arrangement of the 190- and 120-kDa antigens in the surface structure of the rickettsiae awaits further investigation. Subsequent to the sequence analysis, several interesting observations or results were noted. First, a number of attempts to reconstruct the entire gene for the 190-kDa antigen in the multicopy plasmid vectors pUC18 and pUC19 failed, suggesting that continual expression of the full-length protein is detrimental to E. coli. This observation is further supported by the fact that E. coli D1210HP(pBA13) grows poorly and appears to undergo partial lysis after induction. This phenomenon is not unique in that other procaryotic proteins have been reported to be lethal upon transformation into or expression in E. coli (32, 41). Additionally, severe degradation of the full-length 190-kDa antigen appears to increase after induction of expression of the gene (Fig. 6), indicating activation of E. coli proteases in response to production of this foreign protein. Perhaps fusion of the 190-kDa-antigen gene (or portions thereof) to ,B-galactosidase (or another protein) will allow stable expression of the 190-kDa antigenic determinants in E. coli. Second, the molecular mass deduced from the nucleotide sequence (224 kDa) differed by 15% from the apparent molecular mass determined by SDS-PAGE and immunoblotting (190 kDa). Similar results have been noted for other proteins of a hydrophobic nature (10, 19, 48) and may explain the difference between observed and calculated molecular masses for the 190-kDa antigen. Alternatively, the 190-kDa antigen could be posttranslationally modified or processed. This

2768

ANDERSON ET AL.

possibility is consistent with the number of procaryotic glycosylation sites (26) within the deduced 190-kDa protein. Third, the 3.8-kb PstI fragment that is located entirely within the 190-kDa-antigen gene (Fig. 1) is not in frame with the PstI site of pUC8. McDonald et al. have shown that expression of a truncated portion of the 190-kDa antigen from such a plasmid construct, termed pGAM22 (30, 31), confers protection. Thus, expression from pGAM22 must result from internal translation initiation sites present on this cloned fragment or possibly ribosomal slippage from the lacZ alpha peptide reading frame. The possibility of using the nucleotide sequence for the 190-kDa antigen as a starting point for a recombinant or subunit vaccine is attractive. If a synthetic vaccine could be made that had no adverse effects, the use of an RMSF vaccine in certain populations or geographic locations may be warranted (44). The increase in reported cases of Lyme disease, coupled with the emergence of the newly described human disease attributed to members of the genus Ehrlichia, may suggest the feasibility in the future of a multivalent vaccine for tick-borne bacterial diseases. Additionally, the use of recombinant-derived rickettsial proteins or nucleotide sequences as diagnostic reagents for rickettsial diseases may provide a means to avoid the time-consuming and laborious task of cultivating and purifying rickettsiae. Regardless of the applied aspects of the nucleotide sequence analysis, characterization of the highly unusual repetitive structure of the 190-kDa antigen of R. rickettsii should be pursued to understand the role that this protein plays in the rickettsiahost interaction. ACKNOWLEDGMENTS

We thank Brian Holloway for synthesizing the oligonucleotide primers used for nucleotide sequencing and Martha Redus for laboratory assistance. We also thank Joseph McDade, Tom Shinnick, and Daniel Fishbein for helpful comments regarding the manuscript.

1.

2.

3.

4.

5. 6.

7.

8.

LITERATURE CITED Anacker, R. L., R. H. List, R. E. Mann, S. F. Hayes, and L. A. Thomas. 1985. Characterization of monoclonal antibodies protecting mice against Rickettsia rickettsii. J. Infect. Dis. 151: 1052-1060. Anacker, R. L., R. H. List, R. E. Mann, and D. L. Weidbrauk. 1986. Antigenic heterogeneity in high- and low-virulence strains of Rickettsia rickettsii revealed by monoclonal antibodies. Infect. Immun. 51:653-660. Anacker, R. L., R. E. Mann, and C. Gonzales. 1987. Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae. J. Clin. Microbiol. 25: 167-171. Anacker, R. L., G. A. McDonald, R. H. List, and R. E. Mann. 1987. Neutralizing activity of monoclonal antibodies to heatsensitive and heat-resistant epitopes of Rickettsia rickettsii surface proteins. Infect. Immun. 55:825-827. Anders, R. F. 1986. Multiple cross-reactivities amongst antigens of Plasmodiumfalciparum impair the development of protective immunity against malaria. Parasite Immunol. 8:529-539. Anderson, B. E., B. R. Baumstark, and W. J. Bellini. 1988. Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification. J. Bacteriol. 170:4493-4500. Anderson, B. E., R. L. Regnery, G. M. Carlone, T. Tzianabos, J. E. McDade, Z. Y. Fu, and W. J. Bellini. 1987. Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii. J. Bacteriol. 169:2385-2390. Anderson, B. E., and T. Tzianabos. 1989. Comparative sequence

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