FEMS MicrobiologyImmunology89 (1992) 165-174 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier
165
FEMSIM 00201
DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae K.D. Moudgil
1,2,.,
D . L . W i l l i a m s 1 a n d T.P. Gillis 1
1Immunology Research Department, GWL Hansen's Disease Center, Carville, Louisiana, U.S.A., and 2 Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India Received 3 October 1991 Revision received 26 November 1991 Accepted 27 November 1991 Key words: 18-kDa Protein; Mycobacterium leprae; Mycobacteria; D N A hybridization; RFLP; P C R
1. S U M M A R Y D N A hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess D N A sequences homologous to the 18-kDa protein gene of M. leprae. R F L P analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous
Correspondence to: T.P. Gillis, GWL Hansen's Disease Center, Carville, LA 70721-9607, U.S.A. * Present address: Department of Microbiology& Molecular Genetics, University of California, Los Angeles, CA 90024, U.S.A.
sequences in various mycobacteria did not affect the specificity of a previously described P C R test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.
2. I N T R O D U C T I O N Leprosy, a chronic infectious disease caused by Mycobacterium leprae (M. leprae), afflicts about 10-15 million people in the world [2] and continues to present a significant global health problem, particularly for the developing countries. To develop more effective tools for the diagnosis and prevention of this disease, it is important to understand fully the antigens of M. leprae, their distribution among other mycobacteria, and the immune response to these antigens as a result of infection by M. leprae. Numerous protein antigens have been implicated in the immune response to M. leprae. Some of these proteins have been shown to stimulate human T cells [3-6] and are, therefore, likely to be involved in cell-mediated immunity, an impor-
166 tant determinant in protection against leprosy. T h r e e of these antigens with molecular mass of 70, 65 and 18 kilodaltons (kDa) exhibit sequence similarity to known 'stress proteins' [7,8]. The 65-kDa and the 70-kDa proteins of M. leprae are highly conserved in virtually all prokaryotes. The 18-kDa protein, on the other hand, was inferred to be M. leprae-specific, based on immunochemical analysis [9,10]. However, it has recently been reported [1] that the monoclonal antibody (MoAb), L5, previously thought to be specific for the 18-kDa protein of M. leprae [9,11] cross-reacts with a cultivable mycobacterium, M. habana, which is now considered to be M. simiae serovar 1, and that the L5 binding epitope in M. habana is present on an 18-kDa protein, whose expression is increased by heat shock. However, the relationship between the 18-kDa protein of M. leprae and M. habana as well as their corresponding genes is not yet known. The aim of the present study was to determine if mycobacteria other than M. leprae possessed D N A sequences homologous with the 18-kDa protein gene of M. leprae, and should these sequences exist, to determine their relatedness to the M. leprae 18-kDa gene. For this purpose genomic D N A from several mycobacteria was analyzed by D N A hybridization using a D N A probe of approximately 611 base pairs (bp) encoding the entire 18-kDa protein of M. leprae [12] (Fig. 1). Some mycobacteria revealed the pres611
bp 360 212 bp
1
Alu I
bp -
-
I l vu ,l ,u,
Ava II
EcoR I
Sal I
1
Alu
I
Fig. 1. Schematic representation of the restriction map of the 611-bp BamHI-Kpnl M. leprae fragment from the recombinant plasmid pML3 [12]. The open reading frame for the 18-kDa protein gene of M. leprae is shown as a rectangular open box. Forward (F1, F2) and Reverse (R1, R2) primers used for PCR based on 18-kDa gene as the targets are shown as smaller open boxes. F1 and R1 represent primers for generating the M. leprae-specific360-bp product, whereas F2 and R2 generate the 212-bp PCR product probe.
ence of homologous sequences whose relatedness with the 18-kDa protein gene of M. leprae was further analyzed by restriction fragment length polymorphism (RFLP) analysis. In addition, analysis was done to determine the possible impact of the presence of the putative 18-kDa gene homolog(s) in other mycobacteria on the specificity of a previously described polymerase chain reaction (PCR) test for detection of M. leprae [13].
3. M A T E R I A L A N D M E T H O D S
3.1. Bacteria M. intracellulare serotypes 18 and 19 and M. simiae serovars 1 and 2 (Table 1) were grown in 7H12 medium (Difco) at 35°C in an atmosphere of 5% CO 2 (100 ml of 7H12 medium contained 0.47 g 7H9 broth, 0.1 g casitone, 0.4 mg catalase, 0.5 g bovine serum albumin, 0.75 g dextrose and 5 mg oleic acid). Other bacteria were grown and harvested as previously described [13]. Four different isolates of M. leprae from geographically distinct areas of the world were propagated in and purified from tissues of experimentally-infected armadillos as described previously [14]. Armadillo-derived mycobacterium type 2 ( A D M 2) was obtained from Dr. F. Portaels [15]. 3.2. Isolation and purification of chromosomal DNA D N A from several strains of mycobacteria was prepared as previously described [16], with the exception that the recovery of D N A was improved by lyophilization of the bacterial pellets before mechanical grinding and enzymatic lysis. D N A from other bacterial species was purified using the same procedure but without mechanical grinding prior to enzymatic disruption of the cells. D N A from human placenta, Clostridium perfringens and Escherichia coli B was purchased from Sigma Chemical Co., St. Louis, MO. 3.3. DNA probe preparation The gene encoding the 18-kDa protein of M. leprae has been isolated and sequenced [12]. A 611-bp DdeI-DdeI fragment containing the entire 18-kDa coding sequence was subcloned into the
167
SmaI site of pUC 18. The resultant recombinant plasmid (pML3) was amplified in, and purified from, DH5-alpha-competent cells (Pharmacia, Piscataway, N J) using standard procedures described elsewhere [13]. The 611-bp fragment was removed from pML3 by digestion with BamHI and KpnI restriction endonucleases and purified by gel electrophoresis followed by gel electroelution [17]. The BamHI-KpnI 611-bp fragment was radiolabelled with [32p] deoxycytidine triphosphate (Dupont NEN Research Products, Boston,
MA) by nick translation and purified by gel filtration using a NICK column (Pharmacia, Piscataway, N J). The probe was denatured into single strands by addition of 2 N NaOH to a final concentration of 0.2 N. A 212-bp internal fragment of the 611-bp fragment (Fig. 1) was used as a probe for analysis of products from the polymerase chain reaction (PCR). The 212-bp fragment was prepared by PCR using oligonucleotide primers with the following sequences: Forward, (5'-GAACGCAAC-
Table 1 DNA
hybridization and PCR analysis of purified chromosomal
Sample
DNA
Source
Hybridization a
PCR b
65 kDa
Mycobacterial DNA
M. leprae M. tuberculosis ( H 3 7 R v ) M. tuberculosis ( H 3 7 R a ) M. tuberculosis M. bovis ( B C G - P a s t e u r ) M. simiae ( S e r o v a r s l & 2 ) M. kansasii M. scrofulaceum M. gordonae M. flavescens M. avium ( s e r o t y p e 2) M. intracellulare
+
+
+
ATCC 27294
-
+
-
ATCC 25177
-
+
-
Human isolate
-
+
-
A T C C 27291
-
+
-
Clinical isolates d
+
+
_
ATCC
12478
+
+
-
ATCC
19981
+ weak
+
-
ATCC
14470
+ weak
+
-
ATCC
14474
-
+
-
+ weak
+
-
A T C C 25291
( S e r o v a r s 18 a n d 19)
18 kDa
Human isolate c
Clinical isolates d
+
+
_
M. intracellulare M. terrae M. fortuitum M. chelonei M. vaccae M. smegmatis M. phlei M. peregrinum
ATCC
13950
+
+
-
ATCC
15755
Armadillo-derived mycobacterium
M. leprae-infected e
(ADM-2)
+
+
-
A T C C 6841
-
+
-
T M C 1542
+ weak
+
-
ATCC
15483
-
+
-
ATCC
14468
-
+
-
ATCC
11758
-
+
-
ATCC
14467
-
+
-
+
+
-
S i g m a C h e m i c a l Co.
-
-
-
ATCC
-
-
-
-
-
-
ATCC 4277
-
-
-
S i g m a C h e m i c a l Co.
-
-
-
armadillo liver
Nonmycobacterial DNA
Escherichia coli B Clostridium perfringens Staphylococcus epidermidis Rhodococcus erythropolis
13124
Human
isolate
Eukaryotic DNA Human a
placenta
Hybridized with 32p-labeled 611-bp
b Agarose gel and DNA
BamHI-KpnI
c Originally from human borderline lepromatous J F r o m Dr. A. T s a n g . e F r o m D r . F. P o r t a e l s .
fragment encoding the entire 18-kDa gene of
hybridization with 32p-labeled 212-bp probe. lesion m a t e r i a l and grown in a r m a d i l l o s .
M. leprae.
168 GTAGTCACCGT-3') and Reverse, (5'-AACGGAGATCTTGCGCGGTT-3'). The 212-bp PCR product was purified by agarose gel electrophoresis followed by electrolution and concentration by Centricon-30 ultrafiltration (Amicon, Danvers, MA). Nick translation was performed as described above.
3.4. Restriction endonuclease digestion of DNA and Southern blotting Approximately 2 / z g of each DNA sample was treated with restriction endonucleases following the manufacturer's instructions (Bethesda Research Laboratories, Gaithersburg, MD). The restriction endonuclease fragments were separated on 20 cm long 1% agarose (SeaKem GTG agarose, FMC Corporation, Rockland, ME) gels run at 40 V for 16 h using Tris-acetate buffer [17]. One kilobase ladder (Bethesda Research Laboratories, Gaithersburg, MD) was used as a marker. The gel was stained with ethidium bromide (0.05 /zg/ml) and photographed using ultraviolet transillumination. The DNA fragments were transferred from agarose gels to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA) using the alkaline DNA transfer technique of Reed and Mann [18]. Following transfer, the membranes were air dried and then baked for 1 h at 80°C in a vacuum oven. The membranes were prehybridized in plastic bags containing 20 ml of 1 x hybridization solution (Sigma, St. Louis, MO) for ! h at 60°C (low stringency conditions) or 75°C (high stringency conditions) and then hybridized for 18 h at 60°C or 75°C in 8 ml of hybridization solution containing the 32p-labeled 611-bp probe (approx. 100 /zl/blot of 6 x 107 cpm/ml). The blots were washed with light agitation for 90 min either under low stringency conditions (60°C, 3 x SSC and 0.1% SDS) or under high stringency conditions (75°C, 0.3 x SSC and 0.1% SDS). The low and high stringency conditions selected for hybridization and washing, with the 611-bp probe (G + C content 52.38%) were similar to the conditions employed for other mycobacterial DNA hybridization-based studies [16,19,20]. Hybridization signals were detected by autoradiography
with an exposure time of at least 24 h. The blots were exposed for up to 5 days to evaluate those negative and weakly-positive hybridization signals observed by autoradiography at 24 h.
3.5. Reprobing of the Southern blot To determine the relative concentration as well as integrity of the DNA bound to the membrane, the Immobilon-P PVDF membranes hybridized previously with the radiolabeled 611-bp probe, were reprobed with 32p-labeled 0.8 kb and 3.0 kb DNA probes (gift of Douglas Looker) which encode portions of the highly conserved mycobacterial ribosomal RNA (rRNA) operon. The 0.8 kb fragment extends from the 5' end of the 16S rRNA gene to a conserved EcoRI site located approximately 650-bp into the gene of M. auium. The 3.0 kb fragment begins at the same internal EcoRI site of the M. tuberculosis 16S rRNA gene and extends 3' to include a portion of the 23S rRNA gene. These probes hybridize to all mycobacterial DNA tested (data not shown). Prehybridization of the membrane, hybridization with the probe and subsequent washing of the blot was performed under low stringency conditions.
3.6. PCR amplification of M. leprae DNA sequence Analysis of the amplification of the 360-bp fragment (Fig. 1) from M. leprae and other DNA was achieved by using PCR primers and conditions as previously described [13]. Briefly, PCR was performed using a Gene Amp Kit according to instructions of the manufacturer (PerkinElmer-Cetus, Norwalk, CT), and 100 ng of purified chromosomal DNA from M. leprae, other bacterial species, and human placental DNA (Table 1) served as a control. An additional set of primers which have been previously characterized as generating a 200-bp PCR product from DNA of most of the species of mycobacteria tested [21] were also added to the reaction. The amplified products of the PCR were analyzed by agarose gel electophoresis and Southern blotting. Southern blots were analyzed for the presence of low numbers of PCR products using a 212-bp probe found within the 360-bp PCR product (Fig. 1).
169 4. R E S U L T S
4.1. Hybridization analysis D N A hybridization analysis of Southern blots containing mycobacterial D N A digested with the restriction endonuclease E c o R I was performed under low as well as high stringency conditions. U n d e r low stringency conditions the 611-bp probe hybridized to D N A of many species of mycobacteria (Fig. 2, 3A, Table 1). M. leprae, M. intracellulare, M. simiae, armadillo-derived mycobac-
1 2 3 4
5 6
7 8 9
10
kb
6
"5
Fig. 2. Autoradiograph of hybridization between 32p-labeled 611-bp probe and EcoRI digests of chromosomal DNA of mycobacteria under low stringency conditions (60°C, 3 × SSC, 0.1% SDS). EcoRI restriction endonuclease digests of chromosomal DNA were separated by electrophoresis through a 1% agarose gel and blotted onto Immobilon-P membrane using alkaline transfer. By lane: 1, M. intracellulare serovar 19, Darden; 2, M. intracellulare serovar 19, W552; 3, M. intracellulare serovar 18, 4990, O'Connor; 4, M. intracellulare serovar 18, Melnick; 5, M. simiae serovar 2, W58; 6, M. simiae serovar 2, W55; 7, M. simiae serovar 1, Krasnow 7729; 8, M. simiae serovar 1, 1595; 9, M. bouis BCG; 10, M. leprae. DNA size markers (1 kb ladder) are shown on the right margin.
terium ( A D M 2), M. kansasii, and M. terrae gave moderate to strong hybridization signals, whereas M. avium, M. scrofulaceum, M. gordonae and M. chelonei produced only weak hybridization signals. In contrast, several other species of mycobacteria ( e . g . M . boris BCG, M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. vaccae, M. smegmatis, M. fortuitum, M. phlei, M. flauescens and M. peregrinum), other bacteria ( e . g . E . coli, Clostridium perfringens, Staphylococcus epidermidis, Rhodococcus erythropolis) and h u m a n placental D N A gave no hybridization signal with the 611-bp probe. To ensure that appropriate conditions (e.g. D N A concentrations sufficient for digestion of genomic D N A with restriction endonucleases, and for Southern blotting) were met for analysis of the mycobacterial D N A testing negative with the 611-bp probe, the same blot (Fig. 3A) was reprobed with the 0.8 kb and 3.0 kb r R N A gene probes common to all mycobacteria and other bacteria tested in this study. Fig. 3B shows that D N A from various mycobacteria and other bacteria which did not give D N A hybridization signals with the 611-bp probe were at optim u m concentration and were properly digested with the restriction enzyme used. By comparison, out of the 18 species of mycobacteria tested, only M. leprae D N A gave a positive hybridization signal with the 611-bp probe under high stringency conditions. Similar results of hybridization with the 611-bp probe (e.g. mycobacterial species giving positive or negative hybridization signals) were obtained when B a m H I was used to digest the D N A from different sources in place of EcoRI, except that the restriction pattern with B a m H I was different from that obtained with EcoRI.
4.2. R F L P analysis The D N A from three species of mycobacteria, M. leprae, M. intracellulare serovars 18 and 19, and M. simiae serovars 1 and 2 was further subjected to detailed R F L P analysis using the 611-bp probe. Six restriction endonucleases, including AluI, AualI, B a m H I , EcoRI, PcuI and SalI were used in separate reactions to digest the mycobacterial DNA. The restriction endonuclease cleavage sites of these enzymes in the 18-kDa
170 protein gene of M. leprae are depicted in Fig. 1. Of the six enzymes, A l u I is most suited to generate D N A fragments of predictable size from the 611-bp fragment. R F L P patterns of M. intracellulare, M. simiae, and M. leprae using E c o R I and A l u I are given in Fig. 2 and 4, respectively (the data for other restriction enzymes are not shown). A l u l cleaves the 611-bp fragment, which includes the 18-kDa M. leprae gene, at three positions generating at least two predictable fragments of 199 and 321 bp (Fig. 1). Both of these fragments were detected in Southern blots of A l u I - d i g e s t e d M. leprae D N A (Fig. 4). In comparison, A l u I digests of M. intracellulare serovars 18 and 19 D N A revealed one fragment of approx. 260 bp and two other fragments of more than 500 bp, whereas A l u I digests of M. simiae serovars 1 and 2 D N A generated fragments of more than 1.6 kb.
R F L P patterns using the 611-bp probe could discriminate M. intracellulare, M. simiae, and M. leprae from each other with each of the six restriction enzymes tested. R F L P patterns also revealed differences between the two strains of M. simiae serovar 1 with all the restriction enzymes tested, except E c o R I . In contrast, the four different isolates of M. leprae obtained from geographically distinct areas of the world revealed identical patterns with each of the six restriction enzymes used (data not shown). The restriction patterns of the three mycobacterial species revealed that apparently the restriction sites in the M. leprae 18-kDa protein gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare, and that each of these three species of mycobacteria have only one copy of the putative gene.
A
kb
12-9-7-5--
1
2
3
4
5
6
B 7
8
9 10 11 12 13 14
kb
12-I 97-
1
2
3 4
5
6
7 8 9 10 11 12 13 14
=
53-3 D
2~ 1.6--
21.6--
1-1-
0-50.5-
Fig. 3. A: Autoradiograph of hybridization between 32p-labeled 611-bp probe and EcoRI digests of chromosomal DNA of mycobacteria, other bacteria and human placental DNA under low stringency conditions. By lane: 1, M. phlei; 2, M. leprae; 3, armadillo-derived mycobacterium(ADM-2); 4, M. fortuitum; 5, M. kansasii; 6, M. tuberculosis H37Rv; 7, M. boris BCG; 8, M. scrofulaceum; 9, M. gordonae; 10, M. smegmatis; 11, M. caccae; 12, E. coli; 13, Clostridium perfringens; 14, human placental DNA. DNA size markers (1 kb ladder) are shown on the left margin. B: Autoradiograph of hybridization between 32p-labeled 0.8 kb and 3.0 kb rRNA gene probes and EcoRI digests of DNA. The Immobilon-Pmembrane previously hybridized with the 611-bp probe (A) was reprobed with 0.8 kb and 3.0 kb probes under low stringencyconditions.
171 2
3
4
5
6
7
8
9
kb
-12
primers which amplify the highly conserved 65kDa protein gene [21], the characteristic 200-bp product was observed for all mycobacterial species analyzed indicating that the D N A was suitable for P C R analysis.
-9 -7
-5 -3
--2 -1-6
-1
--0"5
Fig. 4. Autoradiograph of hybridization between 32p-labeled 611-bp probe and AluI digests of chromosomal DNA of mycobacteria under low stringency conditions. By lane: 1, M. intracellulare serovar 19, Darden; 2, M. intracellulare serovar 19, W552; 3, M. intracellulare serovar 18, 4990, O'Connor; 4, M. intracellulare serovar 18, Melnick; 5, M. simiae serovar 2, W58; 6, M. simiae serovar 2, W55; 7, M. simiae serovar 1, Krasnow 7729; 8, M. simiae serovar 1, 1595; 9, M. leprae. The size markers (1 kb ladder) are shown on the right margin. The size of the two fragments of M. leprae DNA are indicated as follows, * = 199-bp; ** = 321-bp.
4.3. PCR analysis P C R analysis was performed on 100 ng of chromosomal D N A from M. leprae, other bacterial species and h u m a n D N A (Table 1, Fig. 5). W h e n primers designed for amplification of a portion of the 18-kDa protein gene of M. leprae were used, the 360-bp product was amplified only from M. leprae D N A as detected by agarose gel electrophoresis (Fig. 5A) and Southern blotting followed by hybridization with the radiolabeled 212-bp probe (Fig. 5B). In contrast, using the
5. D I S C U S S I O N The 18-kDa protein of M. leprae has been the subject of various studies directed towards identification of an M. leprae-specific antigen which could confer protective immunity against M. leprae infection. It has been shown that both murine and h u m a n T cells are stimulated by the 18-kDa protein [5,6,22]. Initial characterization of the recombinant 18-kDa protein using an M. lepraespecific MoAb, L7.15, indicated that the protein was unique to M. leprae [10]. However, using Western blot analysis, L a m b et al. [1] have recently reported that M. habana has an 18-kDa heat shock protein which shares an epitope with the 18-kDa protein of M. leprae as defined by a MoAb, L5 (same as L7.15). T cell studies by Dockrell et al. [6] also support the concept that the 18-kDa protein of M. leprae has shared epitopes with another mycobacterium, M. boris BCG. In their studies 70% of M. boris BCG-vaccinated E u r o p e a n donors responded in a lymphoproliferative assay to the M. leprae 18-kDa protein. In addition, they were able to establish 18-kDa antigen-responsive T cell lines from a BCG-vaccinated British donor. In this paper, we provide evidence that M. simiae as well as other mycobacteria possess D N A sequences homologous to the 18-kDa protein gene of M. leprae and suggest that the putative gene homolog in these species has only limited sequence homology with the M. leprae 18-kDa protein gene based on D N A hybridization results and R F L P analysis of genomic DNA. In D N A hybridization experiments using a 611-bp probe, which encodes the entire 18-kDa protein gene of M. leprae, D N A from M. leprae, M. simiae, M. intracellulare, M. avium, M. kansasii, armadillo-derived mycobacterium ( A D M 2), M. terrae, M. scrofulaceum, M. gordonae and M. chelonei gave a positive hybridization signal under
172 low stringency conditions, w h e r e a s u n d e r the same c o n d i t i o n s eight o t h e r species of mycobacteria did not b i n d the p r o b e (Table 1). W h e n hybridization a n d w a s h i n g c o n d i t i o n s were altered to increase the stringency, only M. leprae D N A hybridized with the 611-bp probe. T h e s e o b s e r v a t i o n s suggest the p r e s e n c e of h o m o l o g o u s s e q u e n c e s in the above m e n t i o n e d m y c o b a c t e r i a which exhibit partial homology with the 18-kDa p r o t e i n g e n e of M. leprae.
T h e p r e s e n c e of a n 18-kDa p r o t e i n gene homolog in M. simiae serovars 1 a n d 2 is s u p p o r t e d by observations of L a m b et al. [1], who have r e p o r t e d that M. h a b a n a , c o n s i d e r e d taxonomically to be M. simiae 1, has an 18-kDa heat shock protein, which reacts with M o A b L5, previously t h o u g h t to be specific for the 18-kDa p r o t e i n of M. leprae. T h e i r studies also showed that the 18-kDa p r o t e i n could not be d e t e c t e d in extracts of M. tuberculosis, M. boris BCG, M. smegmatis,
B
1 2 3456
A
1 2 3 4 5 6 7 89101112131415
7 8 9 101112131415
bp
-369 -246 -123
b
-360 -200
0
-360
Fig. 5. A: Analysis of polymerase chain reaction (PCR) amplification products from 45 cycles of PCR starting with 100 ng of purified chromosomal DNA, using two sets of primers, one generating a 360-bp product from the 18-kDa protein gene and the other generating a 200-bp product from the 65-kDa protein gene. 50 ~l of PCR product DNA was separated by gel electrophoresis through 2% agarose gel and stained with ethidium bromide (0.05 p.g/ml), a and b denote upper and lower gel respectively. By lane, a: 1, M. fortuitum; 2, M. flavescens; 3, M. gordonae; 4, M. phlei; 5, Armadillo-derived mycobacterium (ADM 2); 6, M. kansasii; 7, M. intracellulare serovar 18, 4990, O'Connor, 8, M. chelonei; 9, M. peregrinum; 10, M. tuberculosis H37Rv; 11, M. tuberculosis H37Ra; 12, M. terrae; 13, M. intracellulare serovar 19, Darden; 14, M. simiae serovar 1, 1595; 15, 123-bp DNA ladder (BRL); three of the size markers of the 123-bp ladder are shown on the right margin of the figure, b: 1, M. scrofulaceum; 2, M. boris BCG; 3, M. intracellulare ATCC 13950; 4, M. smegmatis; 5, M. caccae; 6, Blank; 7, Clostridium perfringens; 8, E. coli B; 9, Human placental DNA; 10, Negative control (H20); 11, Blank; 12, Blank; 13, M. leprae; 14, Blank; 15, 123-bp DNA ladder. The positions of the 200-bp and 360-bp PCR products are shown on the right margin. B: Autoradiograph of hybridization between 32p-labeled 212-bp probe and the DNA from 45 cycles of PCR. The PCR amplification products from DNA of various bacteria were separated by gel electrophoresis (A) and then subjected to Southern transfer and hybridization.
173
M. phlei, and M. fortuitum, when analyzed by Western blotting using the L5 MoAb. In the present study we have observed that the D N A from the above-mentioned five mycobacteria did not hybridize with the 611-bp probe under either high or low stringency conditions. This indicated that either these bacteria do not have the 18-kDa gene homolog, or that the homology between the probe and the putative 18-kDa protein gene homolog is below the level required for hybridization under the conditions employed. Studies are in progress to analyze the possibility of protein expression by the mycobacteria possessing the 18-kDa gene homologs using antibody probes to the M. leprae 18-kDa protein. The R F L P pattern of different mycobacteria using the 611-bp probe revealed additional information of interest. Most species of mycobacteria gave a characteristic profile, and it was helpful in differentiating, for example, M. intracellulare, M. sirniae, and M. leprae from each other. By using different restriction enzymes, it was further possible to identify one serovar from the other in a given species ( e . g . M . intracellulare Type 18 and 19, and M. simiae serovar 1 and 2), and even to identify one strain from another in a given serovar (e.g. the two strains of M. simiae serovar 1). Though these preliminary results are based on a small number of serovars in the species analyzed by D N A hybridization, R F L P patterns with the 611-bp probe may be useful for taxonomic purposes for mycobacteria as a useful adjunct to other parameters being employed for this purpose. We have previously reported the development of a P C R test for detection of M. leprae, based on amplification of a portion of the 18-kDa protein gene of M. leprae [13]. In the present study we have shown that some mycobacteria possess D N A sequences homologous to the M. leprae 18-kDa gene. Therefore the D N A from all the mycobacteria tested in D N A hybridization experiments were also subjected to P C R analysis. The results from this study indicated that D N A from all mycobacteria except M. leprae were negative for the 360-bp M. leprae specific P C R product. Using the 65-kDa protein gene as a target for amplification, a 200-bp product was obtained from D N A
of all mycobacteria tested, indicating the D N A from different sources was suitable for amplification by PCR. These observations suggest that the presence of homologous sequences in various mycobacteria did not affect the specificity of the previously described P C R test. In the present study D N A hybridization with the 611-bp probe provided evidence that some mycobacteria possessed D N A sequences homologous to the 18-kDa protein of M. leprae. Cloning and sequencing of the putative 18-kDa gene homologs would reveal the precise regions of homology and the extent of relatedness between the genes from different mycobacteria. In addition, the R F L P profiles of mycobacteria using the 611bp probe may prove useful for taxonomic purposes.
ACKNOWLEDGEMENTS The authors wish to thank the Heiser Program for Research in Leprosy and the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and Training in Tropical Diseases (grant no. 890096) for the financial support to carry out this work. We thank Dr. Douglas Looker for r R N A gene probes; Dr. A. Tsang for isolates of M. intracellulare and M. simiae; Dr. F. Portaels for ADM-2; Dr. Rubina Patel and Dr. K. Eisenach for purified D N A from some mycobacterial species; Rita Sanchez for help in cultivation of mycobacteria; and Penne Cason for typing this manuscript.
REFERENCES [1] Lamb, F.I., Singh, N.B. and Colston, M.J. (1990) The specific 18-kilodalton antigen of Mycobacterium leprae is present in Mycobacterium habana and functions as a heat-shock protein. J. Immunol. 144, 1922-1925. [2] Bloom, B.R. and Godal, T. (1983) Selective primary health care: strategies for control of disease in the developing world. Leprosy Rev. Infect. Dis. 5, 765-780. [3] Mustafa, A.S., Gill, H.K., Nerland, A., Britton, W.J., Mehra, V., Bloom, B.R., Young, R.A. and Godal, T. (1986) Human T-cell clones recognize a major M. leprae protein antigen expressed in E. coli. Nature 319, 63-66.
174 [4] Mustafa, A.S. (1988) Identification of T-cell-activating recombinant antigens shared among three candidate antileprosy vaccines, killed M. leprae, M. boois BCG and Mycobacterium w. Int. J. Lepr. 56, 265-273. [5] Ottenhoff, T.H.M., Klaster, P.R., Ivanyi, J., Elferink, D.G., de Wit, M.Y.L. and de Vries, R.R.P. (1986) Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells. Nature 319, 66-68. [6] Dockrell, H.M., Stoker, N.G., Lee, S.P., Jackson, M., Grant, K.A., Jouy, N.F., Lucas, S.B., Hasan, R., Hussain, R. and McAdam, K.P.W.J. (1989) T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae. Infect. Immun. 57, 1979-1983. [7] Nerland, A.H., Mustafa, A.S., Sweetser, D., Godal, T. and Young, R.A. (1988) A protein antigen of Mycobacterium leprae is related to a family of small heat shock proteins. J. Bacteriol. 170, 5919-5921. [8] Young, D., Lathigra, R., Hendrix, R., Sweetser, D. and Young, R.A. (1988) Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85, 4267-4270. [9] Engers, H.D., Abe, M., Bloom, B.R., Mehra, V., Britton, W., Buchanan, T.M., Khanolkar, S.K., Young, D.B., Closs, O., Gillis, T., Harboe, M., Ivanyi, J., Kolk, A.H.J. and Shepard, C.C. (1985) Results of a World Health Organization-sponsored workshop on monoclonal antibodies to Mycobacterium leprae. Infect. Immun. 48, 603605. [10] Young, R.A., Mehra, V., Sweetser, D., Buchanan, T., Clark-Curtiss, J., Davis, R.W. and Bloom, B.R. (1985) Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae. Nature 316, 450-452. [11] Britton, W.J., Hellqvist, L., Basten, A. and Raison, R.L. (1985) Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies. J. Immunol. 135, 4171-4177. [12] Booth, R.J., Harris, D.P., Love, J.M. and Watson, J.D. (1988) Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein. J. Immunol. 140, 597-601. [13] Williams, D.L., Gillis, T.P., Booth, R.J., Looker, D. and
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
Watson, J.D. (1990) The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. J. Inf. Dis. 162, 193-200. Williams, D.L. and Gillis, T.P. (1989) A study of the relatedness of Mycobacterium leprae isolates using restriction fragment length polymorphism analysis. Acta. Leprol. (Geneva) 7 (Suppl 1), 226-230. Portaels, F., Asselineau, C., Baess, I., Daffe, M., Dobson, G., Draper, P., Gregory, D., Hall, R.M., Imaeda, T., Jenkins, P.A., Laneelle, M.A., Larsson, L., Magnusson, M., Minnikin, D.E., Pattyn, S.R., Wieten, G. and Wheeler, P.R. (1986) A cooperative taxonomic study of mycobacteria isolated from armadillos infected with Mycobacterium leprae. J. Gen. Microbiol. 132, 2693-2707. Clark-Curtiss, J.E., Jacobs, W.R., Docherty, M.A., Ritchie, L.R. and Curtiss, R., III. (1985) Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J. Bacteriol. 161, 1093-1102. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Reed, K.C. and Mann, D.A. (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13, 7207-7221. Grosskinsky, C.M., Jacobs, W.R., Jr., Clark-Curtiss, J.E. and Bloom, B.R. (1989) Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: Identification of an M. leprae-specific repetitive sequence. Infect. Immun. 57, 1535-1541. Sela, S., Clark-Curtiss, J.E and Bercovier, H. (1989) Characterization and taxonomic implications of the rRNA genes of Mycobacterium leprae. J. Bacterioi. 171, 70-73. Hartskeerl, R.A., DeWit, M.Y.L. and Klaster, P.R. (1989) Polymerase chain reaction for the detection of Mycobacterium leprae. J. Gen. Microbiol. 135, 2357-2364. Harris, D.P., Backstrom, B.T., Booth, R.J., Love, S.G., Harding, D.R. and Watson, J.D. (1989) The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay. J. Immunol. 143, 2006-2012.
165
FEMSIM 00201
DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae K.D. Moudgil
1,2,.,
D . L . W i l l i a m s 1 a n d T.P. Gillis 1
1Immunology Research Department, GWL Hansen's Disease Center, Carville, Louisiana, U.S.A., and 2 Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India Received 3 October 1991 Revision received 26 November 1991 Accepted 27 November 1991 Key words: 18-kDa Protein; Mycobacterium leprae; Mycobacteria; D N A hybridization; RFLP; P C R
1. S U M M A R Y D N A hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess D N A sequences homologous to the 18-kDa protein gene of M. leprae. R F L P analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous
Correspondence to: T.P. Gillis, GWL Hansen's Disease Center, Carville, LA 70721-9607, U.S.A. * Present address: Department of Microbiology& Molecular Genetics, University of California, Los Angeles, CA 90024, U.S.A.
sequences in various mycobacteria did not affect the specificity of a previously described P C R test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.
2. I N T R O D U C T I O N Leprosy, a chronic infectious disease caused by Mycobacterium leprae (M. leprae), afflicts about 10-15 million people in the world [2] and continues to present a significant global health problem, particularly for the developing countries. To develop more effective tools for the diagnosis and prevention of this disease, it is important to understand fully the antigens of M. leprae, their distribution among other mycobacteria, and the immune response to these antigens as a result of infection by M. leprae. Numerous protein antigens have been implicated in the immune response to M. leprae. Some of these proteins have been shown to stimulate human T cells [3-6] and are, therefore, likely to be involved in cell-mediated immunity, an impor-
166 tant determinant in protection against leprosy. T h r e e of these antigens with molecular mass of 70, 65 and 18 kilodaltons (kDa) exhibit sequence similarity to known 'stress proteins' [7,8]. The 65-kDa and the 70-kDa proteins of M. leprae are highly conserved in virtually all prokaryotes. The 18-kDa protein, on the other hand, was inferred to be M. leprae-specific, based on immunochemical analysis [9,10]. However, it has recently been reported [1] that the monoclonal antibody (MoAb), L5, previously thought to be specific for the 18-kDa protein of M. leprae [9,11] cross-reacts with a cultivable mycobacterium, M. habana, which is now considered to be M. simiae serovar 1, and that the L5 binding epitope in M. habana is present on an 18-kDa protein, whose expression is increased by heat shock. However, the relationship between the 18-kDa protein of M. leprae and M. habana as well as their corresponding genes is not yet known. The aim of the present study was to determine if mycobacteria other than M. leprae possessed D N A sequences homologous with the 18-kDa protein gene of M. leprae, and should these sequences exist, to determine their relatedness to the M. leprae 18-kDa gene. For this purpose genomic D N A from several mycobacteria was analyzed by D N A hybridization using a D N A probe of approximately 611 base pairs (bp) encoding the entire 18-kDa protein of M. leprae [12] (Fig. 1). Some mycobacteria revealed the pres611
bp 360 212 bp
1
Alu I
bp -
-
I l vu ,l ,u,
Ava II
EcoR I
Sal I
1
Alu
I
Fig. 1. Schematic representation of the restriction map of the 611-bp BamHI-Kpnl M. leprae fragment from the recombinant plasmid pML3 [12]. The open reading frame for the 18-kDa protein gene of M. leprae is shown as a rectangular open box. Forward (F1, F2) and Reverse (R1, R2) primers used for PCR based on 18-kDa gene as the targets are shown as smaller open boxes. F1 and R1 represent primers for generating the M. leprae-specific360-bp product, whereas F2 and R2 generate the 212-bp PCR product probe.
ence of homologous sequences whose relatedness with the 18-kDa protein gene of M. leprae was further analyzed by restriction fragment length polymorphism (RFLP) analysis. In addition, analysis was done to determine the possible impact of the presence of the putative 18-kDa gene homolog(s) in other mycobacteria on the specificity of a previously described polymerase chain reaction (PCR) test for detection of M. leprae [13].
3. M A T E R I A L A N D M E T H O D S
3.1. Bacteria M. intracellulare serotypes 18 and 19 and M. simiae serovars 1 and 2 (Table 1) were grown in 7H12 medium (Difco) at 35°C in an atmosphere of 5% CO 2 (100 ml of 7H12 medium contained 0.47 g 7H9 broth, 0.1 g casitone, 0.4 mg catalase, 0.5 g bovine serum albumin, 0.75 g dextrose and 5 mg oleic acid). Other bacteria were grown and harvested as previously described [13]. Four different isolates of M. leprae from geographically distinct areas of the world were propagated in and purified from tissues of experimentally-infected armadillos as described previously [14]. Armadillo-derived mycobacterium type 2 ( A D M 2) was obtained from Dr. F. Portaels [15]. 3.2. Isolation and purification of chromosomal DNA D N A from several strains of mycobacteria was prepared as previously described [16], with the exception that the recovery of D N A was improved by lyophilization of the bacterial pellets before mechanical grinding and enzymatic lysis. D N A from other bacterial species was purified using the same procedure but without mechanical grinding prior to enzymatic disruption of the cells. D N A from human placenta, Clostridium perfringens and Escherichia coli B was purchased from Sigma Chemical Co., St. Louis, MO. 3.3. DNA probe preparation The gene encoding the 18-kDa protein of M. leprae has been isolated and sequenced [12]. A 611-bp DdeI-DdeI fragment containing the entire 18-kDa coding sequence was subcloned into the
167
SmaI site of pUC 18. The resultant recombinant plasmid (pML3) was amplified in, and purified from, DH5-alpha-competent cells (Pharmacia, Piscataway, N J) using standard procedures described elsewhere [13]. The 611-bp fragment was removed from pML3 by digestion with BamHI and KpnI restriction endonucleases and purified by gel electrophoresis followed by gel electroelution [17]. The BamHI-KpnI 611-bp fragment was radiolabelled with [32p] deoxycytidine triphosphate (Dupont NEN Research Products, Boston,
MA) by nick translation and purified by gel filtration using a NICK column (Pharmacia, Piscataway, N J). The probe was denatured into single strands by addition of 2 N NaOH to a final concentration of 0.2 N. A 212-bp internal fragment of the 611-bp fragment (Fig. 1) was used as a probe for analysis of products from the polymerase chain reaction (PCR). The 212-bp fragment was prepared by PCR using oligonucleotide primers with the following sequences: Forward, (5'-GAACGCAAC-
Table 1 DNA
hybridization and PCR analysis of purified chromosomal
Sample
DNA
Source
Hybridization a
PCR b
65 kDa
Mycobacterial DNA
M. leprae M. tuberculosis ( H 3 7 R v ) M. tuberculosis ( H 3 7 R a ) M. tuberculosis M. bovis ( B C G - P a s t e u r ) M. simiae ( S e r o v a r s l & 2 ) M. kansasii M. scrofulaceum M. gordonae M. flavescens M. avium ( s e r o t y p e 2) M. intracellulare
+
+
+
ATCC 27294
-
+
-
ATCC 25177
-
+
-
Human isolate
-
+
-
A T C C 27291
-
+
-
Clinical isolates d
+
+
_
ATCC
12478
+
+
-
ATCC
19981
+ weak
+
-
ATCC
14470
+ weak
+
-
ATCC
14474
-
+
-
+ weak
+
-
A T C C 25291
( S e r o v a r s 18 a n d 19)
18 kDa
Human isolate c
Clinical isolates d
+
+
_
M. intracellulare M. terrae M. fortuitum M. chelonei M. vaccae M. smegmatis M. phlei M. peregrinum
ATCC
13950
+
+
-
ATCC
15755
Armadillo-derived mycobacterium
M. leprae-infected e
(ADM-2)
+
+
-
A T C C 6841
-
+
-
T M C 1542
+ weak
+
-
ATCC
15483
-
+
-
ATCC
14468
-
+
-
ATCC
11758
-
+
-
ATCC
14467
-
+
-
+
+
-
S i g m a C h e m i c a l Co.
-
-
-
ATCC
-
-
-
-
-
-
ATCC 4277
-
-
-
S i g m a C h e m i c a l Co.
-
-
-
armadillo liver
Nonmycobacterial DNA
Escherichia coli B Clostridium perfringens Staphylococcus epidermidis Rhodococcus erythropolis
13124
Human
isolate
Eukaryotic DNA Human a
placenta
Hybridized with 32p-labeled 611-bp
b Agarose gel and DNA
BamHI-KpnI
c Originally from human borderline lepromatous J F r o m Dr. A. T s a n g . e F r o m D r . F. P o r t a e l s .
fragment encoding the entire 18-kDa gene of
hybridization with 32p-labeled 212-bp probe. lesion m a t e r i a l and grown in a r m a d i l l o s .
M. leprae.
168 GTAGTCACCGT-3') and Reverse, (5'-AACGGAGATCTTGCGCGGTT-3'). The 212-bp PCR product was purified by agarose gel electrophoresis followed by electrolution and concentration by Centricon-30 ultrafiltration (Amicon, Danvers, MA). Nick translation was performed as described above.
3.4. Restriction endonuclease digestion of DNA and Southern blotting Approximately 2 / z g of each DNA sample was treated with restriction endonucleases following the manufacturer's instructions (Bethesda Research Laboratories, Gaithersburg, MD). The restriction endonuclease fragments were separated on 20 cm long 1% agarose (SeaKem GTG agarose, FMC Corporation, Rockland, ME) gels run at 40 V for 16 h using Tris-acetate buffer [17]. One kilobase ladder (Bethesda Research Laboratories, Gaithersburg, MD) was used as a marker. The gel was stained with ethidium bromide (0.05 /zg/ml) and photographed using ultraviolet transillumination. The DNA fragments were transferred from agarose gels to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA) using the alkaline DNA transfer technique of Reed and Mann [18]. Following transfer, the membranes were air dried and then baked for 1 h at 80°C in a vacuum oven. The membranes were prehybridized in plastic bags containing 20 ml of 1 x hybridization solution (Sigma, St. Louis, MO) for ! h at 60°C (low stringency conditions) or 75°C (high stringency conditions) and then hybridized for 18 h at 60°C or 75°C in 8 ml of hybridization solution containing the 32p-labeled 611-bp probe (approx. 100 /zl/blot of 6 x 107 cpm/ml). The blots were washed with light agitation for 90 min either under low stringency conditions (60°C, 3 x SSC and 0.1% SDS) or under high stringency conditions (75°C, 0.3 x SSC and 0.1% SDS). The low and high stringency conditions selected for hybridization and washing, with the 611-bp probe (G + C content 52.38%) were similar to the conditions employed for other mycobacterial DNA hybridization-based studies [16,19,20]. Hybridization signals were detected by autoradiography
with an exposure time of at least 24 h. The blots were exposed for up to 5 days to evaluate those negative and weakly-positive hybridization signals observed by autoradiography at 24 h.
3.5. Reprobing of the Southern blot To determine the relative concentration as well as integrity of the DNA bound to the membrane, the Immobilon-P PVDF membranes hybridized previously with the radiolabeled 611-bp probe, were reprobed with 32p-labeled 0.8 kb and 3.0 kb DNA probes (gift of Douglas Looker) which encode portions of the highly conserved mycobacterial ribosomal RNA (rRNA) operon. The 0.8 kb fragment extends from the 5' end of the 16S rRNA gene to a conserved EcoRI site located approximately 650-bp into the gene of M. auium. The 3.0 kb fragment begins at the same internal EcoRI site of the M. tuberculosis 16S rRNA gene and extends 3' to include a portion of the 23S rRNA gene. These probes hybridize to all mycobacterial DNA tested (data not shown). Prehybridization of the membrane, hybridization with the probe and subsequent washing of the blot was performed under low stringency conditions.
3.6. PCR amplification of M. leprae DNA sequence Analysis of the amplification of the 360-bp fragment (Fig. 1) from M. leprae and other DNA was achieved by using PCR primers and conditions as previously described [13]. Briefly, PCR was performed using a Gene Amp Kit according to instructions of the manufacturer (PerkinElmer-Cetus, Norwalk, CT), and 100 ng of purified chromosomal DNA from M. leprae, other bacterial species, and human placental DNA (Table 1) served as a control. An additional set of primers which have been previously characterized as generating a 200-bp PCR product from DNA of most of the species of mycobacteria tested [21] were also added to the reaction. The amplified products of the PCR were analyzed by agarose gel electophoresis and Southern blotting. Southern blots were analyzed for the presence of low numbers of PCR products using a 212-bp probe found within the 360-bp PCR product (Fig. 1).
169 4. R E S U L T S
4.1. Hybridization analysis D N A hybridization analysis of Southern blots containing mycobacterial D N A digested with the restriction endonuclease E c o R I was performed under low as well as high stringency conditions. U n d e r low stringency conditions the 611-bp probe hybridized to D N A of many species of mycobacteria (Fig. 2, 3A, Table 1). M. leprae, M. intracellulare, M. simiae, armadillo-derived mycobac-
1 2 3 4
5 6
7 8 9
10
kb
6
"5
Fig. 2. Autoradiograph of hybridization between 32p-labeled 611-bp probe and EcoRI digests of chromosomal DNA of mycobacteria under low stringency conditions (60°C, 3 × SSC, 0.1% SDS). EcoRI restriction endonuclease digests of chromosomal DNA were separated by electrophoresis through a 1% agarose gel and blotted onto Immobilon-P membrane using alkaline transfer. By lane: 1, M. intracellulare serovar 19, Darden; 2, M. intracellulare serovar 19, W552; 3, M. intracellulare serovar 18, 4990, O'Connor; 4, M. intracellulare serovar 18, Melnick; 5, M. simiae serovar 2, W58; 6, M. simiae serovar 2, W55; 7, M. simiae serovar 1, Krasnow 7729; 8, M. simiae serovar 1, 1595; 9, M. bouis BCG; 10, M. leprae. DNA size markers (1 kb ladder) are shown on the right margin.
terium ( A D M 2), M. kansasii, and M. terrae gave moderate to strong hybridization signals, whereas M. avium, M. scrofulaceum, M. gordonae and M. chelonei produced only weak hybridization signals. In contrast, several other species of mycobacteria ( e . g . M . boris BCG, M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. vaccae, M. smegmatis, M. fortuitum, M. phlei, M. flauescens and M. peregrinum), other bacteria ( e . g . E . coli, Clostridium perfringens, Staphylococcus epidermidis, Rhodococcus erythropolis) and h u m a n placental D N A gave no hybridization signal with the 611-bp probe. To ensure that appropriate conditions (e.g. D N A concentrations sufficient for digestion of genomic D N A with restriction endonucleases, and for Southern blotting) were met for analysis of the mycobacterial D N A testing negative with the 611-bp probe, the same blot (Fig. 3A) was reprobed with the 0.8 kb and 3.0 kb r R N A gene probes common to all mycobacteria and other bacteria tested in this study. Fig. 3B shows that D N A from various mycobacteria and other bacteria which did not give D N A hybridization signals with the 611-bp probe were at optim u m concentration and were properly digested with the restriction enzyme used. By comparison, out of the 18 species of mycobacteria tested, only M. leprae D N A gave a positive hybridization signal with the 611-bp probe under high stringency conditions. Similar results of hybridization with the 611-bp probe (e.g. mycobacterial species giving positive or negative hybridization signals) were obtained when B a m H I was used to digest the D N A from different sources in place of EcoRI, except that the restriction pattern with B a m H I was different from that obtained with EcoRI.
4.2. R F L P analysis The D N A from three species of mycobacteria, M. leprae, M. intracellulare serovars 18 and 19, and M. simiae serovars 1 and 2 was further subjected to detailed R F L P analysis using the 611-bp probe. Six restriction endonucleases, including AluI, AualI, B a m H I , EcoRI, PcuI and SalI were used in separate reactions to digest the mycobacterial DNA. The restriction endonuclease cleavage sites of these enzymes in the 18-kDa
170 protein gene of M. leprae are depicted in Fig. 1. Of the six enzymes, A l u I is most suited to generate D N A fragments of predictable size from the 611-bp fragment. R F L P patterns of M. intracellulare, M. simiae, and M. leprae using E c o R I and A l u I are given in Fig. 2 and 4, respectively (the data for other restriction enzymes are not shown). A l u l cleaves the 611-bp fragment, which includes the 18-kDa M. leprae gene, at three positions generating at least two predictable fragments of 199 and 321 bp (Fig. 1). Both of these fragments were detected in Southern blots of A l u I - d i g e s t e d M. leprae D N A (Fig. 4). In comparison, A l u I digests of M. intracellulare serovars 18 and 19 D N A revealed one fragment of approx. 260 bp and two other fragments of more than 500 bp, whereas A l u I digests of M. simiae serovars 1 and 2 D N A generated fragments of more than 1.6 kb.
R F L P patterns using the 611-bp probe could discriminate M. intracellulare, M. simiae, and M. leprae from each other with each of the six restriction enzymes tested. R F L P patterns also revealed differences between the two strains of M. simiae serovar 1 with all the restriction enzymes tested, except E c o R I . In contrast, the four different isolates of M. leprae obtained from geographically distinct areas of the world revealed identical patterns with each of the six restriction enzymes used (data not shown). The restriction patterns of the three mycobacterial species revealed that apparently the restriction sites in the M. leprae 18-kDa protein gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare, and that each of these three species of mycobacteria have only one copy of the putative gene.
A
kb
12-9-7-5--
1
2
3
4
5
6
B 7
8
9 10 11 12 13 14
kb
12-I 97-
1
2
3 4
5
6
7 8 9 10 11 12 13 14
=
53-3 D
2~ 1.6--
21.6--
1-1-
0-50.5-
Fig. 3. A: Autoradiograph of hybridization between 32p-labeled 611-bp probe and EcoRI digests of chromosomal DNA of mycobacteria, other bacteria and human placental DNA under low stringency conditions. By lane: 1, M. phlei; 2, M. leprae; 3, armadillo-derived mycobacterium(ADM-2); 4, M. fortuitum; 5, M. kansasii; 6, M. tuberculosis H37Rv; 7, M. boris BCG; 8, M. scrofulaceum; 9, M. gordonae; 10, M. smegmatis; 11, M. caccae; 12, E. coli; 13, Clostridium perfringens; 14, human placental DNA. DNA size markers (1 kb ladder) are shown on the left margin. B: Autoradiograph of hybridization between 32p-labeled 0.8 kb and 3.0 kb rRNA gene probes and EcoRI digests of DNA. The Immobilon-Pmembrane previously hybridized with the 611-bp probe (A) was reprobed with 0.8 kb and 3.0 kb probes under low stringencyconditions.
171 2
3
4
5
6
7
8
9
kb
-12
primers which amplify the highly conserved 65kDa protein gene [21], the characteristic 200-bp product was observed for all mycobacterial species analyzed indicating that the D N A was suitable for P C R analysis.
-9 -7
-5 -3
--2 -1-6
-1
--0"5
Fig. 4. Autoradiograph of hybridization between 32p-labeled 611-bp probe and AluI digests of chromosomal DNA of mycobacteria under low stringency conditions. By lane: 1, M. intracellulare serovar 19, Darden; 2, M. intracellulare serovar 19, W552; 3, M. intracellulare serovar 18, 4990, O'Connor; 4, M. intracellulare serovar 18, Melnick; 5, M. simiae serovar 2, W58; 6, M. simiae serovar 2, W55; 7, M. simiae serovar 1, Krasnow 7729; 8, M. simiae serovar 1, 1595; 9, M. leprae. The size markers (1 kb ladder) are shown on the right margin. The size of the two fragments of M. leprae DNA are indicated as follows, * = 199-bp; ** = 321-bp.
4.3. PCR analysis P C R analysis was performed on 100 ng of chromosomal D N A from M. leprae, other bacterial species and h u m a n D N A (Table 1, Fig. 5). W h e n primers designed for amplification of a portion of the 18-kDa protein gene of M. leprae were used, the 360-bp product was amplified only from M. leprae D N A as detected by agarose gel electrophoresis (Fig. 5A) and Southern blotting followed by hybridization with the radiolabeled 212-bp probe (Fig. 5B). In contrast, using the
5. D I S C U S S I O N The 18-kDa protein of M. leprae has been the subject of various studies directed towards identification of an M. leprae-specific antigen which could confer protective immunity against M. leprae infection. It has been shown that both murine and h u m a n T cells are stimulated by the 18-kDa protein [5,6,22]. Initial characterization of the recombinant 18-kDa protein using an M. lepraespecific MoAb, L7.15, indicated that the protein was unique to M. leprae [10]. However, using Western blot analysis, L a m b et al. [1] have recently reported that M. habana has an 18-kDa heat shock protein which shares an epitope with the 18-kDa protein of M. leprae as defined by a MoAb, L5 (same as L7.15). T cell studies by Dockrell et al. [6] also support the concept that the 18-kDa protein of M. leprae has shared epitopes with another mycobacterium, M. boris BCG. In their studies 70% of M. boris BCG-vaccinated E u r o p e a n donors responded in a lymphoproliferative assay to the M. leprae 18-kDa protein. In addition, they were able to establish 18-kDa antigen-responsive T cell lines from a BCG-vaccinated British donor. In this paper, we provide evidence that M. simiae as well as other mycobacteria possess D N A sequences homologous to the 18-kDa protein gene of M. leprae and suggest that the putative gene homolog in these species has only limited sequence homology with the M. leprae 18-kDa protein gene based on D N A hybridization results and R F L P analysis of genomic DNA. In D N A hybridization experiments using a 611-bp probe, which encodes the entire 18-kDa protein gene of M. leprae, D N A from M. leprae, M. simiae, M. intracellulare, M. avium, M. kansasii, armadillo-derived mycobacterium ( A D M 2), M. terrae, M. scrofulaceum, M. gordonae and M. chelonei gave a positive hybridization signal under
172 low stringency conditions, w h e r e a s u n d e r the same c o n d i t i o n s eight o t h e r species of mycobacteria did not b i n d the p r o b e (Table 1). W h e n hybridization a n d w a s h i n g c o n d i t i o n s were altered to increase the stringency, only M. leprae D N A hybridized with the 611-bp probe. T h e s e o b s e r v a t i o n s suggest the p r e s e n c e of h o m o l o g o u s s e q u e n c e s in the above m e n t i o n e d m y c o b a c t e r i a which exhibit partial homology with the 18-kDa p r o t e i n g e n e of M. leprae.
T h e p r e s e n c e of a n 18-kDa p r o t e i n gene homolog in M. simiae serovars 1 a n d 2 is s u p p o r t e d by observations of L a m b et al. [1], who have r e p o r t e d that M. h a b a n a , c o n s i d e r e d taxonomically to be M. simiae 1, has an 18-kDa heat shock protein, which reacts with M o A b L5, previously t h o u g h t to be specific for the 18-kDa p r o t e i n of M. leprae. T h e i r studies also showed that the 18-kDa p r o t e i n could not be d e t e c t e d in extracts of M. tuberculosis, M. boris BCG, M. smegmatis,
B
1 2 3456
A
1 2 3 4 5 6 7 89101112131415
7 8 9 101112131415
bp
-369 -246 -123
b
-360 -200
0
-360
Fig. 5. A: Analysis of polymerase chain reaction (PCR) amplification products from 45 cycles of PCR starting with 100 ng of purified chromosomal DNA, using two sets of primers, one generating a 360-bp product from the 18-kDa protein gene and the other generating a 200-bp product from the 65-kDa protein gene. 50 ~l of PCR product DNA was separated by gel electrophoresis through 2% agarose gel and stained with ethidium bromide (0.05 p.g/ml), a and b denote upper and lower gel respectively. By lane, a: 1, M. fortuitum; 2, M. flavescens; 3, M. gordonae; 4, M. phlei; 5, Armadillo-derived mycobacterium (ADM 2); 6, M. kansasii; 7, M. intracellulare serovar 18, 4990, O'Connor, 8, M. chelonei; 9, M. peregrinum; 10, M. tuberculosis H37Rv; 11, M. tuberculosis H37Ra; 12, M. terrae; 13, M. intracellulare serovar 19, Darden; 14, M. simiae serovar 1, 1595; 15, 123-bp DNA ladder (BRL); three of the size markers of the 123-bp ladder are shown on the right margin of the figure, b: 1, M. scrofulaceum; 2, M. boris BCG; 3, M. intracellulare ATCC 13950; 4, M. smegmatis; 5, M. caccae; 6, Blank; 7, Clostridium perfringens; 8, E. coli B; 9, Human placental DNA; 10, Negative control (H20); 11, Blank; 12, Blank; 13, M. leprae; 14, Blank; 15, 123-bp DNA ladder. The positions of the 200-bp and 360-bp PCR products are shown on the right margin. B: Autoradiograph of hybridization between 32p-labeled 212-bp probe and the DNA from 45 cycles of PCR. The PCR amplification products from DNA of various bacteria were separated by gel electrophoresis (A) and then subjected to Southern transfer and hybridization.
173
M. phlei, and M. fortuitum, when analyzed by Western blotting using the L5 MoAb. In the present study we have observed that the D N A from the above-mentioned five mycobacteria did not hybridize with the 611-bp probe under either high or low stringency conditions. This indicated that either these bacteria do not have the 18-kDa gene homolog, or that the homology between the probe and the putative 18-kDa protein gene homolog is below the level required for hybridization under the conditions employed. Studies are in progress to analyze the possibility of protein expression by the mycobacteria possessing the 18-kDa gene homologs using antibody probes to the M. leprae 18-kDa protein. The R F L P pattern of different mycobacteria using the 611-bp probe revealed additional information of interest. Most species of mycobacteria gave a characteristic profile, and it was helpful in differentiating, for example, M. intracellulare, M. sirniae, and M. leprae from each other. By using different restriction enzymes, it was further possible to identify one serovar from the other in a given species ( e . g . M . intracellulare Type 18 and 19, and M. simiae serovar 1 and 2), and even to identify one strain from another in a given serovar (e.g. the two strains of M. simiae serovar 1). Though these preliminary results are based on a small number of serovars in the species analyzed by D N A hybridization, R F L P patterns with the 611-bp probe may be useful for taxonomic purposes for mycobacteria as a useful adjunct to other parameters being employed for this purpose. We have previously reported the development of a P C R test for detection of M. leprae, based on amplification of a portion of the 18-kDa protein gene of M. leprae [13]. In the present study we have shown that some mycobacteria possess D N A sequences homologous to the M. leprae 18-kDa gene. Therefore the D N A from all the mycobacteria tested in D N A hybridization experiments were also subjected to P C R analysis. The results from this study indicated that D N A from all mycobacteria except M. leprae were negative for the 360-bp M. leprae specific P C R product. Using the 65-kDa protein gene as a target for amplification, a 200-bp product was obtained from D N A
of all mycobacteria tested, indicating the D N A from different sources was suitable for amplification by PCR. These observations suggest that the presence of homologous sequences in various mycobacteria did not affect the specificity of the previously described P C R test. In the present study D N A hybridization with the 611-bp probe provided evidence that some mycobacteria possessed D N A sequences homologous to the 18-kDa protein of M. leprae. Cloning and sequencing of the putative 18-kDa gene homologs would reveal the precise regions of homology and the extent of relatedness between the genes from different mycobacteria. In addition, the R F L P profiles of mycobacteria using the 611bp probe may prove useful for taxonomic purposes.
ACKNOWLEDGEMENTS The authors wish to thank the Heiser Program for Research in Leprosy and the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and Training in Tropical Diseases (grant no. 890096) for the financial support to carry out this work. We thank Dr. Douglas Looker for r R N A gene probes; Dr. A. Tsang for isolates of M. intracellulare and M. simiae; Dr. F. Portaels for ADM-2; Dr. Rubina Patel and Dr. K. Eisenach for purified D N A from some mycobacterial species; Rita Sanchez for help in cultivation of mycobacteria; and Penne Cason for typing this manuscript.
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