INFECTION AND IMMUNITY, Jan. 1985, p. 157-165 Vol. 47, No. 1 0019-9567/85/010157-09$02.00/0 Copyright C) 1985, American Society for Microbiology Cosmid Cloning of Rickettsia prowazekii Antigens in Escherichia coli K-12 DUNCAN C. KRAUSE, HERBERT H. WINKLER, AND DAVID 0. WOOD* Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688 Received 24 May 1984/Accepted 4 October 1984 Rickettsia prowazekii DNA was partially digested with Sau3A or Hindlll, ligated with the cosmid vector pHC79, packaged in vitro, and transduced into Escherichia coli HB101. Cosmid cloning of Sau3A-digested rickettsial DNA yielded 1,288 ampicillin-resistant colonies; 798 cosmid clones resulted with HindlIl-digested rickettsial DNA. Chimeric cosmid DNA was extracted from the latter gene bank, digested to completion with HindIII, and compared by agarose gel electrophoresis with a HindIII digest of rickettsial genomic DNA. The two digestion profiles were quite similar in their overall banding patterns, indicating that the clone bank was significantly representative of the rickettsial genome. When both clone banks were screened for expression of rickettsial antigens by enzyme-linked immunosorbent assay with goat anti-R. prowazekii serum, ca. 20% of the clones reacted positively. Two clones were randomly selected for more detailed analysis. Each contained a large chimeric plasmid (40.2 and 38.1 kilobases) which apparently yielded smaller deletion derivatives (13.6 and 12.6 kilobases) when transformed into an E. coli minicell strain. Each recombinant plasmid directed the synthesis of new protein species not observed in control minicells. One of the clones produced a 51,000-dalton protein in minicells, which comigrated with a protein reactive with anti-R. prowazekii serum. This protein was not present in negative controls. When antibodies to this protein were incubated with a Western blot of rickettsial total protein, they bound to a 52,000-dalton polypeptide. Hence, the cloned rickettsial gene product in E. coli corresponds to a protein of similar size in R. prowazekii. This study demonstrates the feasibility of cosmid cloning of rickettsial antigens in E. coli. Rickettsia prowazekii is an obligate intracellular bacterium 30). Furthermore, interferon gamma levels have been dem- capable of parasitizing a number of different cell types, onstrated to increase in mice immunized with Rickettsia including both professional and nonprofessional phagocytes tsutsugamushi (19). (8, 25, 27, 33, 40). Unlike other intracellular bacteria, both Less is known about the rickettsial moieties responsible facultative and obligate, rickettsiae grow directly within the for eliciting cellular and humoral immune responses. Dasch cytoplasm of their host rather than within phagosomes or and Bourgeois (6) have reported that the species-specific phagolysosomes. Rickettsiae adsorb to the surface of host protein antigens evoke strong cellular and humoral re- cells (20, 39), triggering internalization via "induced phago- sponses in the guinea pig. Burans and Dasch have subse- cytosis" (33). Once inside the cell, the rickettsiae rapidly quently identified additional rickettsial proteins which are escape from the phagosome into the host's cytoplasm, recognized by antibodies from human convalescent typhus presumably by means of a phospholipase A activity (37, 38). sera (Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, I46, p. R. prowazekii possesses a variety of means by which it can 129). Further definition of the role of specific rickettsial exploit the nutrient-rich environment of its host's cytoplasm. proteins (as well as nonproteinaceous moieties) in both the Although some of these mechanisms, such as lysine and host response to and the pathogenesis of typhus are ham- proline transport systems (23, 36), can be found in common pered by difficulties in obtaining sufficient quantities of extracellular bacteria, others, such as the ADP-ATP ex- purified proteins for expanded studies. change system (35) and an adenylate transporter (manuscript The advent of recombinant DNA technology has facili- in preparation), appear to be unique, if not to the genus tated the study of microorganisms difficult to cultivate and Rickettsia, then to obligate intracellular bacteria. manipulate in the laboratory, such as Treponema pallidum It is apparent that host defense against rickettsial infec- and Chlamydia trachomatis (24, 34). Recently, this labora- tions involves both humoral and cell-mediated immunity. tory successfully cloned the R. prowazekii gene for citrate Pretreatment of R. prowazekii with opsonizing antibodies synthase and demonstrated its expression in Escherichia coli results in their destruction after phagocytosis by human (42). Here we report the formation of cosmid clone banks of macrophages; unopsonized rickettsiae survive and multiply the R. prowazekii genome and the identification of clones within macrophages (1). On the other hand, although im- expressing genes for rickettsial antigens. Two antigen-posi- mune serum fails to transfer resistance to intradermal chal- tive clones were selected for more detailed analysis. lenge with Rickettsia typhi in guinea pigs, resistance is (Preliminary reports of this work were presented at the transferred by immune spleen cells (17). In addition, rickett- Fourth National Conference of the American Society for siae are destroyed by both macrophages (18, 30a, 41) and and Diseases, Airlie House, Va., cultured fibroblasts (28, 41) which have been treated with Rickettsiology Rickettsial lymphokines from concanavalin A-treated or rickettsial an- 27 to 30 October 1983, and the Annual Meeting of the tigen-treated mouse spleen cells. The antirickettsial activity American Society for Microbiology, St. Louis, Mo., 4 to 9 has subsequently been attributed to interferon gamma (29, March 1984. At the American Society for Rickettsiology and Rickettsial Diseases Conference, preliminary data were also presented by M. E. Dobson, G. A. Dasch, and J. P. Burans * Corresponding author. on the cloning of R. typhi antigens in E. coli.) 157 158 KRAUSE, WINKLER, AND WOOD INFECT. IMMUN. TABLE 1. Bacterial strains Strain Description Source (reference) E. coli HB101 F- hsdS20 (rB-, mB-) recA13 ara-14 proA2 lacYl galK2 leuB6 rpsL20 xyl-5 mtl-l thi-I ginV44 D. Kopecko" (2) X2687T F- hsdS20 (rB-, mB-) recA13 ara-14 proA2 lacYl galK2 leuB6 rpsL20 xyl-5 mtl-l thi-i ginV44 R. Curtiss 111b X c1857 BHB2688 F- recA [X imm434 clts b2 red3 Eam4 Sam7] R. Curtiss III (5) BHB2690 F-recA [X imm434 clts b2 red3 Daml5 Sam7] R. Curtiss III (5) MOB134 HB101 containing pHC79 R. Welch' (11) X1849 F- tonA53 dapD8 minAl purE41 ginV42 A(gal-uvrB)47 minB2 his-53 nalA25 metC65 oms-i T3r R. Curtiss III (9) iA(bioH-asd)29 ilv-277 cycB2 cycAl hsdR2 X- a Walter Reed Army Institute of Research, Washington, D.C. b Department of Biology, Washington University, St. Louis, Mo. ' Department of Medical Microbiology, University of Wisconsin Medical School, Madison. MATERIALS AND METHODS bromide equilibrium density gradient centrifugations. Rick- ettsial DNA was extracted according to Meyers and Wisse- Bacterial strains and culture conditions. The bacterial man (16), with modifications. Briefly, the pelleted rickettsiae strains used in this study are described in Table 1. R. were suspended in SSC (0.15 M NaCl plus 0.015 M sodium prowazekii Madrid E was cultivated in the yolk sac of anti- citrate [pH 7.0]) to an optical density at 440 nm of 2.0. biotic-free, embryonated hen eggs and purified as described Protease type V was added to a final concentration of 1 elsewhere (35). Purification included Renograffin density gra- mg/ml and incubated at room temperature for 10 min. A dient centrifugation to remove contaminating yolk sac mito- solution of 25% SDS was added to a final concentration of chondria. Rickettsiae were stored as a pellet at -70°C before 0.2% and mixed by gentle inversion. The rickettsial lysate extraction of the DNA. E. coli strains were cultured in LB was incubated for 6.5 h at 37°C. An equal volume of phenol broth and on LB agar plates (7). Where appropriate, ampi- saturated with SSC and containing 0.1% hydroxyquinoline cillin, tetracycline, and streptomycin were added at final was added and incubated for 60 min at 37°C with moderate concentrations of 50, 25, and 100 ,ug/ml, respectively. Except mixing. The lysate was centrifuged (10,000 x g at 20°C for 20 where indicated below, the E. coli strains were grown at min), and the aqueous layer was collected. The DNA was 370C. precipitated with ethanol at -20°C, suspended in 0.1 x SSC, Enzymes and reagents. Ampicillin, tetracycline, strep- and dialyzed against 50 mM phosphate buffer (pH 7.0). tomycin, protease type V, RNase A, Trizma base, sucrose, RNase A was added to a final concentration of 50 ,ug/ml and spermidine hydrochloride, putrescine hydrochloride, 3-mer- incubated for 2.5 h at 37°C. The DNA was further purified by captoethanol, dimethyl sulfoxide, ATP, hydroxyquinoline, CsCl equilibrium density gradient centrifugation and dia- lysozyme, Tween 20, biotin, diaminopimelic acid, adenine, lyzed against TE buffer (10 mM Tris, 1 mM disodium EDTA tryptophan, methionine, cysteine, asparagine, glutamine, [pH 8.0]). and ethidium bromide were purchased from Sigma Chemical Chimeric cosmid DNA was isolated from individual clones Co., St. Louis, Mo. Technical grade cesium chloride was or from a pool of clones according to Hansen and Olsen (10). obtained from Kawecki Berylco Industries, New York, In the latter case, cosmid clones were replica plated from the N.Y. Sequanal grade sodium dodecyl sulfate (SDS) was 96-well microtiter dishes in which they were stored onto LB obtained from Pierce Chemical Co., Rockford, Ill. Agarose, agar plates containing ampicillin. After an overnight incuba- all restriction endonucleases, T4 DNA ligase, and dithiothre- tion at 32°C, the resulting colonies were scraped into phos- itol were purchased from Bethesda Research Laboratories, phate-buffered saline, pH 7.2 (PBS) before extraction of the Rockville, Md. DNase I was obtained as indicated in the plasmid DNA.