Departamento de Inmunologí a, Instituto de Investigaciones Biomé dicas, UNAM, Apartado Postal 70228, 04510 México DF, Mexico1
GBF, German National Research Center for Biotechnology, 38124 Braunschweig, Germany2
Hospital General de México, SSA , Mexico3
MRC Tuberculosis and Related Infections Unit, Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London, UK 4
Author for correspondence: Clara Espitia. Tel: +52 5 6223884. Fax: +52 5 6223369. e-mail: espitia{at}servidor.unam.mx
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ABSTRACT |
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Keywords: Mycobacterium tuberculosis, PGRS family genes
Abbreviations: Fn, fibronectin; PGRS, polymorphic GC-repetitive sequence
The GenBank accession number for the sequence reported in this paper is AF071081.
a Present address: Dept of Molecular Microbiology and Immunology, Johns Hopkins University, School of Hygiene and Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA.
b Present address: Dept of Immunology, Kings College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.
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INTRODUCTION |
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Poulet & Cole (1995) described a polymorphic GC-repetitive sequence (PGRS) in the M. tuberculosis genome. This sequence, called orf3', was cloned and sequenced and showed homology with a group of sequences characterized by the presence of short ORFs. With the M. tuberculosis genome sequence now available, two large unrelated families of putative proteins, PE and PPE, have been identified. These putative proteins have conserved N-terminal sequences, with PE and PPE motifs, respectively, present in the majority of them (Cole et al., 1998
).
PGRSs have been used as a genetic markers of polymorphism for epidemiological studies on M. tuberculosis and M. bovis (van Soolingen et al., 1993 ; Romano et al., 1996
; Chaves et al., 1996
; Torrea et al., 1996
; Skuce et al., 1996
; Cousins et al., 1998
; Strassle et al., 1997
), the highly repeated DNA element (pTBN12) of M. tuberculosis (Ross et al., 1992
) and the PGRS DNA from M. bovis (pMBA2) (Bigi et al., 1995
) being the most frequently used. The knowledge of the PGRS polymorphism could prove useful in at least three aspects of mycobacterial biology: (1) sources of genetic variation in M. tuberculosis and its epidemiological and pathological implications; (2) the biological role that such putative proteins might have if they are really expressed, including their pathological and immunological significance; and (3) the evolution of the genome.
A previous report has suggested that at least one member of the PGRS family is expressed in M. tuberculosis: one coding sequence, isolated from an M. tuberculosis expression library by immunoscreening with an antibody raised against the M. bovis 85 complex, encodes a functional fibronectin (Fn)-binding protein (Abou- Zeid et al., 1991 ). The PGRS member Rv1759c from the genome sequence project has been related to this Fn-binding protein (Cole et al., 1998
).
During the screening of a cosmid library of M. tuberculosis directed to the isolation of the coding sequence of a proline-rich protein (Espitia et al., 1995 ), a clone was isolated containing an insert with an ORF encoding a glycine-rich protein of 81·3 kDa (PE-PGRS81). Here we provide evidence that Rv1759c, a close relative of PE-PGRS81, is a new member of the PE- PGRS glycine-rich protein family, is a functional Fn-binding protein and is expressed in tuberculosis infection. To our knowledge, this is the first direct evidence that a Fn-binding protein member of the PE- PGRS family is expressed in M. tuberculosis.
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METHODS |
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Sera.
Sera from patients with pulmonary tuberculosis diagnosed by smear and/or culture of sputum were obtained from the Hospital General in Mexico City. Rabbit antiserum against M. tuberculosis H37Rv was obtained as described before (Espitia et al., 1991 ). Human Fn from Boehringer was labelled with biotin (Boehringer) following instructions from the manufacturer.
DNA preparations.
DNA from M. tuberculosis H37Rv and M. bovis BCG, and from M. tuberculosis clinical isolates, was isolated as described by van Embden et al. (1993) . In brief, bacteria were killed by heating at 80 °C for 20 min, digested with lysozyme and a mixture of SDS and proteinase K, and then treated with CTAB/NaCl (10% N-acetyl-N,N, N-trimethylammonium bromide, Merck, in 7 mM NaCl). DNA was extracted with chloroform/isoamyl alcohol and precipitated with 2- propanol.
Screening of the cosmid library.
A 30-mer oligonucleotide probe based on the N-terminal amino acid sequence of M. tuberculosis proline-rich protein (Espitia et al., 1995 ) was synthesized: 5'-CGC/G CGC/G CGG/C CAC/G CGG/C CGG/C CGC/G CGG/C CTC CGG/C-3' The oligonucleotide was radiolabelled at its 5' end with T4 kinase (Pharmacia) and [ 32P]ATP (Amersham), and used for screening a cosmid library of M. tuberculosis H37Rv in Tropist3 (De Smet et al., 1993
), by colony plaque blotting on nitrocellulose filters. Filters were prehybridized and probed at 42 °C in 6xSSC (900 mM NaCl, 90 mM sodium citrate; pH 7·0), 1 mM sodium phosphate, 1 mM EDTA, 0·05% skimmed milk, 0·5% SDS, for 2 and 4 h, respectively. Afterwards, filters were washed twice in 2xSSC for 15 min each, and once in 2xSSC, 0·3% SDS for 15 min, and autoradiographed by exposure to an X-ray film (Kodak). Ten positive colonies were picked and grown in overnight cultures in LB-kanamycin (25 µg ml-1). Cosmid DNA was isolated using the Qiagen Plasmid Isolation kit following the manufacturers instructions.
Southern blot assays.
DNA samples from the positive cosmid colonies were initially screened by digestion with EcoRI, separated on 0·8% agarose gels and transferred by blotting onto nylon filters (Amersham). The membrane was probed with the labelled oligonucleotide and autoradiographed as described above. From the three cosmid samples that were positive and contained similar inserts, one (randomly chosen) was digested with other restriction enzymes and probed again. A 3·9 kb SalI DNA fragment was purified by excision of the band from the gel using the Stratagene Gene-Clean kit and subcloned for expansion into pUC19; the resulting plasmid was designated pUC19S/3.9. Since the 3·9 kb SalI insert did not contain the complete gene of PE-PGRS81, a 0·5 kb KpnISalI DNA fragment from the cosmid was subcloned in pUC18. The 3·9 kb and 0·5 kb inserts were sequenced in both directions using an ABI 373 DNA sequencer and Prism dideoxy cycle sequencing kit (Perkin Elmer). The sequence data were analysed using the GeneWorks program (IntelliGenetics).
RFLP analysis.
Southern blot assays were also carried out using restriction-enzyme- digested genomic DNA samples from M. tuberculosis H37Rv and M. bovis BCG, and also from clinical isolates of M. tuberculosis. The DNA probes were labelled with the Amersham direct nucleic acid labelling and detection system. The following probes were used. (1) A 3·9 kb SalISal I fragment from pUC19S/3.9, described above, including derived fragments (Fig. 1): a 693 bp KpnI BamHI fragment, which contains 147 bp of a neighbouring PGRS encoding the C-terminus of Rv0279c, 249 bp of noncoding sequence upstream of the start codon, plus 297 bp of coding sequence for PE- PGRS81; and three consecutive fragments of 339 bp (Bam HIBamHI), 1·2 kb (BamHI KpnI) and 850 bp (KpnIKpnI). (2) A 4·0 kb SalISalI fragment from pUC19S/4.0 (Fig. 1
); this fragment was isolated from an M. tuberculosis
EMBL3 library using the coding sequence for a Fn-binding protein (TB1) as probe (Abou-Zeid et al. , 1991
). A derived fragment of this was also used as a probe, the 647 bp PvuIIBamHI fragment containing 219 bp of noncoding region upstream of the start codon plus 428 bp starting at the putative N-terminus of Rv1759c (see below). (3) A 1·27 kb SphI fragment containing the pstS-1 gene cloned from an M. tuberculosis pYUB328 cosmid library was used as a control.
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SDS-PAGE and immunoblotting.
Rv1759c-C recombinant protein (10 µg) was run in preparative 12% SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Each membrane was cut into strips and incubated for 1 h with sera from tuberculosis patients diluted 1/50 or with biotin-labelled human Fn diluted 1/50 in PBS containing 3% BSA and 0·3% Tween 20. After washing, strips were incubated for 30 min at room temperature with protein A/peroxidase diluted 1/2000 (Sigma) and with streptavidin/peroxidase (Zymed) diluted 1/2000 for detection of biotin- labelled Fn. Peroxidase activity was revealed with 3,3'- diaminobenzidine and hydrogen peroxide in PBS.
Phylogenetic analysis.
Alignment of all distinct PE-PGRS family sequences available in the GenBank database was initially performed with the DNAMAN program (version 2.6, Lynnon BioSoft 199497, Montreal, Canada), and then visually adjusted. The alignment is available from the corresponding author. Phylogenetic analysis of the 82 sequences (see Table 1), was performed using the N-terminal 98 residues that could be reliably aligned. Distances were evaluated using the method of Poisson correction, where gaps and missing information data sites were removed only in pairwise comparisons. The tree was generated by neighbour-joining using MEGA (Kumar et al., 1993
). All M. tuberculosis sequence data used in this work were downloaded from the NCBI database (http://www3.ncbi.nlm.nih.gov/Entrez/Genome/).
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RESULTS |
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BLAST-p searches of the predicted amino acid sequence of PE-PGRS81 showed significant identity to the N-terminus of members of the PE protein family. Moreover, the predicted amino acid sequence showed 100% identity to the partial protein sequence encoded by the previously reported orf3' (Poulet & Cole, 1995 ). At the DNA level, there were only two differences between orf3' and PE- PGRS81 in 647 bp overlap. The amino acid sequence of PE-PGRS81 also showed 96% identity with the 83·8 kDa amino acid sequence deduced from the ORF in Rv0278c. A total of 57 differences were found between the coding sequences of PE-PGRS81 and Rv0278c, including the insertion of 12 amino acids (NGGAGGNGGAGG) in position 464 of Rv0278c, encoded by 36 bp in two identical repeats of 18 bp (5'-CAACGGCGGCGCCGGCGG-3'). This insertion contains the triplet consensus sequences present in PGRSs described elsewhere, CGGCGGCAA (Ross et al., 1992
; Doran et al., 1993
; Poulet & Cole, 1995
).
The putative site for ribosome binding, GGAGG, described by Poulet & Cole (1995) , is present in 38 of the 82 PE glycine- rich protein sequences; 40 show similar sequences and 4 do not have the consensus sequence (Table 1
), raising the question whether these proteins are expressed in mycobacteria. The same consensus sequence has been reported for protein genes of M. tuberculosis, M. leprae and M. paratuberculosis (Dale & Patki, 1990
; Bannantine et al., 1997
), as well as in some Streptomyces protein-coding genes (Strohl et al. , 1992
).
RFLP analysis
RFLP assays were done on membranes blotted with PvuII- restricted genomic DNA of M. tuberculosis, M. bovis BCG and M. tuberculosis clinical isolates, using several PGRS restriction fragments as a probes (Fig. 1). A polymorphic pattern of about 20 positive bands was observed in all cases when with full-length PE- PGRS81 was used as probe (Fig. 2a
). An identical pattern was also observed when blots were probed with a variety of fragments from PE-PGRS81 or with the full-length Rv1759c (not shown). In contrast, only one hybridization band of about 3·3 kb was detected in five out of seven clinical isolates with the 647 bp PvuIIBamHI probe, corresponding to the conserved N-terminal region of Rv1759c (Fig. 2b
). Since Rv1759c contains a fragment of the same size flanked by PvuII sites (Fig. 1
), the 647 bp probe is specifically annealing to the Rv1759c gene. These results indicate that the observed polymorphism is due to the GC- rich region that contains the consensus repeats, and suggest that the same basic sequence is responsible for the polymorphism in all PE- PGRSs. Hybridization of the specific M. tuberculosis complex probe with the clinical isolates confirmed that all of them were M. tuberculosis strains (Fig. 2c
).
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Expression of the C-terminus of Rv1759c
The 1·5 kb (SmaISalI) fragment from pUC19S/4.0 used for expression encodes the putative C-terminus of a Fn-binding protein. The recombinant product was expressed as a fusion protein with polyhistidine purification tags. After purification, the recombinant fragment of about 38·5 kDa (Fig. 3a) was recognized in Western blotting assays by antibodies present in 6 out of 12 sera from tuberculosis patients (Fig. 3b
). In contrast, it was not recognized by a rabbit hyperimmune serum raised against a crude extract of M. tuberculosis H37Rv. The recombinant fusion product on the membrane also bound biotin-labelled Fn (Fig. 3b
).
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Phylogenetic analysis of the 82 sequences, performed on a stretch of 98 amino acids at the N-terminus, that could be reliably aligned, resulted in a tree that suggests a complex evolutionary history for the PGRS family (Fig. 4). Eight groups were arbitrarily defined on the basis of sister branching in the tree. The potential protein PGRS81 fell within group 1 in a small subgroup including orf3', Rv0278c, Rv0279c, Rv1759c, Rv3652 and Rv0747.
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DISCUSSION |
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PGRSs have been widely used as markers of polymorphism in epidemiological studies of the M. tuberculosis complex. Comparison of previously described PGRSs with the sequences in the database showed that they share a very high identity with members of the PE-PGRS family. They only differ from those in the database by deletions/insertions and single nucleotide changes. For example, pTBN12, with an insert of 1111 bp, isolated from genomic DNA of M. tuberculosis H37Rv, showed 93% identity (14 single changes and one insertion of 64 bp in Rv3388) with the C-terminus of Rv3388; pMBA2, a 746 bp fragment, cloned from an Argentinian isolate of M. bovis, has 94% identity (8 single nucleotide changes and a deletion of about 37 bp in Rv0297) with the C- terminus of Rv0297; and MBHR, a 2365 bp fragment, cloned from a genomic library of M. bovis AN5, has 94% identity (14 single nucleotide changes and an insertion of 164 bp in Rv1803c) with Rv1803c. Finally, orf3', a 1435 bp fragment isolated from a cosmid library of M. tuberculosis H37Rv DNA, shows 92% identity with Rv0278c and Rv0279c (which are contiguous sequences). The major changes in these sequences are 15 single changes and a 49 bp insertion following by a 45 bp deletion near to the 3' end of Rv0279c.
From these observations, it is clear that PE-PGRS81, orf3' and Rv0278c on the one hand, and pTBN12 and Rv3388 on the other, all originating from M. tuberculosis strain H37Rv, are highly homologous sequences. The changes (mutations, deletions/insertions) in these sequences could be the result of intergenic or intragenic recombinational events between the repeat regions of these PGRS members (Cole et al., 1998 ). A similar mechanism has been described for protein M in group A of Streptococcus. This protein is a virulence factor and a major surface protein which exhibits size variation in strains of the same serotype. Analysis of variants shows that insertions/deletion mutations arise in a single strain by homologous recombination events. These events can lead to the generation of antigenic variation (Hollingshead et al., 1987
).
The possibility that members of the PE-PGRS family are a source of antigenic variation is relevant in the context of the immune response against mycobacteria. However, no direct evidence on the in vivo expression of PGRS proteins has been obtained. Abou-Zeid et al . (1991) cloned a partial coding sequence from an M. tuberculosis
gt11 DNA expression library, with an antiserum raised against the antigen 85 complex of M. bovis BCG. Proteins from the 85 complex are mycolyltransferases with Fn- binding activity present in all mycobacteria species tested so far (Content et al., 1991
; Soini & Viljanen, 1997
; Belisle et al., 1997
). Recombinant TB1 also binds Fn and is recognized by sera from tuberculosis patients. TB1 appears to be related to Rv1759c, although our restriction enzyme mapping showed that they are distinct sequences (see Methods); the precise identification of TB1 as a PGRS requires further characterization.
It is worthy of note that rabbit polyclonal serum raised against M. tuberculosis H37Rv did not react with the recombinant Rv1759c-C as the patient sera did, suggesting that the protein is expressed in vivo during infection, but not during the in vitro growth of the bacteria against which the rabbit serum was raised.
The relationship between the PGRS and the Fn-binding proteins is also manifested by the annealing of the oligonucleotide designed from a proline-rich protein with the DNA encoding PE-PGRS81. These proline- rich proteins present in M. tuberculosis and other mycobacteria bind Fn (Schorey et al., 1996 ). However, no significant similarity in the amino acid sequence can be found between PGRS and the Fn-binding proteins, although they share short CG-rich stretches in their coding sequence.
There is no homology between the two families of mycobacterial Fn- binding proteins described, including different Fn-binding motifs, FEWYYQ for the 85 complex proteins (Naito et al., 1998 ) and RWFV for the members of the highly homologous Fn attachment protein family (Zhao et al., 1999
). Our finding of Fn-binding activity in Rv1759c suggests that PGRS could constitute a third group of Fn-binding proteins with a distinct Fn- binding motif.
The M. tuberculosis Fn-binding proteins are immunodominant antigens (Huygen et al., 1988 ; Content et al. , 1991
; Espitia et al., 1992
, 1995
) with the capacity to mediate attachment of whole bacteria to Fn- coated surfaces (Ratliff et al., 1988
). Interestingly, antigenic variation in the Fn adhesin has been described in Streptococcus pyogenes (Talay et al., 1994
). Taken together, these observations indicate that putative Fn-binding PGRS proteins could have an important role in hostbacteria interaction.
RFLP analysis using PE-PGRS81 and Rv1759c as probes showed identical polymorphic patterns (number, position and intensity of hybridization bands) for some M. tuberculosis clinical isolates. In contrast, the conserved N-terminus probe derived from Rv1759c identified only one band, indicating that the region of the PGRS which contains the consensus triplet repeats is responsible for the polymorphism, in agreement with previous reports (Poulet & Cole, 1995 ; Cole et al., 1998
). Tandem repeats of CGGCGG, CGGCAA or combinations of both arrangements were found along the coding sequences of PE-PGRS81 and its closest relatives. The polymorphic pattern observed within the 693 bp KpnIBamHI probe coincided with the stretch of 164 bp which is 100% identical to the C-terminus of Rv0279c. Interestingly the deleted region of PE-PGRS81 contains two identical 18 bp repeats, with these repeats indicating that the variation of PGRS could be given by different mechanisms, one involving the presence or not of specific sequences and the other involving changes due to insertions/deletions in the repetitive regions. In terms of the evolutionary relationships among PGRSs it would be interesting to analyse the rate of changes in clinical isolates of M. tuberculosis and its relevance to pathogenicity.
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ACKNOWLEDGEMENTS |
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Received 21 June 1999;
revised 14 September 1999;
accepted 16 September 1999.