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Cloning and Expression of Legionella Pneumophila Antigens in Escherichia Coli N

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Cloning and Expression of Legionella Pneumophila Antigens in Escherichia Coli N INFECTION AND IMMUNITY, May 1984, p. 222-227 Vol. 44, No. 2 0019-9567/84/050222-06$02.00/0 Copyright © 1984, American Society for Microbiology Cloning and Expression of Legionella pneumophila Antigens in Escherichia coli N. CARY ENGLEBERG,' DAVID J. DRUTZ,1'2'3 AND BARRY I. EISENSTEIN1l2* Departments of Medicine' and Microbiology,2 The University of Texas Health Science Center at San Antonio, and the Veterans Administration Hospital,3 San Antonio, Texas 78284 Received 3 October 1983/Accepted 23 January 1984 To isolate and characterize Legionella pneumophila antigens, we constructed a genomic library of L. pneumophila serogroup 1 (strain 130b). L. pneumophila DNA fragments (2.5 to 7.5 megadaltons) obtained by partial digestion with Sau 3A endonuclease and size fractionation on a sucrose density gradient were inserted into the dephosphorylated BamHI site of vector pBR322; CaCl2-treated Escherichia coli cells of strain HB101 were transformed with hybrid plasmids. To detect expression of antigens, 2,559 ampicillin- resistant transformants were transferred to nitrocellulose paper, lysed in situ, and screened by enzyme immunoassay (EIA) with E. co/i-absorbed rabbit anti-L. pneumophila sera. A total of 77 (3%) of the colonies were reactive by EIA; 31 (1.2%) were strongly reactive, and 6 were strongly reactive by EIA without colony lysis. Analysis of 29 stable, strongly reactive clones by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting showed antigenic bands in 18 clones by EIA with E. coli-absorbed antisera. Absorption of antisera with heat- and Formalin-killed L. pneumophila antigen eliminated or dimninished the reactivity of the antigenic bands in representative clones. These studies confirm that several L. pneumophila antigens can be cloned and expressed in E. coli. Since the recognition of Legionella pneumophila as the others have shown that genes from a variety of bacterial causative agent of Legionnaires disease in 1977, vigorous pathogens can be transcribed and translated by Escherichia research has produced an impressive body of information coli (14, 16-18, 20, 23), we chose a simple cloning strategy about the disease, principally in the areas of clinical medi- that did not include specific engineering for gene expression. cine and public health (13). During the same period, howev- We constructed a library of the L. pneumophila genome by er, insight into the immunology and pathogenesis of legionel- using vector pBR322. We then screened the library for losis has developed slowly, and today our understanding of expression of L. pneumophila antigens by using a modified these aspects of infection remains incomplete. In general, filter binding assay (8, 14) in which colonies bound to laboratory-based research has been complicated by difficul- nitrocellulose paper are tested for antigens by in situ enzyme ties in cultivating and handling the organisms and by the immunoassay. In this article, we report the isolation of problem of maintaining their virulence on artificial media. several E. coli clones that express a variety of L. pneumo- In early studies by Wong and co-workers, complex L. phila antigens. pneumophila antigens, prepared by ion-exchange and liquid chromatography, were isolated, and fractions having seroty- MATERIALS AND METHODS pic specificity and cross-reactivity were identified and ana- Bacterial strains. L. pneumophila serogroup 1 (130b) was lyzed (24-26). The serotypic antigen fraction is composed of used for all cloning procedures and antigen preparations. For a pronase-sensitive lipid-protein-carbohydrate complex that both purposes' L. pneumophila was isolated from infected induces active immunity to infectious challenge in mice. guinea pigs and passed only once on buffered charcoal-yeast Cross-reacting antigens, which are almost entirely composed extract agar (7). E. coli K-12 strain HB101 (mk-, rk-, recA) of protein, were found to elicit dermal hypersensitivity. was the recipient for hybrid plasmid transformations. Other workers have studied L. pneumophila antigens by the Enzymes and chemicals. Restriction endonucleases and T4 techniques of crossed immunoelectrophoresis (3, 4, 10), DNA ligase were obtained from Bethesda Research Labora- counterimmunoelectrophoresis (19), and most recently by tories, Bethesda, Md. Calf intestinal alkaline phosphatase, polyacrylamide gel electrophoresis and protein blotting lysozyme (grade I), protein A-Sepharose, 5-aminosalicylic (M. S. Hindahl and B. H. Iglewski, Abstr. Annu. Meet. Am. acid, ampicillin, and tetracycline were purchased from Sig- Soc. Microbiol. 1983, C104, p. 329; W. Ehret, G. Anding, ma Chemical Co., St. Louis, Mo. Horseradish peroxidase- and G. Ruckdeschel, 2nd International Symposium on Le- conjugated goat anti-rabbit immunoglobulin was purchased gionella, Atlanta, Ga., M-8, 1983). Although these studies from Cappell Laboratories, Cochranville, Pa. The color- have confirmed the existence of discrete serotypic and forming reagent 4-chloro-1-naphthol and nitrocellulose paper crossreacting antigens, it will be necessary to purify these for electroblotting were purchased from Bio-Rad Labora- molecules to establish their immunogenicity and to fully tories, Richmond, Calif. Nitrocellulose disks for filter bind- characterize their role in the host response to infection. ing assays (type HA) were purchased from Millipore Corp., To circumvent the problems inherent in working with L. Bedford, Mass.; Whatman 3MM chromotography paper was pneumophila and the difficulties of biochemical purification obtained from American Scientific Products, McGaw Park, of antigens of interest, we chose to isolate antigens at the Il. genetic level by using recombinant DNA technology. Since Construction of the clone bank. DNA was extracted from L. pneumophila cells harvested from buffered charcoal-yeast extract agar plates in phosphate-buffered saline (PBS), pH * Corresponding author. 7.2. To assure against contaminating bacteria, samples of 222 VOL. 44, 1984 CLONING OF L. PNEUMOPHILA ANTIGENS 223 this suspension were plated on buffered charcoal-yeast ex- by an enzyme-linked immunosorbent assay (ELISA) by tract and sheep blood agar and inoculated into brain heart using whole L. pneumophila cells fixed to 96-well microtiter infusion broth. There was no growth on blood agar or brain plates (Dynatech Laboratories, Inc., Alexandria, Va.). FK heart infusion broth. Pure growth of L. pneumophila was cells corrected to optical density at 550 nm of 1.5 with PBS detected on buffered charcoal-yeast extract agar and was (pH 7.2) were further diluted 1:25 with PBS. A 0.1-ml sample confirmed by direct immunofluorescence with fluorescein- of dilute FK cells was added to each well, and the microtiter labeled, polyvalent rabbit antisera (Centers for Disease plates were allowed to dry overnight at 42°C. Plates were Control, Atlanta, Ga.). L. pneumophila DNA was extracted washed three times with PBS containing 0.5% Tween 20, and purified by the method of Nakamura et al. (15). Purified serial dilutions of test sera (0.1-ml samples) were added, and DNA was partially restricted with Sau 3A restriction endo- plates were incubated for 2 h at room temperature on a nuclease, and the digestion fraginents were applied to a 10- Minimix agitator (Fisher Scientific Co., Silver Spring, Md.). ml 5 to 40% sucrose gradient in 1 M NaCl-20 mM Tris- After three washes with PBS-0.5% Tween 20, 0.1 ml of hydrochloride-5 mM EDTA (pH 8.0) and centrifuged at peroxidase-conjugated goat anti-rabbit immunoglobulin 100,000 x g for 21 h. Gradient fractions (0.5 ml) were (1:1,500 dilution) was added to each well, and the plates analyzed by agarose gel electrophoresis, and fractions con- were again agitated for 2 h. After three final washes with taining restriction fragments of 2.5 to 7.5 megadaltons were PBS-0.5% Tween 20, 0.1 ml of a color-forming substrate pooled. solution (0.08% 5-aminosalicylic acid and 0.006% hydrogen Vector pBR322 was prepared for cloning by complete peroxide, pH 6.0) was added to each well. After agitation for digestion with BamHI followed by 5' dephosphorylation 30 min, absorbance at 450 nm was measured in a MR580 with alkaline phosphatase (12). The latter procedure resulted MicroElisa Auto Reader (Dynatech). An absorbance of 0.5 in a 2- to 3-log reduction in recircularization and ligation of or more was considered positive. the vector as compared with untreated linear pBR322. To assess the effectiveness of serum absorption, a similar Size-fractionated L. pneumophila Sau 3A restriction frag- ELISA test was used in which live E. coli whole cells were ments were ligated to dephosphorylated pBR322 with T4 fixed to the microtiter plate wells by the methods described DNA ligase and used to transform E. coli strain HB101 above. rendered competent by treatment with 0.5 M CaCI2 (5). FB-EIA. Apr transformants were spotted onto agar plates Transformants were selected on Louria-Bertani agar con- with sterile toothpicks (ca. 325 colonies per plate), grown taining ampicillin (40,ug/mI) (12). Forty ampicillin-resistant overnight, and then blotted onto dry nitrocellulose filter (Apr) transformants were screened for tetracycline sensitiv- disks; 1 to 2 RI of FK cells was also spotted onto each filter ity (Tcs) on Louria-Bertani medium containing tetracycline as a positive control. Colonies were lysed in situ by the (15,ug/ml). method of Meyer et al. (14). Briefly, filter disks were placed Preparation of L. pneumophila cells for immunization. L. sequentially, colony side up, onto 3MM paper in a series of pneumophila cells from six buffered charcoal-yeast extract four petri dishes saturated respectively with (i) 0.1 N NaOH, agar plates were harvested, pooled, and suspended in 6 ml of (ii) 1.5 M Tris-hydrochloride (pH 7.4), (iii) 300 mM NaCI-30 PBS (pH 7.3).F,ormalin-killed (FK) cells were prepared from mM sodium citrate, and (iv) 70% ethanol for 5 min each. 3 ml of this suspension by adding Formalin to a final Each filter was then dried in a vacuum at 60°C for 2 h.
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