INTRODUCTION

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Tuberculosis (TB) remains a major global health problem and is the most frequent cause of death from a single infectious agent (40). The seriousness of the problem is reflected by the fact that the World Health Organization declared the TB pandemic a global emergency situation in 1993. In the light of the inconsistent efficacy imparted by Mycobacterium bovis bacillus Calmette-Guérin (BCG) (14), the development of an improved TB vaccine is a very high international research priority.

Immunity to TB is mediated by the cellular branch of the immune system, and it has been demonstrated that Mycobacterium tuberculosis culture filtrate proteins are strongly recognized by T cells involved in protection against TB (3, 26). Given as experimental subunit vaccines, culture filtrate proteins promote efficient acquired cellular resistance against the disease (3, 27, 31). Single protective antigens could be included in a future vaccine, as either a subunit or a DNA vaccine or in the form of recombinant BCG expressing these proteins. However, until recently, very limited information on single antigens recognized by T cells was available. By fractionation of extracellular M. tuberculosis proteins into narrow molecular mass fractions we have previously identified two regions that are strongly recognized and stimulate proliferation and gamma interferon (IFN-) production in T cells isolated in the first phase of infection from various species (4, 6, 12, 29). In the low-molecular-mass fraction (5 to 12 kDa), ESAT-6 (for 6-kDa early secretory antigenic target) has been identified as the key target (4). The other mass region (24 to 36 kDa) comprises several well-characterized culture filtrate proteins, including T-cell antigens in the antigen 85 complex (4), which consists of the three proteins, MPT44, MPT45, and MPT59. The present study was undertaken to identify novel T-cell antigens in the 24- to 36-kDa region. This investigation led to the identification and purification of a 29-kDa M. tuberculosis protein, CFP29, which is strongly recognized by mouse memory effector cells. This antigen was characterized, and the corresponding gene was cloned and sequenced. The immunological activity of native as well as recombinant, histidine-tagged CFP29 was evaluated.

	MATERIALS AND METHODS

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Bacterial strains and media. Short-term culture filtrate (ST-CF), which is highly enriched in extracellular M. tuberculosis proteins, was produced in modified Sauton medium (5). Other culture filtrates from mycobacterial species were prepared as described previously (2). Chromosomal DNAs from mycobacterial species were obtained as described in detail elsewhere (39).

Animals and experimental infections. Female C57BL/6J and BALB/c mice were purchased from Bomholtegaard (Ry, Denmark). Memory immune C57BL/6J mice were generated as previously described (7). Briefly, mice received a primary infection with 5 × 10⁴ CFU of M. tuberculosis via the lateral tail vein, after which they were treated with isoniazid (Merck, Rahway, N.J.) and rifabutin (Farmatalia Carlo Erba, Milan, Italy) in their drinking water for 2 months to clear the infection. The mice were rested for a period of 4 to 6 months before challenge with 10⁶ CFU of bacteria intravenously, and the animals were sacrificed on day 4 postinfection.

Protein purification. Narrow molecular mass fractions of ST-CF were obtained by the multielution method (6). Briefly, 6.5 mg of ST-CF was separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was preequilibrated in the elution buffer (25 mM 3-[cyclohexylamino]-1-propanesulfonic acid [CAPS], 37 mM ammonia, pH 10.2). The gel was electroeluted in a Whole Gel Elutor (Bio-Rad, Richmond, Calif.) for 20 min at 40 V, followed by reversal of the current for 10 s. Thirty fractions of approximately 3 ml were harvested from the unit.

MAbs. BALB/c mice were immunized four times at 2-week intervals. MAbs were generated by immunization with SDS-PAGE-purified 26- to 29-kDa proteins from ST-CF in adjuvant R-730 (RIBI ImmunoChem Research Inc., Hamilton, Mont.) (first and second immunizations) or in aluminum hydroxide gel adjuvant (2.0% Alhydrogel; Superfos Biosector, Kvistgård, Denmark) (third immunization and boosting). Spleens were isolated, and spleen cells were fused with the P3-X63 myeloma cell line. Supernatants from wells with visible growth were tested for antibodies by immunoblotting of SDS-PAGE-separated ST-CF. The subclass of the MAb HYB 71-2 was determined with an RPN29 mouse monoclonal antibody isotyping kit (Amersham, Buckinghamshire, United Kingdom) according to the manufacturer's instructions. MAb HBT12, reacting with PstS, has been described previously (24).

SDS-PAGE and 2-DE. Electrophoresis experiments were performed in the Protean IIxi system (Bio-Rad). Standard SDS-PAGE was done with 10 to 20% gradient gels (16 by 16 by 0.075 cm) as described by Laemmli (21). The gels were either silver stained (10) or transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany) as previously described (34). For immunoblot analysis, the nitrocellulose membranes were incubated with mouse MAbs and then with alkaline phosphatase-labeled rabbit anti-mouse antibodies (D314; DAKO, Glostrup, Denmark). Nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate was used as a substrate.

N-terminal sequencing. Separated proteins were blotted to PVDF membranes after Tricine SDS-PAGE. The proteins of interest were excised and subjected to N-terminal sequence analysis by automated Edman degradation with a Procise 494 sequencer (Applied Biosystems) as described by the manufacturer. The EMBL and SWISSPROT databases were searched with the TFASTA and FASTA algorithms (28), respectively.

Cloning and DNA sequencing. Based on the homology to the DNA sequence of the B. linens lin gene (38), the following degenerate primers for PCR cloning of the cfp29 gene were constructed: 5'-CCCGGCTCGAGAACCTYTACCGCGACCTYGCYCC and 5'-GGGCCGGATCCGAYGCYGCGTCCTTYACYGGXTGCCA (where Y is G or C and X is T or C). These primers are derived from nucleotides 6 to 30 and 301 to 327, respectively, of the lin gene.

Interspecies analysis. One microliter of chromosomal DNA (~20 ng/µl) was used as template for PCR with the cfp29 gene-specific primers 5'-GGACGTTCAAGCGACACATCGCCG and 5'-CAGCACGAACGCGCCGTCGATGGC, giving rise to 572-bp DNA fragments. The annealing temperature was 55°C, and the number of cycles was 30. PCR products were analyzed on 1% agarose gels. As positive controls for the PCRs, identical PCRs were set up with 16S rRNA gene primers that give rise to a 1,018-bp DNA fragment with chromosomal DNAs from all the mycobacterial strains tested (11). All chromosomal DNA preparations were positive, as determined by the amplification of the 1,018-bp PCR fragment. Chromosomal DNA preparations that were negative with the cfp29-specific primers were tested again under less stringent conditions by lowering the annealing temperature to 45°C and increasing the number of PCR cycles to 35.

Bacteriocin assay. The spot-on-the-lawn method (9) was used to study the inhibitory effects of the sterile protein samples. To prepare the plates, 0.1 ml of a liquid culture of the indicator organism was inoculated into 6.5 ml of tryptose-soft agar (tryptose broth with 8 g of agar/liter), mixed, and poured into petri dishes (diameter, 10 cm). After solidification of the agar, 10 µl each of sterile filtered ST-CF (4.5 mg/ml), native CFP29 (40 µg/ml), and B. linens Linocin (100 µg/ml) was applied on the agar. The agar plates were incubated at 30°C, and possible inhibition zones were detected by using Henry's lumination (17). The plates were checked for inhibition zones after 24, 48, and 72 h of incubation.

Cellular assays. Spleen lymphocytes were isolated from memory immune mice during the recall of protective immunity as previously described (13). Briefly, cells pooled from three mice were cultured in microtiter wells in a volume of 200 µl of RPMI 1640 medium supplemented with -mercaptoethanol, penicillin-streptomycin, glutamine, and fetal calf serum. Recombinant mouse interleukin-2 (2.5 U/ml; GenZyme, Cambridge, Mass.) was added to all cultures. ST-CF and purified proteins to be tested were all buffer exchanged into compatible buffers before analysis and used at various concentrations (0.5 to 8 µg/ml) in cultures. Supernatants were harvested after 72 h of incubation for detection of IFN- by enzyme-linked immunosorbent assay.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession no. Y12820.

	RESULTS

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Investigation of the highly stimulatory 24- to 36-kDa region of ST-CF. The recall of long-lived memory immunity in the mouse model is characterized by rapid recruitment and activation of memory effector cells directed predominantly to molecules in the 5- to 12- and 24- to 36-kDa regions of ST-CF (4). To identify novel antigens in the 24- to 36-kDa region, ST-CF proteins were separated by SDS-PAGE and divided into narrow fractions by the multielution method, giving rise to six fractions from the selected region (Fig. 1). These fractions were used to stimulate T cells isolated from the spleens of mice during the recall response. Fractions 3 and 4 contained members of the antigen 85 complex as major components, and as expected, stimulation with these fractions elicited a prominent release of IFN- (4). However, large amounts of IFN- were also released in response to fraction 2, indicating that T-cell antigens in the 26- to 29-kDa region were also present (Fig. 1).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   IFN- release from mouse memory effector cells in response to narrow molecular mass fractions of ST-CF. (A) SDS-PAGE, followed by silver staining, of ST-CF and the panel of fractions (lanes 1 to 6). Numbers at the left indicate molecular masses in kilodaltons, and the bands representing the HYB 71-2 antigen (HYB 71-2Ag), MPT51, MPT59, and MPT64 in ST-CF are indicated. (B) The fractions generated were used to stimulate mouse memory effector cells (obtained at day 4 of M. tuberculosis challenge) in culture, and the release of IFN- was measured. All values represent means from triplicate analyses. Standard errors of the mean were not above 10% of the mean.

Purification of a 29-kDa antigen. ST-CF proteins were loaded on an Affi-T gel column for thiophilic adsorption chromatography. Immunoblot analysis with HYB 71-2 demonstrated that the reactive molecule was eluted in the ammonium sulfate concentration range of 0.44 to 0.31 M. The relevant fractions were pooled and applied to a Mono Q column, and this step was very efficient for separation of the 29-kDa protein from other ST-CF proteins; fractions of almost pure 29-kDa protein eluted at between 400 and 450 mM sodium chloride. Final purification was performed by gel filtration on a Superose 6 column equilibrated with PBS, pH 7.4. The reactive molecule eluted near the void volume before any of the marker proteins. In the gel filtration step, minor contaminating bands were removed and the buffer was changed to PBS, which was compatible with the cellular assays. As judged by SDS-PAGE, an at least 90% pure preparation was obtained by this protocol, which provided 40 µg of the purified protein from 400 mg of ST-CF (Fig. 2). The 29-kDa protein was designated CFP29.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Fractions from the different steps used in the purification of CFP29 analyzed by SDS-PAGE followed by silver staining (A) and by immunoblot analysis with HYB 71-2 (B). Lanes: 1, ST-CF; 2, fraction obtained from the Affi-T gel column; 3, fraction obtained after anion-exchange chromatography and gel filtration. Numbers at the left are molecular masses in kilodaltons.

2-DE analysis of CFP29. 2-DE analysis of purified CFP29 revealed a cluster of three distinct spots of the same molecular mass, and all three spots were recognized by HYB 71-2 in immunoblot analysis (Fig. 3A). Tricine SDS--2-DE analysis was performed, and the spots representing CFP29 were excised from the PVDF membrane for sequence analysis. For all three spots, the same two sequences were detected after six cycles: MNNLYR and NNLYRD. This finding confirms the partial cleavage of the N-terminal methionine and suggests that all three spots represent the same gene product.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   2-DE analysis. (A) Immunoblot analysis of the purified CFP29 with HYB 71-2. The three individual spots reacting with HYB 71-2 are marked by arrowheads. (B) Silver-stained gel of ST-CF. The positions of individual proteins are indicated. Numbers at the left of each panel are molecular masses in kilodaltons.

Distribution of CFP29 in subcellular fractions. Many M. tuberculosis culture filtrate antigens are derived from the envelope, from which they are gradually released (5). ST-CF and subcellular fractions of M. tuberculosis were analyzed for the presence of CFP29 and PstS (Table 1). CFP29 was found in ST-CF only in small quantities but was highly enriched in the membrane fraction. In agreement with earlier studies, the PstS homolog, the lipidated 38-kDa protein recognized by HBT12, was located predominantly in the membrane and cell wall fractions. The association between CFP29 and the membrane was evaluated by Triton X-114 phase separation of the cell membrane fraction. CFP29 preferably partitioned in the aqueous phase, whereas the majority of PstS was detected in the detergent phase, in agreement with previously published data (41).

Characterization of the cfp29 gene. Database searches for homology to the N-terminal sequence of CFP29 in EMBL and SWISSPROT by the TFASTA and FASTA algorithms, respectively, identified a B. linens protein, M18 Linocin (accession no. X93588) with 74% identity to the 19 N-terminal amino acids of CFP29. Linocin, encoded by the lin gene, is a potent bacteriocin with activity against Listeria species and other gram-positive bacteria (37).

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Nucleotide sequence of the amplified PCR fragment and deduced amino acid sequence of CFP29. The putative Shine-Dalgarno (SD) site and the amino acid sequence confirmed by N-terminal sequencing are underlined. Alignment of the deduced amino acid sequence of CFP29 and that of B. linens Linocin is shown. A single gap was inserted to improve alignment. Identical amino acid residues are indicated by asterisks.

Comparison of Linocin and CFP29. The deduced amino acid sequence of CFP29 shows 58% identity to that of Linocin (Fig. 4). The deduced molecular mass of CFP29 is 28,860 Da, and the calculated pI is 4.70. In comparison, the calculated mass of Linocin is 28,596 Da, and the pI is 4.41.

Interspecies analyses. The presence of CFP29 in different mycobacterial species was examined at the protein level with HYB 71-2 by immunoblot analysis of mycobacterial culture filtrates. A 29-kDa band reacting with HYB 71-2 was observed in all culture filtrates tested (Table 2).

Immunological activity of CFP29. CFP29 is present in only small amounts in ST-CF, so to obtain CFP29 for immunological studies, recombinant histidine-tagged CFP29 was expressed and purified to homogeneity as described in Materials and Methods.

View this table: [in this window] [in a new window]   TABLE 3.   Mouse IFN- response to ST-CF and antigens from the 24- to 36-kDa regiona

	DISCUSSION

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It is now considered a fact that M. tuberculosis culture filtrate contains targets for protective T cells, and in animal models, experimental vaccines based on culture filtrate proteins induce protective immunity (3, 27).

In the present study, we describe a novel T-cell antigen, CFP29, from the highly stimulatory 24- to 36-kDa region of M. tuberculosis ST-CF. This region contains antigens recognized strongly by memory effector cells in mouse and guinea pig models of TB (4, 16). Furthermore, this region is strongly recognized by Th1 cells in human TB patients with minimal disease (12). Although CFP29 is present in ST-CF in very limited amounts, we were able to isolate enough native CFP29 for a partial biochemical characterization and demonstration of its potent T-cell-stimulatory properties. In cellular assays, native as well as recombinant CFP29 was clearly identified as a T-cell antigen triggering the release of large quantities of IFN- from memory effector cells isolated during the recall of protective immunity in the mouse model of TB infection. However, given the complexity of the reactive 26- to 29-kDa fraction in SDS-PAGE, our identification of CFP29 does not exclude that other, as-yet-unidentified T-cell antigens may also contribute to the prominent release of IFN- in response to this fraction.

It has been suggested that the extracellular proteins most relevant for novel vaccines are the ones appearing in abundant quantities in culture filtrates (19). Since intracellular growth of M. tuberculosis in macrophages induces a significant change in protein expression compared to extracellular growth in culture medium (1, 23), the search for new M. tuberculosis antigens should not be biased by their relative quantities in vitro. The identification of the highly reactive T-cell antigens CFP29 and ESAT-6 (34), both of which are present in small amounts in culture filtrates, demonstrates that important antigens may be overlooked if only the most abundant molecules are characterized. For both antigens, immunoblot analysis with specific MAbs has proven to be a sensitive technique for identification and isolation of proteins which constitute less than 1% of the total protein quantity.

Investigation of subcellular fractions of M. tuberculosis showed that CFP29 is present in the membrane and culture filtrates but not in the cytosol fraction. This leads to the conclusion that CFP29 is exported and probably gradually released from the envelope, making it available for immune recognition in the early stage of infection. The localization of CFP29 emphasizes the relevance of defining antigens from various subcellular fractions of M. tuberculosis, and this matter is currently being pursued as part of our attempts to identify new M. tuberculosis T-cell antigens.

A consensus signal sequence is absent from the cfp29 gene, a phenomenon also observed for other extracellular mycobacterial proteins, like ESAT-6 and superoxide dismutase (34, 42), suggesting the existence of signal sequence-independent mechanisms for translocation of proteins across the bacterial membrane. The partitioning of CFP29 in the aqueous phase indicates a peripheral association of the protein with the membrane, and in agreement with this finding, no transmembrane segments in the amino acid sequence of CFP29 were identified by the algorithm of Klein et al. (20).

2-DE analysis of CFP29 showed three different spots in the pI range of 5.0 to 5.2 with the same N-terminal sequence. The cause of the heterogenity of CFP29 observed in 2-DE is not known, but it could be explained by deamidation, oxidation of amino acids, or posttranslational modifications such as phosphorylation. Our data suggested that glycosylation was not a possible explanation.

CFP29 is widely distributed in mycobacterial species, and its expression was demonstrated in culture filtrates from pathogenic species as well as nonpathogenic species of environmental origin, suggesting that CFP29 could play an important physiological role in mycobacteria. Comparison of the amino acid sequences, calculated pIs, and molecular masses of CFP29 and B. linens Linocin indicated that CFP29 is a mycobacterial homolog of Linocin, and it was therefore relevant to investigate whether CFP29 possesses bacteriocin activity. Twenty-four L. monocytogenes strains and M. diernhoferi were tested for growth inhibition in the presence of purified native CFP29 or ST-CF. However, no inhibition was observed with any of the strains tested. CFP29 may not share this property with Linocin, due to either significant differences in the amino acid sequences or lack of posttranslational modifications which may be important for the bacteriocin activity. Alternatively, CFP29 may have lost its activity during the purification process.

In this regard, M. tuberculosis is known to possess bacteriocin activity against several rapidly growing mycobacterial strains, including M. diernhoferi (35). The inhibitory activity was sensitive to heat treatment and proteinase digestion, implying that the responsible molecule is of proteinaceous origin. To our knowledge, no detailed characterization of the molecule responsible for this mycobacteriocin activity has been published. However, the expression of a 29-kDa protein which reacts with HYB 71-2 in the rapidly growing, nonpathogenic species M. diernhoferi further supports that CFP29 is not the molecule responsible for this inhibition observed by Takeya and Tokiwa (35). It therefore still remains to be established which physiological role CFP29 may play in mycobacteria.

Work is in progress to test the recognition of recombinant CFP29 by human TB patients in different stages of disease. The results obtained in the mouse model of TB infection are encouraging and suggest that CFP29 should be added to the list of important T-cell antigens derived from culture filtrates.