Moiecutar Microbiology (1992) 6(2), 153-163

Molecular and immunological analysis of a fibronectinbinding protein antigen secreted by Mycobacterium leprae J. E. R. Thole/* R. Schdningh,' A. A. M. Janson,' T. Garbe,^' Y. E. Cornelisse,' J. E. Clark-Curtiss,^ A. H. J. Kolk," T. H. M. Ottenhoff/ R. R. P. De Vries^ and C. Abou-Zeid^ ^Department of Immunohaematotogy and Bloodbank, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands. ^MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital. Ducane Road. London W12 OHS. UK. ^Department of Biology and Moiecutar Bioiogy, Washington University, St Louis. Missouri 63130, USA. "A/. H, Sweltengrebet Laboratory of Tropical Hygiene. Royal Tropical Institute. 1105 AZ Amsterdam, The Netherlands. ^Department of Medicat Microbiotogy, University College and Middlesex Schoot of Medicine, London W1P 7PN, UK. Summary By screening a Mycobacterium leprae ?.gt11 genomic DNA library with leprosy-patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et ai, 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectinbinding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327-amino-acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of muitipie genes of the 85 complex in the genome of Af. leprae. The amino acid sequence displays 75-85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and Af. kansasH. Furthermore, antibodies to the antigen 85 complex of Af. tuberculosis and Af. Received 5 July, 1991: revised 7 October, 1991. 'For correspondence. Tel. (71) 261737; Fax (71) 216751,

bovis BCG reacted with two fusion proteins containing the amino acid regions 55-266 and 265-327 of the Af, leprae protein. The Af. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T ceils derived from healthy Individuals and leprosy patients. Amino acid regions 55-266 and 265-327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin-bindJng sites on the Af. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may aiso have a biological role in the interaction of this bacillus with the human host. Introduction Mycobacterium leprae is the aetiologic agent of leprosy, and a spectrum of clinical symptoms is seen amongst those individuals who develop the disease, reflecting differences in their cell-mediated immune response to the pathogen. The availability of individual antigens of M. leprae is one of the prerequisites for identifying and characterizing the antigenic determinants that may be involved in immunopathological and/or protective responses during infection. By probing a A.gt11 expression library with monoclonal antibodies against M. leprae proteins (Engers et ai. 1985). a number of antigens have been isolated (Young et ai, 1985). These antigens have been characterized and a number of B- and T-cell epitopes were defined; their specific role(s) in protection or immunopathology, however, remain to be established (Young et ai, 1990; Thole efa/.. 1990). Although the use of mouse monoclonal antibodies has been successful for the identification of proteins recognized by the human immune system, sera from leprosy patients have been shown to recognize a larger array of M. teprae antigens (Chakrabarty et at.. 1982: Klatser et ai. 1984; Ehrenberg and Gebre, 1987), These differences may arise because monoclonal antibodies generafed in BALB/c mice recognized only the antigens which are immunodominani in this mouse strain. Thus, the proteins which were identified do not necessarily represent

154 J.E.R. Thole e\a\.

1 CroLac2 E

327 30/31 kDa gene E E

PIHB1004

ANTIGEN EXPRESSION

(129kDa}

55

266 E

PIHB1006

(120 kDa)

265 327 E plHBiOII 266

Fig. 1. Map of recombinant expression plasmids containing various parts of the 30^31 kDa antigen 85 complex gene of M leprae The open box represents the crolacZganB of the vector pEX2, Pq and P| denote Ihe left and righl promoter of bacteriophage \. present in the vectors pEX2 (ptHBIOCM and 1006) and pPLc236 (plHBIOl 1), re^jectively. The arrow indicates the direction of Iranscriplion, The numbers in ihe 30/31 KDa antigen 85 complex gene (stippled box) represent the amino acid residues in the protein sequence, which delimit Ifie Iruncaied parts ol the gene. E indicates the restnction nuclease f coRI, The molecular masses (in kllodallons) of the expressed antigens are indicated on the rtghl.

0.5 kb

every singie antigenic component of M. teprae that participates in human immune responses. Furthermore, secreted proteins are essentially absent from the mycobacterial extracts used for mice immunization and may not be identified by these monoclonal antibodies. Antigens secreted by mycobacteria during growth may become available for immune recognition at an early stage of infection and may make an important contribution to protective immunity. Several protein antigens secreted by mycobacteria have been identified by analysis of culture filtrates (Verbon etai, 1990; Nagai etai, 1991). Major proteins found in these filtrates are the so-called antigen 85 complex proteins, which consitute a group of similar proteins of approximately 30 kDa. Several components of this complex are secreted by individual mycobacterial cells, and they may also be localized at the cell surface (Abou-Zeid etai. 1988; Ratliff etai, 1988; Barnes etai, 1989; Nagai etai. 1991), Three components of Mycobacterium tubercutosis and Mycobacterium bovis BCG, designated 85A, 85B and 85C. have been purified and characterized in more detail (Wiker et ai, 1986a.b: 1990; Nagai et ai, 1991), and the genes coding for the A and B components have been cloned and sequenced recently (Borremans et ai. 1989; Matsuo e^ ai. 1988: De Wit et ai, 1990). An interesting feature of the antigen 85 complex is its ability to bind to fibronectin (Abou-Zeid et ai. 1988: Ratliff etai, 1988). Obviously, the antigen 85 complex components of M. teprae have been difficult to define so far because of the inability to grow this mycobacterium in vitro. We have recently selected from an M. leprae X ^ I I library 50 recombinant clones that expressed antigenic determinants recognized by pooled serum samples from lepromatous (LL) or tuberculoid (BT and TT) leprosy patients (Sathish etai. 1990). These recombinant dones were divided into 22 different groups, each representing an antigen(s) not previously identified. Here we report further molecular, immunological and functional studies of an antigen expressed by recombinants from one of these

groups, designated group IV. and show that this antigen is a member of the M. teprae 30/31 kDa antigen 85 complex. Results Motecutar anatysis of group IV recombinants and expressed antigens Subctoning and DNA sequence anatysis. As previously described by Sathish et ai (1990). group IV clones isolated from the M. teprae ?igt11 library consisted of four recombinant phages — L7. L27, L35 and L44. These clones were shown to be related to each other on the basis of DNA hybridization of their EcoRI inserts of 0.5. 1.4. 1.9 and 2.1 kb respectively, and all expressed seroreactive proteins of 129.120. 38/42 and 120 kDa. In order to characterize this group of clones, we have subcloned EcoRI recombinant inserts into expression plasmids, after amplfication by the polymerase chain reaction {Fig, 1). Analysis of the amplified DNA insert of phage L44 after digestion with the restriction endonuclease EcoHl revealed the presence of 1.3 and 0.8 kb fragments, indicating the presence of an internal EcoRI site within the original 2.1 kb DNA insert of this phage clone. The amplified 1.9 kb DNA fragment of phage L35 consisted of fragments of 1.3 and 0.6 kb after digestion with EcoH\ (data not shown). The 0.5 and 1.3 kb EcoRI DNA fragments derived from >.gt11 recombinants L7 and L44 were subcloned into the EcoR\ site of pEX2. resulting in the construction of plasmids ptHB1004 and plHB1006. Sodium dodecyl sulphate/poly acrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis showed that, after induction of the thermo-inducible PR promoter, both plasmids gave high expression of the fusion proteins of the expected size (129 and 120 kDa) that were recognized by a pool of serum samples from LL leprosy patients (data not shown). A 1.3 kb EcoRI DNA fragment derived from recombinant L35 was subcloned into the

Fibronectin-binding protein antigen secreted by Mycobacterium leprae

TC COT CTC a/K

CCA C f f CTG GOC TAT TCG CAG (Wg A l i ^ r c TOC CO« QAO CAC COA CAA CAT

S/O ia ATT A M TTT ATG ATT OAC OTO AOC 0 0 0 AAO ATC COA OCC TOO OOO -H i P V S f K I K A I f Q 152 TOO CTT TTG OTO GOT GCA OCT OCO ACT CTO CCO AOC CTA ATC AOC CTT OCT QOC OOA

COC K 1 » OCO

155

Fig. 2. Nucleolide and derived amino add sequence of the M. leprae 30/31 kDa aniigen 85 compiex gene. Boxes indicate potennal -10 and -35 promoter, and Shine-Dalgarno sequences. The underlined pan represents the putative signal sequence. These sequence dala will appear in the EMBL/GenBank/DDBJ Nucieotide Sequence Data Libraries.

212 24, GCG ACC OCA AOC CCC TTC TCA COA CCA OOC CTA CCC OTC OAO TAC CTA CAO OTTl CCO TCO A T A S A F S R P O L P V t r L a v P S JT2 OAO OCG ATO OOO COC AGC ATC AAO GTO CAO CTT CAA AAC OOC OGA AAC OOC TCT CCO E A H O R S I K V Q L Q N O O H O S P A 332 GTO TAT CTG CTG GAT GGT TTG CGT OCO CAO GAC GAC TAT AAC GOC TOO OAC ATC AAC V Y L L D G L R A Q D D r H O W D I H T

38!

302 GCO 3et ACC

„,

TCC OCA TTC GAG TOO TAC TAT CAO TCG GOA CTC TCO OTC OTO ATO CCO OTC OOT 000 CAA S A F E W Y Y f l S G L 3 V V H P V 0 0 < » 452 4a, TCC AGC TTC TAC AOC GAC TOO TAC AOC CCA GCO TOC OOC AAO OCA OOT TOC ACO ACC TAC S S P y S O W V S P A C O K A G C T T T 512 542 AAG TGG GAA ACA TTC CTT ACT AGC GAG CTG CCT AAA TGC OTA TCC OCC AAT AOG AOT OTC K W E T F L T S E L P K C V 8 A H R S V

B72

eo»

AAA TCC ACC OGC AGC CGC GTG GTC GOC CTC TCO ATG OCC OOT TCC TCG OCC CTA ATA CTO X S T G S 8 V V 0 L S H A G S 8 A L I L

"2

Ml

GCA OCT TAT CAC CCC OAT CAO TTC ATC TAT OCT OOC TCO TTO TCO OCG CTG ATO OAC TCC A A Y H P D Q F i r A O S L S A L H D a 092 Tit TCC CAG GGG ATA OAA CCC CAO CTA ATC OOC TTO OCG ATO OOT OAT OCT GGT GOC TAC AAS S Q G I e P Q L I O L A H O D A G G Y E 182 in OCC GCG GAC ATO TOO OOA CCA CCA AAT OAC CCG OCC TWl CAA COA AAC GAC CCC ATT CTO A A D N N O P P H D P A H Q B H O P I L •13 %M CAG GCT 0 0 0 AAO CTO GTC GCC AAC AAC ACC CAC CTA TOO OTT TAC TOT OOT AAC OOC ACA 9 A G K L V A N H T K L H V Y C 0 H 0 T «TI ,0, CCG TCA GAG TTG GGT OGA ACC AAC GTA CCC GCO OAA TTC CTO OAO AAC TTC OTO CAC OOC P S S L O O T N V P A l F L l M f V H O a32 SIS AOC AAC CTA AAO TTC CAO OAC OCC TAC AAC OOT OCT QOT OOC CAC AAC OCT OTO TTC AAC S

H

L

I

F

Q

O

A

T

N

O

A

O

O

I

H

Iti

A

V

F

H

loas

CTC AAT OCC OAC OOA ACG CAC AOC TOO OAO TAC TOO OOA OCC CAO CTC AAC OCC ATO AAO L » A D G T H S " « V « * O A Q L I I A N I 1091 lOII CCC GAC CTA CAG AAC ACC TTO ATG OCT OTA CCC COC AOC OOT TAO CCT OOC CCT OTC TOT P D L Q N T L H A V P i t O

FcoRI site of pPLc236, resulting in plasmid plHBIOII. Temperature induction of the inducible PL promoter did not result in detectable expression of the expected products (of approximately 38/42 kDa; data not shown), and this recombinant was used only for DNA sequence analysis. A set of synthetic oligonucleotides was used to sequence the complete 0.5 kb M. teprae DNA fragment of plHB1004. the 5' 204 bp of the 1.3 kb fragment of plHB1006, and the 3' 881 bp of the 1.3 kb fragment of piHB1011 (Fig. 1). The assembled sequence of 1088 bp of these partly overlapping DNA sequences is depicted in Fig. 2. The 0.5 kb fragment of plHB1004 in fact consisted of 636 bp. and was identical to the 3' 636 bases of the 1.3 kb fragment of plHB1011. Both sequences have an

EcoRI site at their 3' end. Comparison with DNA sequences from other mycobacteria (see below) indicates that this EcoRI site is at the same position as the EcoRI site at the 5' end of the 204 bp sequence from the 1,3 kb fragment in plHB1006, Thus, the insert DNA of p l H B I o n (and p!HB1004) is contiguous with fhe insert DNA of plHB1006. only overlapping at this EcoRI site. Since the EcoRI site was found at the same position in three independent clones, we concluded that this EcoRI site must be present in the M. teprae genome and could not have been added when the library was constructed. An open reading frame corresponding to the reading frame of the expressed cro/acZgene fusions of plHB1004 and plHB1006 was found beginning at position 3 and ending at position 1064 (Fig. 2). It encodes a polypeptide

156 J.E. R. Thole elal M I O V S * G K I R A W G R W L > * * L -QL R - A V T G M S - R - V V G A

20

A Y H P O O F I Y A G S L S A L M O S S I - . - Q . - V A M - G - L - P -

200

V I -

40

QGI - A M - - M - - M

220

BO

A D M W G P P N D P A N O R N O P I

G -

A T -

A -

A L -

T « A A

« « V A

« L P S L I » V S G - V V - G A - - G - V

S G V G

L A G -

A V -

G G A A - - T - - - - - - - -

T A S A F S R P G L P V E V L O V P S E

— - G - - . - . - - - - — --...-..--p - - G - - - - - - - - - - - - - - - - P --G A

E P O G - T G - S G - S

L -

I -

G -

L -

A -

H G D A G G Y K A - - - - - - - - - - - - - - T L - - - - - - T L LQ

2«0

S - - T - - S E - - - T - - - - - L - N S S - - - W E - - - - T O S . - s s - . _ K - R - - - S - H

AHGRSIKV01.QNQGNGSPAV S D F-S--A'N L S D-L-'F-S D-.__ - - - - - - L - - F - S - - D O - - - -

80

V L L O G L R A Q O D Y N G W O I N T S --FS P - _ . . . . _ - . - _ . ____p _p

100

A F E W Y Y Q S G L S V V M P V G G Q S

120

A G K L V A N N T H L W V Y C G N G T P V - - - 1 - - - - P V - - - - - - - K ip

l«0

M E T F L T S E L P K C V S A N R S V K

ISO

R--I

S E L G G T N V P A E F L E N F V H Q S ..DN - L - - K - - - G - - R T N - - - - A - I - - - - - R S - - - - - A - - - - - - - - - - - R S -

200

N I K F O D A Y N G A G G H N A V F N L

300

_ _ _ _ - _ _ _ L P S F Y S D M r S P A C G K A G C T T Y K . Q - . - _ . 0---

280

N P P D

....p

A O G T H S W E Y W G A Q L N A H K P D S - - - - - - P N - - - - - - - -Q -N-.._.Q

D L O N T L H A V P R S G « » « » « « - - - R A - G - T - N T - P A P Q G A

320

340

S T G S R V V G L S M A G S S A L I L A P - - - A - - - - - - - A - - - - T - P---AAI -----M P ..AA--I----------S Rg. 3. Comparison of amino acid sequences of the 30/31 kDa antigen 85 complex of mycobaderia. The sequence derived from the M. leprae gene is depicted in the single-letter code (A) and aligned with the sequences for the A components of M. tuberculosis/M. bovis BCG (8: De Wil etal., 1990). and the Bcomponent of M, bows BCG (C; Matsuo e/a/,. 1988) and M./(a/isaSH{D; Matsuo e/a/.. 1990), Identical residues are given by dashes, and gaps Ihat had to be introduced to maximize sequence alignment are indicated by asterisks.

of 354 amino acids. ATG start codons corresponding to this reading frame were found at positions 84. 249. 425. 578, 654, 698, 731, 1017 and 1044, At position 11 and position 35 the sequences TCGACA and TTAAATT, very similar to -10 and -35 consensus promoter sequences, were found. A purine-rich sequence (AAGGGG) with similarity to the consensus Shine-Dalgarno motif, was found at position 68. Identity with antigen 85 complex molecules from other mycobacterial species. Comparison with DNA sequences from other mycobacteria revealed extensive identity with the genes encoding the 30/31 kDa antigen 85 complex of /W, tubercuiosis. M. bovis BCG and Mycobacterium kansasii. in M. tuberculosis and M. bovis BCG, this complex comprises three components designated A, B (also referred to as the aipha antigen), and C, which are highly similar but also have distinct structural differences (Wiker et ai. 1990). The derived amino acid sequences for the 85A component of M. tubercutosis/bovis BCG, the 85B components of M. bovis BCG and M. kansasii, and the M. teprae homologue described here are compared in Fig, 3. On the basis of this comparison, we assume that the ATG at position 84 is the most probable start codon for translation of the M. teprae homologue. This indicates that the M. leprae gene encodes a sequence of 327

amino acids, consisting of an W-terminal consensus signal peptide of 38 amino acids and a mature protein of 289 amino acids. The calculated molecular weight of the mature protein is 31 029 and this value corresponds to those reported for other antigen 85 complex components. The amino acid sequence data indicated that there is approximately 75-85% identity with the antigen 85 complex from other mycobacteria. and the highest identity value (84%) was found with the alpha antigen of M. kansasii. Most differences were found among the signal peptides. and at the C-terminal ends of the sequences. The lengths cf the mature proteins vary up to 10 amino acids between the different members of the 30/31 kDa antigen 85 complex. The identification of an M. leprae homologue of the 30/31 kDa antigen 85 complex was confirmed by Western blotting analysis using antibodies to the 85 antigen complex of M. tx}vis BCG (Fig. 4). Polyclonal antibodies to the BCG 85 complex recognized the fusion proteins expressed by plHBiOO4 and plHB1006 that span amino acid regions 55-266 and 265-327 of the M. teprae protein (Fig, 4A). With antisera to the purified A. B or C components of M. bovis BCG. a variable reactivity was found. Antibodies to component B recognized both fusion proteins (Fig. 4C). whereas antibodies to component A reacted only with the fusion protein expressed by

Fibronectin-binding protein antigen secreted by Mycobacterium leprae

157

Fig. 4. Westem blot analysis of expression of the M. teprae 30/31 kDa protein in E coh using ar)tibQdles to the M. bovis BCG antigen 85 complex. Lanes 1, 2 and 3 in all panels are protein extracts from £ C0//P0P2136 carrying pEX2, plHB1004, and plHB1006, respectively, Immunoblots were incitbated wilh poiyclonal antiserum raised againsi the BCG 85 complex (A), the A component (8), the B component (C), or the C componenl (D) of this complex. Molecular size markers (in kiiodallons} are indicated on the left.

kDa 205-

11697.466 -

plHB1006 (Fig. 4B), Antibodies to component C oniy reacted with the fusion protein expressed by plHB1004 {Fig, 4D), These results indicate a difference in the B-ce!i epitopes shared between the M. teprae homoiogue and the A, B and C components of the antigen 85 compiex of M. bovis BCG. Southern biotting analysis. In M. tuberculosis and in M. bovis BCG. three separate genes are proposed to encode the three components of the antigen 85 complex. In order to investigate the possible existence of multiple genes in M. teprae. Southern blotting was applied using the 636 bp EcoRI fragment from plHB1004 as a probe for genomic DNA from M. tubercuiosis and armadillo-derived M. leprae that were digested with a variety of restriction endonucleases (Fig. 5). The M. teprae DNA probe hybridized under conditions of high stringency to single fragments of M. leprae DNA resulting from digestion with Pst\, Nco\. or EcoRI (Fig, 5A), suggesting that the M. teprae genome contains only a single copy of this gene. However, if washing was carried out under reduced stringency (Fig.

5B), one or two additional fragments hybridized but the signal was at least 10 times weaker, according to densitometric analysis. This suggest that there are other M. teprae genes related to the 30/31 kDa protein gene sequenced in this study, interestingly. Southem hybridization with M. tubercutosis DNA gave three strong hybridizing fragments with most restriction digests, under identical reduced stringency washes (Fig, 5B, lane 3. and 5C). These data indicate that the M. teprae gene described here is more homologous to these genes in M. tubercutosis than to other members of the antigen 85 complex in M. teprae.

Functionat anatysis of the M. leprae 30/3 / kDa antigen 85 comptex Binding to fibronectin. We have previously shown that fibronectin bound to all three purified components of the antigen 85 complex of M. tubercutosis and M. bovis BCG (Abou-Zeid etai, 1988; C, Abou-Zeid. unpublished). To test whether the M. leprae homologue also has this

ttb

23.19.46.64.42.3O

0.6-

12 3 4

5 6 7

12 3 4 5 6 7

2 3 4

5 6 7 8 9

10

Rg. 5. Identification of the At, leprae 30/31 kDa protein gene on genomic DNA from Af, leprae and Af. tuberculosis. Plasmid DNA from plHB1004 and genomic DNAs from M. leprae and M. tuberculosis were restricted, loaded on a 0.7% agarose gel and electrophoresed overnight at 30 V. The gels were blotted and hybridized wiih the ^^P-labelled, 623 bp EcoRI fragment from plasmid plH6i004 representing an internal DNA fragment from the M. leprae 30/31 kDa protein gene. Washing was carried out under high stringency (A) or under less stringent conditions (B and C). A and B, The DNA samples were Ihe following: EcoRI-digestedplHB1004{!ane 1). H/ndllldigested bacteriophage X (lane 2), EcoRIdigested M. tutierculosis (lane 3). two preparations from Af. leprae digested with EasRI (lanes 4 and 5). A/col digested Af, leprae (lane 6), and Ps(l-digested M. leprae DNA (lane 7). C. The two DNA preparations from Af. tuberculosis shown in adjacent lanes were digested with Not\ (lanes 1 and 2). Nco\ (lanes 3 and 4), Xho\ (lanes 5 and 6), Sph\ (lanes 7 and 8), and EcoRI (lanes 9 and 10).

158

J.E. R. Thole ex al data indicate that this antigen contains major B-cell epitope(s) which were recognized by antibodies in sera from LL patients.

Rg. 6. Binding of fibronectin to the M. lepraa 30/31 kDa recombinant fusion proteins. Western blots prepared from £ co'(POP2136 carrying pEX2 (lane 1), plHB1004 (lane 2), and plHB1006 (lane 3) were incubated with (ibronectin (25 \IQ ml"') and probed with peroxidase-conjugated rabbit anti-fibronectin antibodies (1/1000). No reacbvity was seen in control immunoblots probed with only peroxidase-conjugated antifibronectin antibody.

fibronectin-binding capacity, we have probed Western blots with fibronectin, as shown in Fig, 6. Both fusion proteins expressed by piHB1004 and by plHB1006 reacted with fibronectin, with no binding being seen in control extracts, indicating that the M. teprae protein carries at least two fibronectin-binding sites (one in the 55-266 amino acid sequence and one in the 265-327 amino acid sequence). Immunotogicat analysis of the M. leprae 30/31 kDa protein

T-cett response. To investigate whether this apparently major B-cell antigen of M. teprae is able to elicit T-cell responses, we tested the proliferation of a panel of M. /eprae-reactive T-cell lines derived from healthy individuals and TT leprosy patients in the presence of the fusion protein expressed by plHB1004. In healthy individuals and in TT leprosy patients reactivity was found with the M. teprae recombinanf antigen as well as with the 30/31 kDa antigen purified from culture filtrate of M. tuberculosis, and in Table 1 the responses of two representative lines are shown. In addition, a T-cell clone derived from the line from the healthy individual was found to respond to these two antigens. These data indicate that the 55-266 amino acid region of the M. leprae and the M. tuberculosis 30/31 kDa antigen carry cross-reactive T-ce!l epitope(s) recognized by patients and healthy individuals.

Discussion The 85 complex of mycobacterial antigens was designated according to the BCG crossed immunoelectrophoresis reference system (Closs et ai, 1980}. The complex consists of a group of closely related proteins with molecular masses of approximately 28 to 32 kDa (Wiker etai, 1986a,b) that are able to bind to fibronectin (Abou-Zeid ef ai. 1988). Two-dimensional gel eiectrophoresis indicated that in culture fiitrates of M. bovis BCG and M. tuberculosis up to five components of this

Table 1. Proliferative responses of a Af, leprae reactive T-cell line derived Irom a TT leprosy patient, and a T-cell tine and a T-cell clone derived from a tiealttiy individual with the 30/31 kDa antigen. Heallhy Individual

B-cell response. To analyse the B-cell response to the fusion protein expressed by >.gt11 clone L7 in leprosy patients. Western biotting was employed using serum samples from 20 LL, 7 BT. to TT patients and from four normal individuals. As shown in Fig. 7. the majority (18 out of 20) of the serum samples from LL patients reacted with the recombinant antigen. A reactivity witfi bands whose molecular weights were smaller than the fusion protein was observed and was probably the result of some recognition of degradation products, since this pattern was not found in plaque lysates prepared from other Xgti 1 dones. The protein product was not recognized (or only very weakly so) by the samples from BT and TT patients or from normal individuals. Although the expressed fusion protein (amino acids 55-266) does not represent the enfire M. leprae 30/31 kDa protein, these

Stimulated by: M. leprae M. tuberculosis 30/31 kDa antigen Irom M. tuberculosis E. CO//expressing AA 5&-266 Ot M. /eprae 30/31 kDa pnatein E. CO//control

TT-patient T-cell line

T-cell line

T-cell clone

25,8

21.6 56.1

35.0 60.3

9.2

28.6

24.7

5,2

5^

0,3

0,2

23.8 5.6

9.6

Antigens were: M. leprae sonicate (1 ug ml '), M. tuberculosis sonicate (5 ug ml"'), the 30/31 kDa antigen trom M. tuberculosis (20 jig ml"'), Ihe insolubie traction ot induced E. coli cells carrying plHB1004 enriched tor the fusion protein containing the amino acids 55-266 of the M, leprae 30/31 kOa protein (0,025 to 10 ug ml"'), and the insoluble fraction of induced E. coli cells carrying the vector pEX2 enriched in the cro-belagalactosidase control protein (0.025 to 10 ug ml '), The numbers give the incorporation of pH]-thymidine m 10^ counts per minute. The standard error of the mean was less than 20%.

Fibronectin-binding protein antigen secreted by Mycobacterium leprae

01 03 05 07 09 II 13 15 17 19 02 04 0606 10 12 14 16 18 20

2t 23 25 27 29 31 33 35 37 22 24 26 28 30 32 34 36

LL-patients

BT/TT-patients

159

39 41 38 4O Controls

Fig. 7. Reactivity ot serum samples from leprosy patients and healthy irdividuais with recombinant fusion protein expressed by >.gt11 cione L7, Western blots were incubated with serum samples from 20 lepromatous (LL) leprosy patients (1/500), from 17 tuberculoid (BT/TT) leprosy patients (1/100), and from tour healthy controls (1/100), Theserumsamples were absorbed wilh whole E. co//POP2i36 cells ot an induced culture to remove anti-E co'i and anti-|)galactosidase reactivity.

complex may exist within this complex {Pessolani et ai. 1989). The deduced amino acid sequences determined for A and B components of these species correspond to a protein preceded by a consensus signal peptide. further confirming that these proteins are secreted {Borremans et al., 1989; Matsuo era/.. 1988; De Wit era/,, 1990). The M. leprae gene and derived amino acid sequence described here display strong similarity to previously characterized members of the antigen 85 complex (including the presence of a potential signal peptide). Antibodies to the M. bovis BCG 85A, 85B and 85C antigen components reacted with the fusion proteins expressed in pEX2. On the basis of the antibody reactivity and the sequence identity, the M. teprae 30/31 kDa protein is more closely related to component B of the antigen 85 complex than component A. Furthermore, the M. ieprae protein was found to bind human fibronectin. We conclude from the sequence identify, antibody reactivity. and fibronectin-binding affinity, that group IV serumdefined ?.gf11 recombinants described previously by Sathish etai (1990) encode an M. teprae homologue of the mycobacterial antigen 85 complex. Southern blot analysis with two independent preparations of genomic DNA from M. teprae digested with various enzymes indicated that the M. leprae genome only contains a single copy of the 30/31 kDa protein gene analysed in this study. However, two weak bands were revealed (by using less-stringent washing conditions), that could correspond to at least two more copies of genes expressing less-homologous proteins. The protein products of these genes may correspond fo other

members of the antigen 85 complex or to proteins like MPT51. a 27 kDa protein of M. tubercutosis which crossreacts with the antigen 85 complex, and whose /V-terminal amino acid sequence revealed 60% homology (Nagai et ai, 1991). Preliminary nucieotide sequence analysis of a novel recombinant clone isolated from the M. teprae X.gt11 library using leprosy-patient sera revealed approx. 70% homology to the 30/31 kDa gene described here (T. Rinke de Wit. personal communication). In contrast to the findings with M. /eprae genomic DNA, the FcoRI fragment derived from within the M. teprae 30/31 kDa gene hyridized strongly with three fragments in M. tubercutosis genomic DNA under less-stringent conditions, and these bands probably correspond to the three genes of the antigen 85 complex of M. tuberculosis. It is clear that further molecular analysis is required fo resolve the genetic organization of the antigen 85 complex and related protein genes in M. leprae and in M. tubercutosis. The M. teprae protein shares with other members of the 85 complex the ability to bind to fibronectin (Abou-Zeid ef at.. 1988). In fact, two /W. ieprae protein fragments, the 55-266 and 265-327 amino acid regions, were shown fo bind fibronectin. indicating the presence of at ieast two binding sites. These sites will be furfher mapped by sfudying the interaction of fibronectin with peptide sequences of the 30/31 kDa protein of M. leprae. Regarding the binding domain of fibronectin, preliminary studies using cathepsin D digests of fibronectin indicate that the domain for the 30/31 kDa proteins of M. tuberculosis and M. bovis BCG is close to fhe heparin-binding domain at the carboxyl terminus of fibronectin (C. Abou-Zeid and T. Raftiff.

160 J.E. R. Thole etal unpubiished). Furfher definifion of fhe mycobacterial antigen-binding site within fibronectin is currently under investigation. Live M. teprae bacilli do bind fo fibronectin-coated surfaces (Abou-Zeid et ai. 1988), and the identification of fibronectin binding site sequences could be of particular significance for adhesion of M. teprae to phagocytic and other host cells. Fibronectin has been shown to be involved in the entry of trypanosoma parasites into phagocytic as well as non-phagocytic cells (Ouaissi and Capron. 1989). and to mediate attachment of bacteria like streptococci and Staphytococcus aureus to human tissues (Beachey and Courtney, 1987; Froman etat., 1987). The fibronectin-binding proteins exposed on the ceil surface may play a role in the adherence and uptake of M. teprae by phagocytic or non-professional phagocytic cells, but local accumuiation of secreted fibronecfin-binding molecules could interfere wifh the attachment to cells. Furthermore, mycobacterial fibronectin-binding molecules could infiuence the ability of mycobacteria to survive intracellularly in the host in vivo, since fibronectin was shown to increase adherence and chemotaxis of phagocyfes help mainfain the oxidative bactericidal capacity of macrophages (Proctor, 1987). The recombinant clones studied here were selected from an M. ieprae A.gt11 library using a pool of sera from LL patients, and the majority of the individual serum samples were shown to recognize the 55-266 amino acid region of the M. ieprae 30/31 kDa antigen. Almost no antibodies were detected in serum samples from TT leprosy patients and healthy subjects. This is the firsf report on antibody and T-cel! (see below) reactivity in leprosy patients using the M. teprae 85 compiex antigen. Our findings confirm the data obtained in previous studies in which culture filtrates of M. tubercutosis and M. bovis BCG were used as antigens (Rumschlag et ai, 1988; Pessolani etai, 1989; Das etai, 1990). A doublet protein band of 30/31 or 28/30 kDa, probably corresponding to the antigen 85 complex, was recognized by antibodies from lepromatous patients but not by antibodies from fuberculoid patients or from healthy individuals in close contact with leprosy patients. Furthermore, Britton et ai (1988) reported that antibodies in the sera of LL patients precipitated antigens of 32/33 kDa from radiolabelled extracts of M. teprae. whereas antibodies in the sera of TT patients did not. Other studies have reported on the immunoreacfivity with component A from M, bovis BCG, also designated P32, in tuberculosis patients and tuberculin-positive or negative healthy individuals (Huygen et ai. 1988). They discovered that IgG levels to P32 were found in the majority (77%) of tuberculosis patients but that no P32 antibodies were found in tuberculin-positive or negative volunteers. The highest IgG levels were found in patients with advanced tuberculosis. These data

suggest that both in leprosy and in tuberculosis a massive mycobacterial load and extensive infection seem to be prerequisites for fhe inducfion of antibodies againsf molecules of the antigen 85 complex. Perhaps other factors, too. may play a role in the induction of antibodies fo fhis antigen, since if was reported in one study that no correlation existed between the bacteriological index of leprosy patients at the fime of serum collection and the levei of binding to the 28/30 kDa doublet of the antigen 85 complex (Pessolani et ai. 1989), it might be that the antibody response to this protein correiates with iive persisting baciili, and this wouid be of parficular interest with respect to the use of M. ieprae 30/31 kDa antigen to monitor leprosy treatment. T-cell responses to the antigen 85 compiex in ieprosy patients and in healthy contacts could be detected. We showed that the amino acid 55-266 region of the M. leprae 30/31 kDa antigen and the highly homologous M. tubercutosis 30/31 kDa antigen was recognized by M. /eprae-reactive T ceiis from both healthy individuals and TT patients. We will investigate these responses further at the cionai level to see if different epitope(s) are recognized by T ceiis derived from patients and healthy contacts. The outcome of these studies may indicate the possible role(s) of particular epifopes of the M. teprae 30/31 kDa antigen in pathoiogicai or protective immune responses induced by infection wifh M. leprae.

Experimentai procedures Bacterial strains, phages and plasmids Tfie bacterial strains, phages and plasmids used in this study are listed in Table 2. Strain POP2136 was used as a host for plasmids pEX2 and pPLc236 and their derivatives, Bacteriophage ;*^t11 recombinants were propagated on strain Y1090. LB medium and LB agar were used for growing Escherichia coli K-12 strains (Maniatis ef ai, 1982), Ampiciilin was added as previousiy described (Thole etai, 1988).

Mycobacterial antigens A sonicate of M. leprae (CD135) was kindiy provided by Dr R, Rees (National Institute for Medical Research, London. UK) and a sonicate of M. tuberculosis was a kind gifi from Dr P, Klatser (Royal Tropical'Institute, Amsterdam. The Netherlands), The 30/31 kDa antigen 85 complex was purified from the culture filtrate of M. tuberculosis as described previousiy (Abou-Zeid efa/., 1988).

Antibodies and fibronectin Serum sampies from leprosy patients have been described by Sathish atai. 1990, Control sera were from four healthy Dutch individuals, of wfiom two were BCG vaccinated, Polyclonal rabbit anfibodies against the antigen 65 complex of M. bovis BCG

Fibronectin-binding protein antigen secreted by Mycobacterium leprae and rabbit antisera to tfie purified A, B and C components were a kind gift of Dr M. Harboe, Institute of Immunology and Rheumatology, University of Osio. Norway. Affinity-purified human plasma fibronectin was purchased from the New York Blood Center, New York. USA, Horseradish peroxidaseiabelled anti-rabbit and anti-human IgG immunogiobuiin were from Sigma Chemicals Co,, and peroxidase-label led rabbit anti-fibronectin immunogiobuiin was from DAKO (P246).

DNA analysis and sequencing Restriction endonucleases and T4 DNA-ligase (Promega) were used as specified by fhe manufacturers. Standard procedures were used for the preparation of phage and plasmid DNA. transformation, and transduction (Maniatis etal., 1982), Southern biotting and hybridization were performed as described by Maniatis etat. (1982) using nylon membranes (Hybond-N, Amersham). Washing under reduced conditions was carried out as described by Garbe ef ai (1990), Chromosomal DNA from M. tuberculosis H37Rv was isolated as described by Thole ef al. (1985). Two independent preparations of M. teprae genomic DNA were generously provided by Dr J. Colston (National Institute for Medical Research. London, UK). EcoRI DNA inserts from }jgt11 recombinant clones L7. L35 and L44 were subcloned into the EcoRI site of the expression vector pEX2 using poiymerase chain reaction (PCR)ampiified DNA fragments. The primers GTAATGGTAGCGACCGGCGC (90^98) and GGCGACGACTCCTGGAGCCCG (90-499) were used for amplification of M. teprae DNA inserts from the A,gt11 recombinants. PCR was performed as follows: one isolated plaque was picked with a toothpick, resuspended in 5 |ii distilled water, and boiied for 5 min. To 1 \i\ of the pfiage suspension was added 49 \i\ of a solution of 50 mM KCI, 2.0 mM MgCla. 10 mM Tris/HCi pH 8,3. with 2 mg mi^^ bovine serum albumin. 0,25 mM dNTPs, and 20 pmoi of the primers Taq enzyme (0.5 units; Perkin Elmer Cefus) was finally added, and PCR was performed in a PCR-processor using 35 cycles as follows: 90 s at 9 5 ^ . 120 s af 60-"'C. and 180 s at 72°C, The amplified fragments were piienoi-extracted, digested with EcoRI, and subcloned into the EcoRI site of the expression vector pEX2, Nucieotide sequence analysis was performed on plasmid DNA that had been purified on QIagen Pack 500 columns (Diagen) by the dideoxy chain-termination method (Sanger ef a/,. 1977) using fhe T7 sequencing kit (Pharmacia LKB) according to tfie instructions of the manufacturers. The synthetic oligonucleotide primer 8625-2 that is complementary tc the DNA sequence 12 to 26 bp upstream (8625-2) of the Eco RI site of pEX2 was used fo sequence the M. teprae region fused to the cfo/acZ gene in the pEX2 derivatives plHB1004 and piHBI 006 (Thole ef ai, 1988). A set of primers based on the M. teprae 30/31 kDa gene sequence was consecutively synthesized (GCAAGCGCGTTCTCACGA. TCGTGAGAACGCGCTTGC, CAGCCGTTATAGTCGTCCTG. GGAAACATTCCTTACTAGCG, GATAAGCTGCCAGTATTAGG. GCCAACAACACCCACCTATG. CATGAAGCCCGACCTACA, TGTAGGTCGGGCTTCATG) to determine tfie sequence of the entire 30/31 kDa gene using plasmids plHBI 004. plHB1006 and plHBlOl 1. All primers were synthesized on a Cyclone DNA synthesizer (Biosearch inc.).

161

Production of recombinant fusion proteins Expression of cro/acZhybrid genes from piHB1004, plHB1006 was induced as described (Zabeau and Stanley, 1982). induced cells were suspended in 100 mM Tris-HC! pH 8,0 containing 10 mM EDTA to an absorbance of 20 at 600 nm. freezethawed once, and sonicated at 80 W (tfiree bursts of 30 s) using a Branson sonifier to give complete iysis. For T-cell proliferation assays, iysates of E. coti were centrifuged (10 min, 12000 X g) and the pellets, enriched in fusion proteins, were resuspended in Iscove's modified Dulbecco's medium (IMDM) and stored at -20°C untIi used.

SDS-PAGE and immunoblotting Production of recombinant fusion protein in E.co//lysates was analysed by SDS-PAGE and Western blotting on 6% acrylamide gels. SDS-PAGE was done according to Laemmli (1970). Western blotting of proteins expressed by Xgtii recombinant L7 was done as described by Verbon ef a/. (1990). Processing of nitrocellulose blots with polyclonai rabbit antisera was carried out as described (Abou-Zeid etal., 1988). For the identification of fibronectin-binding fusion proteins, immunoblots were incubated with fibronecfin (25 ng mP') for 2 h at 37"C and probed with peroxidase-labelied rabbit antifibronectin immunogiobuiin at a 1:1000 dilution.

T-celt tines and dones T-cell lines were generated by stimulation of 1 x 10^ peripheral biood mononuclear cells (PBMCs) from ieprosy patients or healthy individuals as described previously (Ottenhoff et ai. 1985). In brief, PBMCs were stimulated with 1 ng mP^ of a M. leprae sonicate; 5d later 20% T-cell growth factor (Biotest, Frankfurt. FRG) was added, and T cells were frozen at day 11 or 12, T-celt clones derived from a T-ceil line from a healthy individual were generated as described previously (Ottenhoff andMutis, 1990),

Protiferation assays T celis (1-1,5 X 10") and 5 x 1 0 " irradiated (20 GY) autologous or HLA-DR-matched PBMCs as antigen-presenting cells (APCs) were cultured in 96-well flat-boftomed microtitre plates (Greiner) in IMDM containing 10% pooled human serum and an optimal concentration of antigen. Control wells contained phytohaetnagglutinin (PHA; 2 ^g ml"'; Wellcome Diagnostics) or IMDM without antigen. Cultures were set up in triplicate and incubated at 37''C in a fully fiumidified atmosphere containing 5% COs for 72 h. For the last 18 h, 1.0 ^Ci of [^H)-thymidine (specific activity. 5.0 |iCi mmoP'; Radiochemical Centre, UK) was added and cells were finally harvested onto glass-fibre filters with a semiautomatic sample harvester. [^H]-thymidine incorporation was assessed by liquid scintillation spectroscopy.

Acknowledgements We are grateful to J. van Leeuwen and S, Gelani for perfect technical assistance, Drs R, Rees and P, Klatser for providing mycobacterial sonicates, Dr M. Giphart for synthezising

162

J.E. R. Thole ex al

Table 2. Bacterial strains, phages and plasmids. Strain/Phage/ Plasmid

Relevant properties

Reference/Origin

Strain £, CO//K-12 lacU169 Ion E. CO//POP2136 ci857 M. tuberculosis Clinical isolate H37RV Ecoft VI090

Young and Davis eraM1983) A. Raibaud Garbe efa/, (1990)

Phage L7 L27 L35 L44

Xgti 1 recombinant containing 0.5 kb M. leprae DNA insert Xgtii recombmant containing 1 A kb M. leprae DNA insert X,gt11 recombinant containing 1,9 kb M. leprae DNA insert ^ g t i i recombinant containing 2,1 kb M. /epraeDNA insert

Sathish e( a/. (1990)

Ap", contains the P, promoter of bactertophage X and cro/acZ genef usion pEX2 recombinant containing 0,5 kb M. leprae EcoRI fragment of L7 pEX2 recombinant containing 0 9kb/W, leprae EcoR\ fragment of L44 Ap'^, contains P, promoter ot bacteriophage X pPlc236 recombinant containing 1,8 kb M. leprae EcoRI fragmeni of L35

Stanley and Luzio (1984)

Sathish e( a/. (1990) Sathish efa/. (1990) Sathish efa/,, 1990

Plasmid pEX2

plHBiOO4

plHBi006

pPtc236 plHBion

This Study

This study

Remaut efa/. (1981) This study

Ap'', ampicillin resistance.

oligonucleotides. Dr M. Harboe for polyclonal rabbit antibodies, and Dr J. Colston for chromosomal DNA from M. teprae. We would also like lo thank Drs G. Rook and D, Young for their helpful discussions. This research was supported by the Dutch Leprosy Relief Association (NSL). the Britisfi Leprosy Relief Association (LEPRA). the Royal Netherlands Academy of Arts and Sciences, the UNDPAVORLD BankAfl/HO Special Programme for Research and Training in Tropical Diseases (TDR) and by Public Health Service grants AI-26186 and AI-23470 from the US National Insfitutes of Health awarded to J.E,C,-C.

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