Mycobacterium avium - Journal of Bacteriology - American Society for ...

JOURNAL OF BACTERIOLOGY, Nov. 1991, p. 6991-6997 0021-9193/91/216991-07$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 21

Isolation and Expression of a Gene Cluster Responsible for Biosynthesis of the Glycopeptidolipid Antigens of Mycobacterium avium JOHN T. BELISLE,' LISA PASCOPELLA,2 JULIA M. INAMINE,' PATRICK J. BRENNAN,' AND WILLIAM R. JACOBS, JR.2* Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523,1 and Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 104612 Received 7 June 1991/Accepted 27 August 1991 Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS. Serovars of the M. avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces. A genomic library of DNA from serovar 2 of the M. avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M. avium serovar 2-specific glycopeptidolipid. The responsible gene cluster was mapped to a 22- to 27-kb functional region of the M. avium genome. The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M. smegmatis, whereas the oligosaccharide segment arose from the cloned M. avium genes. This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes.

Since the time of their recognition until recently, Mycobacterium avium organisms have seldom been associated with severe life-threatening, disseminated infections in humans (30). However, it is now recognized that the most frequently encountered bacterial infections in patients with AIDS are due to members of the M. avium complex (10, 23). The single most distinguishing feature of M. avium is the copious amounts of highly antigenic glycolipids, the glycopeptidolipids (GPLs), present on the bacterium's surface (5, 20). Other than these, the cell walls of M. avium apparently display an architecture that is common to the genus (11). The GPLs which distinguish the 30-odd serovars of the M. avium complex and bear responsibility for their serotypic differences are multiglycosylated at the threonine substituent of an otherwise invariant lipopeptide core (Fig. 1). An extraordinary array of sugars within this variable oligosaccharide region confer serological specificity; the structure of the serospecific GPL of serovar 2 (ssGPL-2) (9) is shown in Fig. 1A. In addition, all members of the M. avium complex contain simpler, nonspecific GPLs (nsGPLs) which dilfer from the ssGPLs in that they are singly glycosylated at the threonine substituent with either 6-deoxytalose or 3-0methyl-6-deoxytalose (5, 6). While considerable effort has been devoted to structural elucidation of the GPLs, their biosynthesis and roles in drug permeability and disease pathogenesis have barely been addressed (7). The fact that Mycobacterium smegmatis, unlike M. avium, is now amenable to genetic transfer systems (26, 27) provides a unique opportunity to clone the DNA responsible for the synthesis of complex secondary gene products associated with the cell *

walls of pathogenic M. avium. The added fact that M. smegmatis contains the simpler, singly glycosylated nsGPLs (Fig. 1B) and is devoid of the multiglycosylated ssGPLs (1, 12) also presented the opportunity to express in another mycobacterium the particular gene cluster responsible for the biosynthesis of the serovar 2-specific sugar segment of its ssGPL. These experiments have set the stage for a new generation of studies of the biosynthesis and function of not only the GPLs but also those other mycobacterial cell wall products that distinguish pathogenic from nonpathogenic

mycobacteria. MATERIALS AND METHODS Bacterial strains and other materials. Escherichia coli

X2764 and X2819 were used for transduction and amplification of the M. avium cosmid library (15). The M. avium serovar 2 strain TMC 724 was the source of genomic DNA. M. smegmatis mc2 155 (27) was used for electroporation and subsequent expression of the recombinant serovar 2-specific GPL (R-ssGPL-2). Restriction endonucleases, calf intestine alkaline phosphatase, and T4 ligase were purchased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.), New England Biolabs, Inc. (Beverly, Mass.), and Bethesda Research Laboratories (Gaithersburg, Md.). Lysozyme, lipase, and proteinase K were purchased from Sigma Chemical Co. (St. Louis, Mo.). The Genius kit from Boehringer Mannheim Biochemicals was employed in the preparation of nonradioactive DNA probes. DNA and cosmid isolation. Genomic DNA was isolated from Mycobacterium spp. by using slight modifications of the method of Whipple et al. (29). Cell pellets stored at

Corresponding author. 6991

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BELISLE ET AL. Serovar Specific GPL-2 from M. avium Serovar 2

A

Fatty acyl - NH - D - Phe - D - aThr - D - Ala - L - Alaninol - 0 - (3,4 - Me2 - Rha) 0 U

6-dTal - Rha - 2,3 - Me2 - Fuc Non-Specific GPL from M. smegmatis

B

Fatty acyl - NH - D - Phe - D - aThr - D - Ala - L - Alaninol -0 -(2,3,4 -Me3 - Rha) 0 U

6-dTal FIG. 1. Structures of native GPLs. (A) Structure of the multiglycosylated M. avium ssGPL-2 (9). (B) Structure of one of the singly glycosylated nsGPLs of M. smegmatis (analogous structures with different fatty acyl and sugar substitutions are present in all serovars of the M. avium complex) (22).

-70°C were thawed and washed once in 50 mM Tris-HCI (pH 8.0)-10 mM EDTA-100 mM NaCI. The washed cells were suspended at a concentration of 200 mg (wet weight) of cells per ml of buffer. Lipase was added to a final concentration of 8,000 U/ml, and the mixture was incubated at 37°C for 2 h on a rocking platform. The addition of lysozyme to a final concentration of S mg/ml was followed by incubation for 1.5 h at 37°C. Proteinase K and sodium dodecyl sulfate (SDS) were added to final concentrations of 0.2 mg/ml and 1% (wt/vol), respectively. The suspension was incubated for 6 to 8 h at 55°C and chilled for 15 min. The lysates were extracted three times with phenol-CHC13-isoamyl alcohol (25:24:1) and then once with CHCl3-isoamyl alcohol (24:1). The DNA was precipitated and treated with RNase A according to the method of Maniatis et al. (19). The pYUB18 cosmid and recombinant derivatives were isolated from E. coli by the standard alkaline-SDS method (19). Recombinant cosmids from M. smegmatis were isolated in like fashion except that lysozyme digestion lasted for 1 h at 37°C and the NaOH-SDS treatment lasted for 45 min at 600C. Construction of the M. avium cosmid library. An E. coliMycobacterium shuttle cosmid (pYUB18) was constructed by inserting the lambda cos sequence into the E. coliMycobacterium shuttle plasmid pYUB12 (1Sa, 26). Genomic DNA from M. avium serovar 2 strain TMC 724 was subjected to partial Sau3A digestion. DNA fragments ranging from 32 to 40 kb were isolated from a preparative 0.4% agarose gel by electroelution and ligated into the BamHI site of the pYUB18 vector (15a, 19). The ligation mixture was packaged into lambda phage particles by using the Giga Pack Plus system from Stratagene (La Jolla, Calif.). The phage particles were subsequently used to transduce E. coli X2819 cells (15). Infected cells were plated on Luria-Bertani plates containing 25 jig of kanamycin per ml and grown at 30°C. Kanamycin-resistant colonies were pooled and grown to mid-log phase at 30°C for isolation of the cosmid library as plasmid. For storage, in vivo packaging was induced in the X2819 cells and the library was isolated as transducingparticle lysate (15). Screening of the M. avium serovar 2 cosmid library. The M. avium serovar 2 cosmid library isolated as plasmid DNA was electroporated into M. smegmatis mc2155 (26, 27), and the cells were plated on 7H10(+) agar plates containing 10 ,ug of kanamycin per ml. Approximately 104 clones per gLg of DNA were obtained. After 5 days of incubation at 37°C, individual

colonies of kanamycin-resistant M. smegmatis were patched onto 7H10(+) agar plates containing 25 ,ug of kanamycin per ml and allowed to incubate for 4 to 5 days. These clones were picked and placed on Whatman no. 40 cellulose filter paper. The filters were then subjected to colony dot blot enzyme-linked immunosorbent assay (ELISA) (2) employing the serovar 2-specific CS-17 monoclonal antibody (MAb)

(24). Mapping of the ser2 gene cluster. The pJTB21 cosmid was digested with the restriction enzymes EcoRI, BamHI, and PstI (19). DNA fragments corresponding to the M. avium insert were purified by electroelution (13) and labeled with digoxigenin by using a Genius kit. A second probe was constructed by digesting pJTB21 with DraI plus Sacl and labeling the isolated insert fragment with 32P via random oligonucleotide primers. E. coli X2764 cells were infected with the phage particles containing the M. avium library and grown at 30°C on Luria-Bertani plates containing 25 gg of kanamycin per ml (15). Approximately 7,000 E. coli recombinant colonies were lifted onto nitrocellulose filters, lysed, and hybridized with the pJTB21 probes (19). Positive clones were isolated and grown in superbroth with 25 ,ug of kanamycin per ml at 30°C. The recombinant cosmids were isolated as circular DNA and mapped with restriction enzymes DraI, ClaI, HindIII, and SspI. Additionally, each recombinant cosmid was electroporated into M. smegmatis mc 2155 and the M. smegmatis transformants were screened for serovar 2 GPL expression by colony dot blot ELISA and gas chromatography (GC) of the GPL-associated sugars. Isolation and purification of GPLs. M. avium serovar 2 was grown as previously described (20). M. smegmatis:pYUB18 and M. smegmatis:M. avium recombinant clones were grown on 7H10(+) agar plates (15 by 150 mm) containing 25 ,ug of kanamycin per ml for 5 to 6 days at 37°C. Cells were scraped from plates, autoclaved, and lyophilized. Lipids were extracted with CHCl3-CH30H (2:1) and subjected to a mild alkali treatment which, by destroying nonspecific acylglycerols, served as a purification step for the GPLs (20). The resulting lipids were applied to columns (25 by 1.5 cm) of silicic acid-Celite (2:1) and eluted with increasing concentrations of CH30H in CHCl3 (8); the total GPL population from each strain was collected in the 25% CH30H eluate. Total GPL (10 mg), in CHCl3, was loaded onto an Alltech Econosphere high-pressure liquid chromatography (HPLC) column (4.6 by 250 mm) packed with 5 ,um of silica. The individual ns- and ssGPLs were eluted with increasing

VOL. 173, 1991

_ "_

GENES ENCODING MYCOBACTERIAL GLYCOLIPIDS

A

6993

B

-1'

-

+

FIG. 2. Screening of M. smegmatis transformants with the M. avium serovar 2-specific MAb CS-17. (A) Initial screening of M. smegmatis transformed with the pYUB18:M. avium library, resulting in identification of the MA21-21 clone. (B) Secondary screening of M. smegmatis transformed with pJTB21. The negative (-) and positive (+) controls are M. smegmatis:pYUB18 and M. avium serovar 2, respectively.

concentrations of CH30H in CHCI3 (20). Fractions were examined for purity by thin-layer chromatography in CHCl3CH30H (9:1) (3). Other analytical methods. The amino acid compositions of the various total and purified GPL fractions were determined by GC-mass spectrometry (MS) of the N,(O)-heptafluorobutyryl isobutyl derivatives (14, 17) on an HP-1 capillary column by using a Hewlett-Packard model 5890 gas chromatogram and model 5970 mass detector (20). The total and purified GPL fractions were also analyzed for their sugar compositions by GC-MS of the alditol acetate derivatives (20) on a DB-23 capillary column. Fast atom bombardment (FAB)-MS was performed on 10-gg samples of selected GPL fractions on a 3-nitrobenzyl alcohol matrix spiked with 1 ,lI of 0.1 M KCl by using a VG 7070 extra-high-frequency mass spectrometer and an ion saddle field gun operated at 7 to 8 kV and 1 mA with xenon gas (16, 21).

RESULTS

Construction and screening of the M. avium cosmid library. A major obstacle in expressing recombinant secondary gene products in mycQbacteria has been the inability to clone the required complex of genes. In the present instance, the capability to clone genes for glycosyl- and methyltransferases and possibly for enzymes responsible for de novo synthesis of specific sugar residues had to be developed. In order to clone the expected large multigene segment, we chose the E. coli-Mycobacterium shuttle cosmid vector pYUB18 (26, 27). The genomic library of M. avium serovar 2 DNA was constructed first in E. coli by utilizing bacteriophage X-mediated packaging. The transduction of E. coli produced approximately 6,700 kanamycin-resistant E. coli clones containing recombinant cosmids with M. avium inserts of 32 to 40 kb; the efficiency of this transduction was ser2 Gene Cluster

C

ELISA Results GC Results

S C HC SH

H I

l

a

l l

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l l

IIL +

l l

L

+

C

I

II

N/D

* a

+

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I

_

l l

H

l a

m

_

l l

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I

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1

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a

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pJTB 211

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pJTB 21

pJTB 22

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.3)-a-L-rhamnopyranosyl [2,3-Me2-a-L-Fucp-(1--3)-a-L-Rhap] disaccharide (8, 9, 24). Mapping of the ser2 gene cluster. In order to map the minimal contiguous M. avium DNA fragment necessary for expression of the serovar 2-specific oligosaccharide, probes were derived from the M. avium insert of pJTB21. Applica-

tion of these probes yielded 22 positive E. coli transformants containing individual pYUB18:M. avium cosmids. After isolation from E. coli, each cosmid was transformed into M. smegmatis, and the resulting M. smegmatis:M. avium clones were screened with the CS-17 MAb. Of the 22 clones, 3 (pJTB22, pJTB41, and pJTB211) produced a positive reaction. However, as summarized in Fig. 3, the degree of reactivity with the CS-17 MAb varied among these clones, suggesting altered levels of oligosaccharide expression. The alkali-stable GPLs were isolated from each clone and examined by GC for the presence of the serovar 2-specific sugars. As expected, the clones containing pJTB21, pJTB22, and pJTB41 possessed the serovar 2-specific sugars, Rha and 2,3-Me2-Fuc (Fig. 3). Small quantities of these sugars were also observed in the GPLs from a clone that did not react with the CS-17 MAb; this clone contained cosmid pJTB11 (Fig. 3). Functional mapping of the DNA of pJTB21 and the additional 22 cosmids resulted in the recognition of an M. avium genomic region of approximately 60 kb, of which a 48-kb segment is depicted in Fig. 3. The insert in the original serovar 2 GPL-producing cosmid, pJTB21, was estimated to


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nsGPL-2 A

|nsGPL-M.smeg. j-R-ssGPL-2

SsGPL-2 -

4 1 2 3 FIG. 5. Thin-layer chromatography of the native and recombinant GPLs. Lanes: 1, purified M. avium ssGPL-2; 2, total GPL population from M. avium serovar 2; 3, purified major R-ssGPL-2 from MA21-21; 4, total GPL population, both native and recombinant, from MA21-21. The lipids were resolved on a silica gel thin-layer chromatography plate in CHCl3-CH30H (9:1).

be 32 kb, and, of this, 22 to 27 kb appeared to be necessary for the production of the serovar 2-specific segment of its GPL molecule (Fig. 3). We have termed this region the ser2 gene cluster. A comparison of the ELISA and GC results with maps of the inserts (Fig. 3) indicates that pJTB11, pJTB22, and pJTB211 are devoid of those sequences on the right side of the gene cluster deemed necessary for increased expression of the ssGPL-2. Expression seems not to be influenced by vector sequences, since the two inserts of pJTB22 and pJTB211 were cloned in opposite orientations in pYUB18. Thus, the right segment of this gene cluster may be a regulatory region. Chemical evidence for the generation of a recombinant GPL. In order to lend chemical weight to the immunological evidence for the presence of the serovar 2 determinant in recombinant M. smegmatis, the total GPLs from M. avium serovar 2, the transformed M. smegmatis containing pJTB21 (MA21-21), and control M. smegmatis containing pYUB18 were isolated and analyzed by a number of chemical means. GC-MS revealed that the GPLs of the recombinant MA2121 possessed those sugars that typify the M. smegmatis nsGPLs, namely, 2,3,4-Me3-Rha, 3,4-Me2-Rha, and 6-dTal, as well as 2,3-Me2-Fuc and Rha (Fig. 4), i.e., the sugars characteristic of the serovar 2-specific oligosaccharide. Also, large amounts of 3-Me-Rha were evident, the origin of which is not known at this time. Individual GPLs from each organism were purified by HPLC. The presence of the tetrapeptide core was confirmed by the observation that all contained D-Phe, D-allo-Thr, D-Ala, and L-alaninol in equimolar quantities (5, 6). Thin-layer chromatography demonstrated that the purified ssGPL-2 migrated directly below its nonspecific relatives (Fig. 5, lanes 1 and 2), consistent with previous observations (8, 9). The purified R-ssGPL-2 from MA21-21 was shown to be less polar than the corresponding M. avium ssGPL-2 (Fig. 5, lanes 1 and 3), most likely reflecting differences in the fatty acyl content. GC-MS

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analysis of the sugars from the purified R-ssGPL-2 confirmed the presence of 2,3,4-Me3-Rha, 6-dTal, Rha, and 2,3-Me2Fuc (data not shown) and thus the presence of the serovar 2-specific oligoglycosyl unit in the recombinant glycolipid. The presence of the 2,3,4-Me3-Rha unit in R-ssGPL-2 indicated that the core originated in M. smegmatis (Fig. 1B), whereas chemical proof of the presence of Rha and 2,3-Me2Fuc supported the antigenic evidence that the distal segment of the oligosaccharide originated in the M. avium serovar 2 genome (Fig. 1A). In order to confirm the proposed structure of the R-ssGPL-2, FAB-MS, with its potential to provide structural information about the holistic molecule, was employed (16, 21). A (M+ K)+ pseudomolecular ion at mlz 1509 was observed (Fig. 6A), supporting the evidence for a GPL endowed with the serovar 2-specific oligosaccharide: a 2,3,4Me3-Rha unit linked to L-alaninol and an amino-terminallinked saturated C28 mono-hydroxy fatty acid (1). One of the major nsGPLs isolated from the host M. smegmatis also contained 2,3,4-Me3-Rha and 6-dTal and showed an (M+K)+ ion of mlz 1189 (Fig. 6B), demonstrating the presence of an nsGPL with an amide-linked saturated C28 mono-hydroxy fatty acid in M. smegmatis, presumably the template for serovar 2-directed glycosylation. DISCUSSION This landmark study represents the first reported cloning of genes responsible for the synthesis of complex cell wall structures of mycobacteria. Thus, genes from a member of the M. avium complex were successfully expressed in M. smegmatis, and the resulting products were shown to reflect the serotypic identity of the parental M. avium. The responsible genes were mapped to a 22- to 27-kb contiguous M. avium genomic fragment, the so-called ser2 gene cluster. Biochemical and immunochemical analyses of M. smegmatis clones possessing the pJTB21 and pJTB208 cosmids indicate that the left terminus of ser2 is contained within a 4-kb region, whereas, according to analysis of pJTB11 and pJTB22, the right terminus of ser2 mapped to a 1.5-kb region. Also, the decreased expression of the serovar 2-specific oligosaccharide with increased deletions of the right terminus of the ser2 gene cluster points to a possible region of regulation for gene expression. Although the ser2 cluster must possess the genes required for serovar 2-specific oligosaccharide biosynthesis, the 22- to 27-kb fragment is larger than expected for the two to three glycosyltransferases and methyltransferases expected to facilitate synthesis of the oligoglycosyl unit. Thus, this gene cluster may contain multiple operons or it may encompass the genes required for the de novo synthesis of fucose and rhamnose, perhaps analogous to the Salmonella rfb operon (4, 28, 31). Conceivably, the ser2 gene cluster could contain the genes required for synthesis of the GPL core. However, if this was so, the R-ssGPL-2 should possess an M. aviumtype core rather than an M. smegmatis-type core. Chemical analysis of the R-ssGPL-2, by GC-MS and FAB-MS, demonstrated the presence of a 2,3,4-Me3-Rha unit and a saturated C28 mono-hydroxy fatty acyl unit, the same substituents attributed to the simple GPLs of M. smegmatis (1, 12, 22) but not to those of M. avium (3, 6, 22). The chemical characteristics of the R-ssGPL-2 also indicate that the simplier monoglycosylated GPLs act as biosynthetic precursors of the multiglycosylated GPLs of M. avium, a relationship that had been assumed but never demonstrated. The chemical evidence also suggests that the genes encoding the synthesis of the components of the GPL core, namely,

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BELISLE ET AL.

H53CH(OH)CO - NH - Phe - aThr - Ala - Aino -

-

2,3,4 - Me 3Rha|

0

A

6d Tai: - Rha - 2, 3 -

2Fuc

1509 (M+K)+

1493 (M+Na)'

m/z |

CHWCH(OH)CO-NH-Phe-aThr-aAla-nnol-O-2,3,4-Me3Rha O

B

1189 (M+K)+

6d Tal

Sequence analysis, once specific genes within the ser2 gene cluster are identified, should be of considerable benefit in this sense. M. smegmatis, with a growth rate 10 times higher than that of mycobacterial pathogens (25), was chosen in the present study as the preferred host for cloning M. avium genes because its cell wall is similar in several key respects to that of M. avium, and, unlike M. avium, it is genetically manipulable. Application of this strategy to other major mycobacterial pathogens, Mycobacterium leprae and Mycobacterium tuberculosis, should greatly facilitate an analysis of the pathways leading to the biosynthesis of their own distinctive cell wall entities (7, 20). In addition, this initial study of genetic mechanisms underlying the biosynthesis of the exceptionally complex glycoconjugates of mycobacterial cell walls should provide new approaches to the age-old issue of the roles of such entities in pathogenesis and to the problem of intractable chemotherapy of M. avium infections. ACKNOWLEDGMENTS This work was supported by grants AI-26170, AI-18357, and AI-30189 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Barry R. Bloom and Michael R. McNeil for encouragement and intellectual and technical input.

1. 2.

3.

1173 (M+Na)t 4.

5. mlz

FIG. 6. FAB-MS of purified GPLs. (A) The FAB-MS of RssGPL-2 from MA21-21. The depicted structure of the major R-ssGPL-2 isolated from MA21-21 has a formula weight of 1,470, in accordance with observed molecular ions. The structural components outlined in the solid box originate in the M. smegmatis genome, whereas those in the dashed box originate in the M. avium serovar 2 genome. (B) FAB-MS of an nsGPL purified from M. smegmatis:pYUB18 lipid. The depicted structure of this nsGPL has a formula weight of 1,150, in accordance with observed molecular ions.

6.

7.

8.

unusual fatty acids and amino acids, are located within an operon other than ser2. The separation of operons responsible for the synthesis of individual components of a complex molecule is not uncommon; for instance, the rfa operon responsible for the synthesis of the lipopolysaccharide core is separate from the rjb operon for 0-antigen synthesis (18). Clearly, elucidation of the relationship of genes encoding serovar-specific oligosaccharides to those governing the expression of the communal core requires further work.

9. 10. 11.

REFERENCES Asselineau, C., and J. Asselineau. 1987. Lipides specifiques des mycobacteries. Ann. Microbiol. (Inst. Pasteur) 129A:49-69. Belisle, J. T., and P. J. Brennan. 1989. Chemical basis of rough and smooth variation in mycobacteria. J. Bacteriol. 171:34653470. Bozic, C. M., M. McNeil, D. Chatterjee, I. Jardine, and P. J. Brennan. 1988. Further novel amido sugars within the glycopeptidolipid antigens of Mycobacterium avium. J. Biol. Chem. 263:14984-14991. Brahmbhatt, H. N., P. Wyk, N. B. Quigley, and P. R. Reeves. 1988. Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the 0 antigen of Salmonella typhimurium LT2. J. Bacteriol. 170:98-102. Brennan, P. J. 1988. Mycobacterium and other actinomycetes, p. 203-298. In C. Ratledge and S. G. Wilkinson (ed.), Microbial lipids, vol. 1. Academic Press, London. Brennan, P. J., and M. B. Goren. 1979. Structural studies on the type-specific antigens and lipids of the Mycobacterium avium-

Mycobacterium intracellulare-Mycobacterium scrofulaceum serocomplex. J. Biol. Chem. 254:4205-4211. Brennan, P. J., S. W. Hunter, M. McNeil, D. Chatterjee, and M. Daffe. 1990. Reappraisal of the chemistry of mycobacterial cell walls, with a view to understanding the roles of individual entities in disease processes, p. 55-75. In E. M. Ayoub, G. H. Cassell, W. C. Branch, Jr., and T. J. Henry (ed.), Disease processes in microbial determinants of virulence and host response. American Society for Microbiology, Washington, D.C. Camphausen, R. T., R. L. Jones, and P. J. Brennan. 1985. A glycolipid antigen specific to Mycobacterium paratuberculosis: structure and antigenicity. Proc. Nati. Acad. Sci. USA 82:30683072. Camphausen, R. T., R. L. Jones, and P. J. Brennan. 1986. Structure and relevance of the oligosaccharide hapten of Mycobacterium avium serotype 2. J. Bacteriol. 168:660-667. Chaisson, R. E., and P. C. Hopewell. 1989. Mycobacteria and AIDS mortality. Am. Rev. Respir. Dis. 139:1-3. Daffe, M., P. J. Brennan, and M. McNeil. 1990. Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass

VOL. 173, 1991 spectrometry and by 'H and `3C NMR analyses. J. Biol. Chem. 265:6734-6743. 12. Daffe, M., M. A. Laneelle, and G. Puzo. 1983. Structural elucidation by field desorption and electron impact mass spectrometry of the c-mycosides isolated from Mycobacterium smegmatis. Biochim. Biophys. Acta 751:439-43. 13. Gobel, U., R. Maas, U. Bantel-Schual, and A. Clad. 1987. Rapid and quantitative electroelution and electrodialysis of DNA from gels. J. Biochem. Biophys. Methods 14:245-260. 14. Hirschfield, G. R., M. McNeil, and P. J. Brennan. 1990. Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis. J. Bacteriol. 172:1005-1013. 15. Jacobs, W. R., Jr., J. F. Barrett, J. E. Clark-Curtiss, and R. Curtiss M. 1986. In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries. Infect. Immun. 52:101-109. 15a.Jacobs, W. R., Jr., G. V. Kalpana, J. D. Cirillo, L. Pascopella, S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol. 204:537-555. 16. Jardine, I., G. Scanlan, M. McNeil, and P. J. Brennan. 1989. Plasma desorption mass spectrometric analysis of mycobacterial glycolipids. Anal. Chem. 61:416-422. 17. Mackenzie, S. L., and L. R. Hogge. 1977. Gas chromatographymass spectrometry of the N (O)-heptafluorobutyryl isobutyl esters of the protein amino acids using electron impact ionization. J. Chromatogr. 132:485-493. 18. Makela, P. H., and B. A. D. Stocker. 1984. Genetics of lipopolysaccharide, p. 59-137. In E. T. Rietschel (ed.), Chemistry of endotoxin, vol. I. Elsevier, New York. 19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. McNeil, M., D. Chatterjee, S. W. Hunter, and P. J. Brennan. 1989. Mycobacterial glycolipids: isolation, structures, antigenicity, and synthesis of neoantigens. Methods Enzymol. 179:215242. 21. McNeil, M., A. Y. Tsang, and P. J. Brennan. 1987. Structure and antigenicity of the specific oligosaccharide hapten from the glycopeptidolipid antigen of Mycobacterium avium serotype 4, the


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