INFECTION AND IMMUNITY, Jan. 1990, p. 17-20

Vol. 58, No. 1

0019-9567/90/010017-04$02.00/0 Copyright © 1990, American Society for Microbiology

Isolation and Characterization of Recombinant Xgtll Bacteriophages Expressing Four Different Mycobacterium intracellulare Antigens SHELDON L. MORRIS,* DAVID A. ROUSE, DAVID HUSSONG, AND SOTIROS D. CHAPARAS Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received 18 July 1989/Accepted 20 September 1989

Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a Agtll library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracelulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracelulare derivation of the inserts. The physical maps of the inmunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.

The Mycobacterium avium-M. intracellulare complex (MAC) acid-fast bacilli represent a group of opportunistic pathogens which can cause severe disease in immunocompromised patients (18, 23). In the past decade, the prevalence of MAC disease has increased dramatically primarily due to the acquired immunodeficiency syndrome (AIDS) epidemic (7, 15, 26, 28). About 45% of AIDS patients are infected with MAC bacilli. In a significant proportion of these individuals, MAC infections result in disseminated life-threatening disease. Recent hospital-based studies have suggested that at least 10% of AIDS patients may also have tuberculosis. The clinical management and preventative treatment of MAC disease and tuberculosis require different approaches. It has been suggested that human immunodeficiency virus (HIV)-infected persons who are tuberculin positive be placed on a preventative regimen in order to abort the disease (20). Preventative treatment for MAC disease may also be a prudent measure in HIV-infected persons. The skin test is an invaluable aid in the diagnosis of tuberculosis. However, because of cross-reactivity among mycobacteria and the lack of specificity of current skin test reagents, the interpretation of skin test reactions is often confounded (4). The development of new immunological reagents such as specific skin test antigens should accelerate and improve the diagnosis of mycobacterial infections. Early detection and differentiation of mycobacterial disease in HIV-infected individuals should facilitate clinical management strategies. Recent advances in molecular biology have provided methods that allow the production of mycobacterial antigens in Escherichia coli which may be useful as diagnostic reagents or as components in a multivalent mycobacterial vaccine (2, 5, 6, 13, 21, 27). We have previously described the isolation from a M. intracellulare Agtll library of a bacteriophage expressing an immunologically active M. intracellulare fusion protein that reacts with absorbed polyclonal anti-M. intracellulare serum (17). We now describe the isolation and characterization of four unique phages from this expression library which encode immunoreactive antigens that are recognized by four different anti-M. intracellulare monoclonal antibodies.

*

MATERIALS AND METHODS Production of hybridomas. The procedures for hybridoma production have been published elsewhere (3) and are described briefly here. BALB/c mice were injected intraperitoneally with 10 to 100 ,ug of a dissolved ammonium sulfateprecipitated fraction of M. intracellulare sonic extract emulsified with an equal volume of 70% (vol/vol) Drakol 6VR mineral oil-30% Aracel A. Three days prior to the collection of spleens for fusions, a 5- to 10-,ug intravenous boost of the same material was given. Spleens from immunized mice were collected, washed with RPMI 1640 medium, and treated with RBC lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA [pH 7.4]) to remove erythrocytes. The intact cells were then fused to equal numbers of SP2/0 myeloma cells by treatment with 50%o polyethylene glycol 1000 dissolved in RPMI 1640 medium with 0.01 M HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid). Fused cells were then suspended in RPMI 1640 medium containing 20% fetal bovine serum, a 1% nonessential amino acids solution (Flow Laboratories, Inc., McLean, Va.), 2 mM glutamine, 1 mM sodium pyruvate, 50 ,ug of gentamicin sulfate per ml, and HAT selection supplement (Sigma Chemical Co., St. Louis, Mo.). The cultures were kept at 37°C with 5% CO2 in a humidified incubator for 1 to 3 weeks. Antibody-producing hybrids were detected by enzyme-linked immunoabsorbent assay and Western (immuno-) blotting. Reactive cultures were cloned by limiting dilution. Bacterial sonic extract preparation. The M. intracellulare species used in this study were grown in completely synthetic glycerol-asparagine medium and sonicated as described previously (5). The E. coli sonic extracts were prepared by briefly exposing the organisms to a 90-s sonication pulse in a Heat Systems Ultrasonics apparatus (model 375) at 150 W. The Bordetella pertussis sonic extract was a gift from Michael Brennan, Center for Biologics Evaluation and Research. Screening the M. intracellulare Agtll expression library. Preparation of the Agtll expression library has been discussed previously (17). The method utilized for screening the library was developed by Young et al. (27). Typically, the Agtll library was plated with approximately 10,000 to 20,000 phage per plate. After 2 to 3 h at 42°C, nitrocellulose filters saturated with 10 mM isopropylthio-I-galactosidase (IPTG) were placed on the plates. The incubation was continued at

Corresponding author. 17

18

MORRIS ET AL.

37°C for 3 to 4 h. Subsequently, the filters were rinsed for 1 h with TBS (150 mM NaCl, 10 mM Tris [pH 8]) and blocked for 16 h at 4°C with TBS-5% nonfat dry milk. The next morning, the filters were incubated with the appropriate monoclonal antibody for 2 h at room temperature. After the filters were washed three times with TBS-0.05% Tween 20, goat anti-mouse alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, Mo.) was applied for 1 h at room temperature. The filters were then washed with TBS-Tween for 20 to 30 min. The filters were developed with NBT-BCIP alkaline phosphatase substrate (Bio-Rad Laboratories, Richmond, Calif.) per the instructions of the manufacturer. Positive phages were purified twice by this method to ensure

homogeneous populations. Preparation and characterization of bacteriophage DNA. Phage DNA was prepared from plaque-purified phage by the plate lysate method (16). The mycobacterial insert fragments were separated from Xgtll DNA by restriction digestion with EcoRI, followed by fractionation on 0.8% low-meltingpoint agarose gels. Amplification of insert fragments was achieved by cloning the M. intracellulare-derived fragment into the E. coli plasmid pUC18. Radioactive probes for the insert fragments were prepared by nick translation of the recombinant pUC18 plasmids (19). These probes were hybridized to Southern blots of EcoRIdigested M. intracellulare genomic DNA at 650C in 5 x SSPE (750 mM sodium' chloride, 50 mM sodium phosphate [monobasic] 5 mM EDTA [pH 7.4])-5x Denhardt solution (0. 1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin [Pentax Fraction V])-0.1% sodium dodecyl sulfate (SDS)-100 ,ug of denatured salmon sperm DNA per ml. After incubation at 65°C for 16 h, the nitrocellulose filters were washed in 2x SSC (0.3 M sodium chloride, 0.33 M sodium citrate [pH 7.0])-0.2% SDS'at room temperature for 60 min. These washes were followed by more stringent washes in 0.2x SSC-0.2% SDS at 65°C for 3 h. The filters were air dried and exposed to Kodak XAR-2 film for 4 to 16 h at -20°C prior to their development. Preparation of recombinant M. intracelulare antigens. Lambda lysogens of recombinant Xgtll clones were made in E. coli y1089 (11). The induce the recombinant antigens, the lysogens were inoculated into LB medium (16) and grown at 32°C. When the cultures reached an optical density of 0.5, the temperature was shifted to 42°C. After 20 min, 0.5 M IPTG was added to a final concentration of 10 mM. The cultures were subsequently incubated at 38°C for an additional 60 min. The cells were harvested at 25°C by centrifugation, suspended in 1/25 the original volume in 10 mM Tris (pH 8)-i mM EDTA-2 mM phenylmethylsulfonyl fluoride, and frozen in liquid nitrogen. The cells were then thawed and sonicated as described above. Cellular proteins were precipitated with 'an equal volume of saturated ammonium sulfate and dialyzed against phosphate-buffered saline. The antigen preparations were evaluated by SDS-polyacrylamide gel electrophoresis and Western blotting, using the Mini-Protean II Gel and Transfer System (Bio-Rad). Western blots were developed with the alkaline phosphate substrate system (Bio-Rad). RESULTS Specificity of four anti-M. intracellulare monoclonal antibodies. Three of the four anti-M. intracellulare monoclonal antibodies (IE5, 6B8, and 7F7) were prepared as described in Materials and Methods. Preparation of the fourth monoclonal antibody'(HGT-6) has been discussed previously (12).

INFECT. IMMUN.

TABLE 1. Characterization of specificity of four anti-M. intracellulare monoclonal antibodiesa Sonic extract

7F7

(22 kDa) M. intracellulare M. avium M. bovis BCG M. chelonei M. fortuitum M. kansasii M. marinum M. phlei M. scrofulaceum M. smegmatis M. tuberculosis M. vaccae M. xenopi

+ + -

E. coli B. pertussis

Reactivity with: 6B8 IE5 (40 kDa) (43 kDa) + +

+ + + + -

-

+ -

-

-

-

+

-

+

HGT-6 (54 kDa)

+ + + + + + + + + + + + +

a Specificity was evaluated by Western blot analysis.

The specificity of these antibodies was evaluated by Western blot analysis. The four monoclonal antibodies recognized different M. intracellulare antigens at 54, 43, 40/38, and 22 kilodaltons (kDa) (Table 1 and Fig. 1). The IE5 antibody bound to a 43-kDa protein in M. intracellulare and M. avium sonic extracts but did not react with antigens in sonic extracts of 11 other mycobacterial species, E. -coli, or B. pertussis. Hence, the epitope recognized by the IE5 antibody may be specific for MAC. This will need to be confirmed for a large number of strains. The 6B8 monoclonal

B.

A. 2

1

1 2

3 -137

3 -10-0

_

-

--40

_n- 22

D.

C. 1 -

1 2

2 3

3

-155 -54 -43

FIG. 1. Western blots of lysogen sonic extract and M. intracellulare sonic extracts. Lanes: 1, corresponding A lysogen sonic extract; 2, E. coli control; 3, M. intracellulare sonic extracts. The blots were incubated with the following antibodies: 7F7 (A), 6B8 (B), IE5 (C), and HGT-6 (D).

VOL. Vgtll PHAGES EXPRESSING M. INTRACELLULARE ANTIGENS 58, 1990 N

PS

I

ii

22kDa X E X

4OkDa

I

I

X

1 lac z B

X

i

i

43kDa

Sp

lac z E

i

i

X

PX

_

X

P

4

X

9.4-

tac z 54kDa

3

Kb) 23.1-

l

X E

2

19

1 kb

6.6 -

I I

am

46

lac z

FIG. 2. Restriction maps of four immunoreactive phages. B,

BamHI; E, EcoRI; N, NruI; P, PstI; S, SaII; Sp, SphI; X, XhoI.

antibody also did not react with the sonic extracts of the 11 other mycobacterial species, E. coli, or B. pertussis. This 6B8-reactive epitope was found on 40- and 38-kDa MAC proteins. The 7F7 antibody recognized a 22-kDa protein cross-reactive with several mycobacterial sonic extracts including those of M. tuberculosis and M. bovis BCG. Finally, the HGT-6 monoclonal antibody seems to recognize a common myobacterial epitope. This antibody reacted with all 13 mycobacterial sonic extracts but did not react with E. coli or B. pertussis proteins. Characterization of immunoreactive bacteriophages isolated from the M. intracellulare Agtll expression library. The M. intracellulare Xgtll library was screened with the four monoclonal antibodies. Unique recombinant bacteriophages were identified that expressed immunoreactive epitopes recognized by the anti-M. intracellulare antibodies. These recombinant phages were characterized by restriction mapping (Fig. 2). EcoRI restriction endonuclease digestion of phage DNA indicated that the mycobacterial insert fragments were the following sizes: 3.8 kilobases (kb) for the phage reacting with 7F7 (22 kDa), 6.8 kb for the 6B8 (40 kDa)-reactive phage, 4.4 kb for the IE5 (43 kDa)-reactive phage, and 2.1 kb for the phage recognized by HGT-6 (54 kDa). Further restriction mapping allowed the determination of insert orientation in the recombinant phage with respect to lacZ (Fig. 2). It is of interest that none of the restriction patterns shown are similar to those published for M. leprae or M. tuberculosis genes (2, 10, 14, 22). To confirm the M. intracellulare derivation of the recombinant phage insert fragments, we hybridized nick-translated probes generated from the insert fragments to EcoRI-digested M. intracellulare chromosomal DNA. Each of the insert probes hybridized to a single fragment in the EcoRIrestricted M. intracellulare DNA (Fig. 3). These hybridization patterns substantiate the M. intracellulare origin of the insert fragments and strongly suggest a simple genetic map with a single chromosomal locus within the M. intracellulare genome for each of these antigen-encoding genes. Western blot analysis of A lysogen sonic extracts. Overproduction of recombinant M. intracellulare antigens was achieved by preparing E. coli y1089 X lysogens from the immunoreactive phage. The recombinant proteins were evaluated by immunoblotting IPTG-induced X lysogen sonic extracts and M. intracellulare sonic extracts against the anti-M. intracellulare antibodies. Sonic extracts derived from a previously described A lysogen (17) expressing a P-galactosidase fusion protein recognized by a M. intracellulare polyclonal serum served as controls. The 7F7, 6B8, and IE5 monoclonal antibodies recognized M. intracellulare-p-galactosidase fusion proteins in the appropriate k lysogen sonic extract that were not present in the E. coli controls (Fig. 1A, B, and C). These hybrid proteins are much

4.4

-

2.3 2.0

-

FIG. 3. Southern blot hybridization of insert fragment probes to EcoRI-digested M. intracellulare DNA. The inserts were derived from Xgtll phages recognized by the following antibodies: 7F7 (lane 1), IE5 (lane 2), 6B8 (lane 3), and HGT-6 (lane 4). The sizes of the EcoRI fragments used to prepare the hybridization probes were 3.8 kb (7F7), 2.3 kb (IE5), 6.0 kb (6B8), and 2.1 kb (HGT6).

larger than the immunoreactive antigens from which they derived because the 3-galactosidase portion of the polypeptide is 115 kDa. However, the HGT-6-reactive antigen synthesized in this E. coli k lysogen is not a ,B-galactosidase fusion protein, since it has a molecular mass similar to that of the native 54-kDa antigen. Although the Xgtll expression system is designed to detect antigenic determinants as ,3-galactosidase fusion proteins, the expression of nonfusion proteins which often are similar in size to the native antigens has been previously demonstrated (2, 25). were

DISCUSSION The importance of nontuberculous mycobacterial disease has been magnified dramatically by the AIDS epidemic. At least 50% of AIDS patients are infected with nontuberculous mycobacteria; about 90% of these infections are derived from the MAC (7). Disseminated MAC disease is a primary cause of death in a significant number of these individuals (15, 26, 28). Additionally, patients with HIV infections are likely to develop tuberculosis from dormant M. tuberculosis acquired prior to the development of their immunocompromised state. Clinical differentiation of an infecting mycobacterial species is necessary in order to determine the proper treatment. Since cross-reactivity among mycobacteria is extensive, monospecific antigens and antibodies may serve as useful diagnostic reagents for the laboratory and for skin tests.

The evolution of the Xgtll cloning system has provided a methodology for expressing potentially clinically important mycobacterial antigens in E. coli (27). Previously, we described the preparation of a M. intracellulare Xgtll library and the isolation from the library of a 190-kDa M. intracellulare hybrid antigen, the fusion product of P-galactosidase and an 85-kDa immunologically active M. intracellulare protein (17). In this paper, we describe the isolation of bacteriophages expressing four new M. intracellulare recombinant antigens. Sonic extracts of K lysogens derived from these immunoreactive bacteriophages stimulate sensi-

20

MORRIS ET AL.

tized T cells in vitro (S. Morris and D. Rouse, unpublished results). The in vivo cell-mediated reactivity of these sonic extracts is currently being tested. The HGT-6-reactive antigen may be significant because of its apparent common mycobacterial epitope. All 13 of the mycobacterial sonic extracts tested reacted with the HGT-6 antibody. At least three common mycobacterial antigens, of 12, 65, and 70/71 kDa, have been shown to have structural homologies with known heat shock proteins (24). DNA and protein sequencing will be required to assess whether the 54-kDa antigen recognized by the HGT-6 antibody is also a stress protein. Of particular interest are the recombinant antigens that were isolated with monoclonal antibodies IE5 and 6B8. These antibodies recognized M. intracellulare and M. avium proteins but did not react with sonic extracts of 11 other mycobacterial species, E. coli, or B. pertussis. Although several M. leprae- and two M. tuberculosis-specific protein epitopes have been defined (8, 9), only one M. avium- (1) and no M. intracellulare-specific antigen epitopes have been identified. We are currently trying to delineate the DNA sequences encoding the IE5 and 6B8 epitopes by genetic modification of the immunoreactive bacteriophages. Hopefully, MAC-specific synthetic peptides inferred from the appropriate DNA sequence can then be generated for evaluation in serological assays and in skin tests.

1.

2.

3.

4.

5.

6. 7.

8.

9.

LITERATURE CITED Abe, C., H. Saito, H. Tomoika, and Y. Fukasawa. 1989. Production of a monoclonal antibody specific for Mycobacterium avium and the immunological activity of the affinity-purified antigen. Infect. Immun. 57:1095-1099. Andersen, A. B., A. Worsaae, and S. D. Chaparas. 1988. Isolation and characterization of recombinant Xgtll bacteriophages expressing eight mycobacterial antigens of potential immunological relevance. Infect. Immun. 56:1344-1351. Campbell, A. M. 1984. Monoclonal antibody technology, p. 101-184. In R. H. Burdon and P. H. van Knnipenberg (ed.), Laboratory techniques in biochemistry and molecular biology, vol. 13. Elsevier Science Publishing, Inc., New York. Chaparas, S. D. 1984. Immunologically based diagnostic test with tuberculin and other mycobacterial antigens, p. 196-220. In G. P. Kubica and L. G. Wayne (ed.), The Mycobacteria. Marcel Dekker, Inc., New York. Chaparas, S. D., T. Brown, and I. Hyman. 1978. Antigenic relationships among species of Mycobacterium studied by fused rocket immunoelectrophoresis. Int. J. Syst. Bacteriol. 28:547560. Cherayel, B. J., and R. A. Young. 1988. The 28 kDa protein from Mycobacterium leprae is a target of the human antibody response in lepromatous leprosy. J. Immunol. 141:43704375. Collins, F. M. 1988. AIDS-related mycobacterial disease. Springer Semin. Immunopathol. 10:375-391. Engers, H. D., M. Abe, B. R. Bloom, V. Mehra, W. Britton, T. M. Buchanan, S. K. Khanolkar, D. B. Young, 0. Class, T. Gills, M. Harboe, J. Ivanyi, A. H. J. Kolk, and C. C. Shepherd. 1985. Results of a World Health Organization-sponsored workshop on monoclonal antibodies to Mycobacterium leprae. Infect. Immun. 48:603-605. Engers, H. D., V. Honba, J. Bennedsen, T. M. Buchanan, S. D. Chaparas, G. Kadival, 0. Class, J. R. David, J. D. A. van Embden, T. Gadal, S. A. Mustafa, J. Ivanyi, D. B. Young, S. H. E. Kaufmann, A. G. Khomenko, A. H. J. Kolk, M. Kubin, J. A. Louis, P. Minden, T. M. Shinnick, L. Trivka, and R. A. Young. 1986. Results of a World Health Organization-sponsored workshop to characterize antigens recognized by mycobacterium-specific monoclonal antibodies. Infect. Immun. 51:718-720.

INFECT. IMMUN.

10. Husson, R. N., and R. A. Young. 1987. Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen. Proc. Natl. Acad. Sci. USA 84:1679-1683. 11. Huynh, T. V., R. A. Young, and R. W. Davis. 1985. Construction and screening cDNA libraries in XgtlO and Agtll, p. 49-78. In D. M. Glover (ed.), DNA cloning, vol 1. IRL Press, Oxford. 12. Kadival, G. C., and S. D. Chaparas. 1987. Production, characterization and species specificity of five monoclonal antibodies to Mycobacterium tuberculosis. J. Clin. Microbiol. 25:76-80. 13. Kingston, A. E., P. R. Salgami, N. A. Mitchison, and M. T. Colston. 1987. Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tuberculosis H37Rv. Infect. Immun. 55:3149-3154. 14. Lu, M. C., M. H. Lein, R. E. Becker, H. C. Heine, A. M. Buggs, D. Liprovesk, R. Gupta, P. W. Robbins, C. M. Grosskinsky, S. C. Hubbard, and R. A. Young. 1987. Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG. Infect. Immun. 55:2378-2382. 15. Macher, A. M., J. A. Kovacs, V. Gill, G. D. Roberts, J. Ames, C. H. Park, S. Straus, H. C. Lane, J. E. Parillo, A. S. Fauci, and H. Masur. 1983. Bacteremia due to Mycobacterium aviumintracellulare in the acquired immunodeficiency syndrome. Ann. Intern. Med. 99:782-785. 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Morris, S. L., D. A. Rouse, D. Hussong, and S. D. Chaparas. 1988. Isolation and characterization of a recombinant Agtll bacteriophage which expresses an immunoreactive Mycobacterium intracellulare protein in Escherichia coli. Infect. Immun.

56:3026-3031. 18. O'Brien, R. J., L. J. Geiter, and D. E. Snider. 1987. The epidemiology of non-tuberculous mycobacterial diseases in the United States: results from a national survey. Am. Rev. Respir. Dis. 135:1007-1014. 19. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. 20. Selwyn, P. A., D. Hartel, V. A. Lewis, E. E. Schoenbaum, S. H. Vermund, R. S. Klein, A. T. Walker, and G. H. Freiland. 1989. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N. Engl. J. Med. 320:545-550. 21. Shinnick, T. M. 1987. The 65-kilodalton antigen of Mycobacterium tuberculosis. J. Bacteriol. 169:1080-1088. 22. Shinnick, T. M., C. Krat, and S. Shadon. 1987. Isolation and restriction maps of the genes encoding five Mycobacterium tuberculosis proteins. Infect. Immun. 55:1718-1721. 23. Wolinsky, E. 1979. Nontuberculosis mycobacteria and associated diseases. Am. Rev. Respir. Dis. 119:107-159. 24. Young, D., R. Lathigia, R. Hendrix, D. Sweetser, and R. A. Young. 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85:4267-4270. 25. Young, D. B., L. Kent, and R. A. Young. 1987. Screening of a recombinant mycobacterial DNA library with polyclonal antiserum and molecular weight analysis of expressed antigens. Infect. Immun. 55:1420-1425. 26. Young, L. S., C. B. Inderleid, 0. G. Berlin, and M. S. Gottlieb. 1986. Mycobacterial infections in AIDS patients, with an emphasis on the M. avium complex. Rev. Infect. Dis. 8:1024-1033. 27. Young, R. A., B. R. Bloom, C. M. Grosskinsky, J. Ivanyi, D. Thomas, and R. W. Davis. 1985. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci. USA 83:2583-2587. 28. Zakowski, P., S. Fligiel, G. W. Berlin, and B. L. Johnson. 1982. Disseminated Mycobacterium avium-intracellulare infection in homosexual men dying of acquired immunodeficiency syndrome. J. Am. Med. Assoc. 248:2980-2982.