Bacterial strains, plasmids, primers, and cloning
E. coli XL-1 Blue (Stratagene) was used as host strain for transformations of the vectors pGEX-2T (Pharmacia), p19Kpro (a gift from Koen De Smet, Imperial College Medical School at St Mary's, London, UK), and pBluescriptSK (pBS) (Stratagene). pGEX-2T is an E. coli expression vector for the expression of a heterologous protein with an N-terminal GST fusion partner, and p19Kpro is a mycobacterial expression vector derived from p16R1 with a constitutive 19 kDa antigen promoter [3]. M. tuberculosis clinical isolate GSH-3052 [7] and the laboratory strain H37Rv were maintained on Lowenstein-Jensen slopes and grown as liquid cultures in Kirchner's medium, at 37°C with shaking (200 rpm). M. smegmatis (mc2155) was grown as described [3]. All DNA manipulations were carried out using standard techniques [8]. Full-length mycP1 and mycP1 lacking the signal peptide-encoding sequence (mycP1-) were amplified using 5'-ACA GGA TCC GTC AAG TGG CCC AAG GTA GCA-3' (corresponding to nucleotides -71 to -51) and 5'-CGT TCT AGA GGA TCC CCC GCA TCG GCC ATC ACG-3' (corresponding to nucleotides 52–69) as the forward primers, respectively, and 5'-AA AGA ATT CTG CAG TCA TCG GCG GCT CAG CG-3' as the reverse primer. Amplification was performed with the Expand™ High Fidelity PCR System (Roche) using approximately 100 ng of M. tuberculosis cosmid Y15F10 DNA as template (Genbank accession no. Z94121, obtained from the Sanger Centre, UK). The mycP1 (containing ~70 bp upstream of the putative start site to incorporate any putative translation signals) was cloned into p19Kpro (p19K-P1), andmycP1- (lacking the signal peptide sequence) was cloned in-frame with the glutathione S-transferase (GST) gene in pGEX-2T (pGEX-P1). Electroporation of M. smegmatis was performed as described [9].
Antibody production and detection of mycosin-1 in M. smegmatis and M. tuberculosis
The GST-mycosin-1 fusion protein was expressed and purified essentially as described [10]. One hundred micrograms of purified GST-mycosin-1 in incomplete Freund's adjuvant was injected subcutaneously into two female New Zealand White rabbits. Three additional booster injections were given at 3-week intervals. Approximately 10 ml of blood was taken from each rabbit at 1 and 2 weeks after the last booster injection and used as anti-mycosin-1 antiserum. To enhance its specificity, the antiserum was depleted of non-specific antibodies by consecutive passage over Sepharose 4B columns containing E. coli and M. smegmatis cellular lysates.
To obtain mycobacterial cellular lysates, wild-type and transformed M. smegmatis were grown in liquid broth, cells were harvested by centrifugation, washed twice in PBS, resuspended in 2% SDS, and then sonicated (W-385 sonicator, Heat Systems-Ultrasonics, Inc; output level 3; 80% duty cycle) on ice for 3 periods of 4 min each. After extraction of protein at 60°C for 2 h, the extract was centrifuged at 12 000 × g for 20 min to remove insoluble material. SDS extraction of M. tuberculosis GSH-3052 proteins was performed as above, except that prior to sonication the bacteria were heat-killed at 80°C for 45 min. In addition, culture filtrates from various stages of growth were harvested, dialyzed against sterile water, and then lyophilized.
Immunodetection of mycosin-1 during growth in macrophages
P388D1 macrophages were grown and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and were infected with the M. tuberculosis clinical strain GSH-3052 at a ratio of 50 bacilli/cell for 5 h at 37°C. After washing in PBS, fresh RPMI-1640 medium was added and the infected cells were incubated at 37° C for 6 days. The cells were then washed with PBS to remove any extracellular bacteria, detached in 5-mM EDTA, sonicated for three periods of 4 min on ice, and centrifuged at 100 000 × g for 3 h. The pellet was resuspended in SDS loading buffer, resolved by SDS-PAGE, and analyzed by Western blotting, using anti-mycosin-1 antiserum (1:1 000) as the primary antibody.
Subcellular localization
To obtain subcellular fractions of M. smegmatis, bacteria were isolated after two days of growth in culture, resuspended in PBS and sonicated, as described above. The lysate was then centrifuged at 1 000 × g for 30 min, to remove debris and intact bacteria. The supernatant was incubated with DNAse I and RNAse at 4°C overnight and then centrifuged at 20 000 × g to collect the cell wall fraction (pellet). The supernatant was then centrifuged at 100 000 × g for 3 h to isolate the cellular membranes (pellet) and cytoplasm (supernatant), which was further concentrated 2–3 fold using 10 kDa Amicon spin columns. The cell membrane and cell wall fractions were resuspended in 2% SDS and heated at 60°C for 2 h, after which insoluble components were again removed by centrifugation. Subcellular fractionation of M. tuberculosis GSH-3052 proteins was performed as above, except that cultures were grown for 3 weeks and prior to sonication the bacteria were heat-killed at 80°C for 45 min.
For immunoelectronmicroscopy, whole bacteria were incubated with anti-mycosin-1 or preimmune antiserum, fixed in 2% paraformaldehyde / 0.05% gluteraldehyde in 0.1 M phosphate buffer (pH 7.4), embedded in 2% low-melting-point agarose, sucrose-infiltrated by serial passage through a 50–100% sucrose gradient, and then cryosectioned at -100 to -110°C. Ultrathin sections were placed onto formvar-coated nickel grids. Non-specific antibody binding was blocked with 0.02 M glycine followed by 1% BSA, and the grids were then incubated on goat anti-rabbit IgG conjugated to 5 nm-diameter gold particles. After washing with 1% BSA and PBS, postfixing in 1% gluteraldehyde and staining with uranyl acetate/methylcellulose, the sections were viewed by transmission electron microscopy (Zeiss EM 109, 80 kV).
Assay of M. tuberculosis culture filtrates for protease activity and inhibition by class-specific protease inhibitors
Mycobacterial culture supernatants were harvested by centrifugation, filtered (0.22-μm filter), adjusted to 0.02% Na azide, and concentrated 20-fold (Amicon, 10 kDa cut-off). Mycobacterial culture filtrates, bacterial whole cell lysates containing recombinant mycosin-1, and purified GST-mycosin-1 fusion protein were assayed for proteolytic activity by incubation with 125I-labeled fibrinogen, prepared as described [11]. Assays consisted of 200 μl of concentrated filtrate made up to a final volume of 500 μl in 75 mM Hepes, pH 7.3, containing 1.5–2.0 × 106 c.p.m. 125I-fibrinogen, and were incubated at 37°C with rotation for 16–18 h. Fibrinogen degradation was determined quantitatively by trichloroacetic acid (TCA) precipitation as described [11]. The inhibition of proteolytic activity of M. tuberculosis culture filtrates by class-specific protease inhibitors was investigated by addition of each of the following inhibitors to the reaction: (A) Serine protease specific inhibitors: 3, 4-DCI (3, 4-dichloroisocoumarin) (1 mM), aprotinin (2 μg/ml) and pefabloc (1 mM). (B) Mixed serine and cysteine protease inhibitors: chymostatin (0.2 mM), ALLN (N-acetyl-Leu-Leu-norleucinal or Calpain Inhibitor I) (200 μg/ml), ALLM (N-acetyl-Leu-Leu-methional or Calpain Inhibitor II) (100 μg/ml), PMSF (phenylmethanesulfonyl fluoride) (1 mM), leupeptin (0.4 mM), TPCK (L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone) (100 μg/ml) and TLCK (L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone HCl) (100 μg/ml). (C) Cysteine protease specific inhibitor: E-64 (L-trans-epoxysuccinyl-leucylamido-[4-guanidino]-butane) (0.05 mM). (D) Aspartic protease specific inhibitor: pepstatin A (50 μg/ml). (E) Metallo-protease inhibitors: EDTA (ethylenediaminotetraacetic acid) (10 mM), 1, 10-phenanthroline (2 mg/ml) and phosphoramidon (0.09 mM).
