Mycobacterium tuberculosis H37Rv was grown at 37°C in Middlebrook 7H9 broth supplemented with 10% oleic acid-albumin-dextrose (OAD), 0.5% glycerol, and 0.05% Tween 80. Escherichia coli strain XL10-gold (Stratagene) was used for cloning and plasmid propagation. The recombinant protein was expressed in E. coli BL21 DE3 strain (Invitrogen). E. coli strains were maintained in Luria-Bertani (LB). Plasmid selection was performed with addition of kanamycin (50 μg/mL) or ampicillin (100 μg/mL) to the medium. Solid medium was prepared by the addition of 1.5% agar to the LB medium or 7H11 supplemented with 10% OAD.

Male specific-pathogen-free C57BL/6 or interferon-gamma knockout (IFN-γ KO) mice aged 4–8 weeks, obtained from the animal care facility of the Institute of Tropical Pathology and Public Health at Federal University of Goiás (UFG), were maintained in isolators in class 3 biosafety level (BL3) cabinets with water and food provided ad libitum. Temperature was monitored and maintained around 22°C, relative humidity 60%, and 12 h light/dark cycles. The use of mice was conducted in accordance with the guidelines of the Brazilian Society of Animal Science Laboratory (SBCAL/COBEA). This study was carried out in accordance with the recommendations of Committee on the Ethics of Animal Experiments of Universidade Federal de Goiás (Approved protocol: 027/14).

Mycobacterium tuberculosis LAP gene (Rv2213¹) was subcloned into PCR2.1 TOPO vector (Invitrogen) using a PCR amplified product from the M. tuberculosis H37Rv genome. The recombinant PCR2.1 vector was digested to release the gene that was ligated into the pET28a expression vector (Novagen), submitted to DNA sequencing and inserted into the expression host E. coli BL21. Bacteria containing the recombinant expression vector were grown at 37°C. When the bacterial cells reached OD₆₀₀ measurements of 0.6, the expression of the recombinant protein was induced by the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and the incubation continued at 20°C during 16 h. Bacterial cells were collected by centrifugation (10,000 × g for 5 min), and suspended in 4 mL of binding buffer (20 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl pH 7.9). After sonication, cell lysate was centrifuged (20,000 × g for 15 min), and the supernatant was applied onto a resin column (Novagen) charged with NiSO₄ and equilibrated with binding buffer. Proteins were eluted with a concentration gradient of imidazole (40–000 mM) and protein concentration of the eluted fractions was determined by Bradford protein assay. The purified MtLAP was dialyzed against Tris-HCl pH7.5 with 250 mM NaCl and stored in 50% glycerol at -20°C. The purity of the protein was analyzed by Coomassie-stained 12% SDS-PAGE.

H37Rv lysates and culture filtrate protein (CFP) were prepared as previously described Correa et al. (2014). Bacterial cultures (200 mL) were grown in Sauton media to late log phase and then pelleted by centrifugation at 2,000 × g for 20 min. Supernatant was transferred to a fresh tube, centrifuged again, and then filtered through a 0.2 μm filter unit. Supernatant was concentrated by protein acetone precipitation. Bacterial pellet was used for H37Rv lysates by sonication. Samples were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% powdered milk in PBS and probed with the anti-MtLAP or anti-Zmp1 polyclonal serum produced in mice, followed by incubation with the anti-mouse IgG-alkaline phosphatase secondary antibody (Sigma–Aldrich). Antigen-antibody complex was detected via BCIP/NBT liquid substrate (Sigma–Aldrich).

Sedimentation velocity experiments were performed using a Beckman XL-I analytical ultracentrifuge and an AN-60 TI rotor (Beckman Coulter). The experiments were carried out at 20°C for MtLAP at 18.2 μM in 25 mM Tris pH 7.5, 500 mM NaCl. A volume of 400 μL was loaded into 12 mm path cells and centrifuged at 130,000 × g (42,000 rpm). Scans were recorded every 3 min, overnight, at 280 nm. We used the Sednterp software² to estimate the partial specific volume of the polypeptide chain, v ^- = 0.741 mL/g, the solvent density, ρ = 1.01920 g/mL, and the solvent viscosity, η = 1.0556 mPa.s, at 20°C. Sedimentation profiles were analyzed by the size-distribution analysis of Sedfit³. In Sedfit, finite element solutions of the Lamm equation for a large number of discrete, independent species, for which a relationship between mass, sedimentation and diffusion coefficients, s and D, is assumed, are combined with a maximum entropy regularization to represent a continuous size-distribution. We used 200 collected sets of data using fitted frictional ratio for sedimentation coefficients comprised between 0 and 45 S and a 0.68 confidence level.

The activity of MtLAP was assayed by incubating 300 ng of the purified enzyme with 20 μM of L-Leu-7-amido-4-methylcoumarin (Leu-AMC), Met-AMC, Pro-AMC, Arg-AMC, or Asp-AMC (Sigma–Aldrich) in 100 μL reaction buffer (25 mM Tris-HCl pH 7.5 and 1.5 mM NiSO₄). Substrate hydrolysis was determined by AMC release measured fluorometrically using SpectraMax M5 Microplate Reader with SoftMax Pro Data Acquisition and Analysis Software (Molecular Devices) as described previously (Cadavid-Restrepo et al., 2011). Enzyme cation preference was performed using different concentrations of CaCl₂, CoCl₂, CuSO₄, FeCl₂, MgCl₂, MnCl₂, NiSO₄, or ZnSO₄ in Tris-HCl pH 7.5 at 25°C containing Leu-AMC as substrate. The optimal pH for activity was determined as described above in 25 mM AMT buffer (25 mM acetic acid-25 mM MES-25 mM Tris-HCl) adjusted to pHs ranging from 5 to 10. To assess the effect of temperature on MtLAP activity, enzyme reactions took place at temperatures ranging from 10 to 100°C. The Michaelis–Menten constant (Km) of MtLAP were determined according to the hyperbolic regression method using Prism software version 5.03 (GraphPad). The oligomeric structure of the enzyme was evaluated by electrophoresis as described previously (Cadavid-Restrepo et al., 2011). Briefly, purified protein was subjected to 8% SDS-PAGE in the presence 0.01% SDS under non-reducing conditions with or without previous boiling of the sample.

Different concentrations of tosyl-lysylchloromethane (TLCK), bestatin, tosyl phenylalanyl chloromethyl ketone (TPCK), EDTA, L-trans-epoxysuccinylleucylamido-4-guanidino butane (E-64), phenylmethylsulfonyl fluoride (PMSF), 1,10-phenanthroline, leupeptin, or pepstatin A were incubated with 300 ng of purified MtLAP in 100 μL reaction buffer for 5 min at room temperature, before the substrate was added. Enzymatic reactions were monitored as described above. All inhibitors were from Sigma–Aldrich. Bestatin half-maximal inhibitory concentration (IC50) and 95% confidence interval (CI) were calculated by non-linear regression log (inhibitor) vs. normalized response with variable slope method.

To study the effect of bestatin on M. tuberculosis growth, bacterial cultures were adjusted, and 100 CFU were deposited into each well of a 96-well microplate that contained a serial dilution of bestatin, ranging from 1600 to 25 μg/mL in Middlebrook 7H9 supplemented with 10% OAD, 0.5% glycerol and 0.05% Tween 80. The bestatin 50% growth inhibitory concentration (GI50) value was determined by non-linear regression log (inhibitor) vs. normalized response with variable slope method.

Alveolar macrophages were obtained from C57BL/6 mice bronchoalveolar lavage (BAL). Mice were euthanized and the pulmonary cavities were opened. Using an 18-gage needle, the trachea was cannulated, and 1.0 mL of ice-cold 5 mM EDTA in PBS was slowly injected into the lungs and then withdrawn. This procedure was repeated five times and a total of 4 mL of BAL fluid was collected from each mice. The single-cell suspension was then washed and centrifuged at 1,000 × g. Red blood cells were lysed upon 5 min incubation in 2 mL of Gey’s solution (155 mM NH₄Cl and 10 mM KHCO₃) at room temperature. Macrophages were then washed with PBS and resuspended in RPMI medium 1640 (Gibco) supplemented with 1% glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 1% penicillin-streptomycin and 10% FBS.

The alveolar macrophages were plated in 96 well plates (100 μL/well) at 10⁶ cells/well and incubated for 24 h at 37°C. Macrophage monolayers were then washed and incubated with 100 μL infection media (RPMI medium 1640 supplemented with 1% glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 10% FBS) containing 5 × 10⁶ CFU of M. tuberculosis H37Rv (MOI = 5) at 37°C under CO₂ for 3 h. Then the cells were washed twice with warm PBS to remove extracellular bacteria and supplemented with RPMI free of antibiotics and containing a range of 0, 100, or 200 μg/mL of bestatin. Forty-eight hours after infection, media was aspirated from infected macrophage wells, and 100 μL of ice-cold sterile lysis solution (0.05% SDS, w/v in H₂O) was added to each well. The lysates were transferred to a new 96 well plate for serial dilutions. Triplicate wells were plated on 7H11 agar supplemented with OAD. The plates were cultured in a CO₂ incubator for 3–4 weeks and the number of CFUs counted.

IFN-γ knockout mice were intranasally infected with 10⁴ M. tuberculosis H37Rv and treatment was performed as previously described (Lenaerts et al., 2003). In brief, 1 day post infection, three mice were euthanized by cervical dislocation to verify bacterial uptake per mouse. Each treated group consisted of four mice. Treatment was started 18 days after infection and lasted up to 28 days post infection. Another three infected mice were euthanized to determine the bacterial load at the beginning of the treatment (day 18 post infection). Lyophilized bestatin was dissolved in PBS and 100 μL administered by intranasal instillation in eight treatments for 5 days/week at 1 mg/kg of body weight while Isoniazid (INH) was administered at 25 mg/kg as positive control. A negative control group of infected but untreated mice received 100 μL of intranasal PBS. After the cessation of treatment (day 28 post infection), viable bacteria in the mice lungs were evaluated. Right and left cranial lung lobes were removed and homogenized in PBS containing 0.05% Tween 80, and tissue homogenates were serially diluted in PBS and plated in duplicate onto Middlebrook 7H11 agar. Colonies were counted after 3–4 weeks of incubation at 37°C in a 5% CO₂ incubator.

The caudal left lung lobe was excised from all animals, stored in 10% formalin, then embedded in paraffin and sections were prepared and stained with hematoxylin and eosin (H&E). Lung lesions were determined using the measurement tool in the AxioVision software (Carl Zeiss). Total lesion area was calculated by the sum of all areas of lesions in a lung section per mouse.

Statistical significance between groups was determined by the two-tailed unpaired Student’s t-test or One-way ANOVA with Dunnett or Tukey post-test using Prism software version 5.03 (GraphPad).

The sequencing of M. tuberculosis H37Rv genome revealed a small number of genes coding for putative peptidases that mediate aminopeptidolytic activities https://merops.sanger.ac.uk/index.shtml. We identified only one copy of a putative LAP gene (Rv2213⁴), which has thus far not been characterized. Rv2213 is an open reading frame (ORF) that encodes a protein of 515 amino acids. Multiple amino acid sequence alignments revealed that the C-terminal conserved region presents the typical signature sequence NTDAEGRL of the M17 family (Burley et al., 1990) as well as multiple conserved amino acids within predicted functional domains for metal binding and catalytic activity conserved across different taxa (Figure 1A). LAP of M. tuberculosis shares high identity with other pathogenic mycobacterial LAPs, over 80% with M. leprae, M. bovis, and M. avium paratuberculosis. Although it contains conserved functional domains, MtLAP shares less than 32% identity with species out of the Mycobacterium genus, diverging from LAPs from other bacteria, protozoa and higher eukaryotes, including only 28.1% identity with human LAP (Figure 1A and Table 1). The phylogenetic analysis of LAP from the Mycobacterium genus shows a relationship between LAP sequence similarities and bacterial physiology and pathogenesis. The phylogenetic tree shows divergent groups of proteins that cluster according to the bacterial rate of growth and pathogenicity with a strong bootstrap support (Figure 1B). Sequences are divided in two defined clades; one branch includes slow growing mycobacteria and the other cluster, the rapid growers. Moreover, for slow and rapid growing mycobacteria, LAP sequences are divided into three groups. One branch includes true pathogenic mycobacteria (TP), another branch groups opportunistic bacteria (OP) and the other branch contains non-pathogenic mycobacteria (NP). Similarly, true pathogenic slow growing mycobacteria belong to the M. tuberculosis complex (MTC) grouped together in a very close phylogenetic clade (Figure 1B). These findings suggest that MtLAP may be involved in physiological and pathogenicity processes in tuberculosis, thus predicted to be essential for in vivo survival and pathogenicity (Ribeiro-Guimarães and Pessolani, 2007).

To establish the kinetic parameters of MtLAP, the Mycobacterium tuberculosis gene Rv2213 was cloned into the prokaryotic expression vector pET28a. The recombinant protein was expressed and purified by nickel affinity chromatography under non-denaturing conditions with increasing imidazole concentrations. SDS-PAGE analysis revealed a strong band, with high purity, eluted with imidazole at a concentration higher than 200 mM, corresponding to the expected protein molecular mass of approximately 55 kDa (Figure 2A). For detection and identification of the native protein, anti-MtLAP polyclonal serum was produced in mice. We verified by western blot the presence of the protein in the H37Rv cell fraction extract, but not in the culture supernatant (CFP, Figure 2B). To confirm that MtLAP was not released to the supernatant, mycobacterial metalloprotease Zmp1, previously described as a secreted protein (Master et al., 2008; Correa et al., 2014), was detected in the same culture supernatant preparations (Figure 2B). Since LAPs exist naturally as oligomeric-dependent active proteases (Matsui et al., 2006), we decide to test MtLAP oligomerization. Therefore, non-boiled nor reduced purified protein was subjected to 8% SDS-PAGE in the presence of 0.01% SDS. Bands about 100 kDa and over 220 kDa were detected indicating an oligomeric protein complex, although the 55 kDa monomer was predominant in gel (Figure 2C, lane 1). Sample boiling resulted in complete monomerization of MtLAP (Figure 2C, lane 2).

The results obtained from sedimentation velocity experiments corroborate the calibrated exclusion size chromatography findings. The experimental and fitted sedimentation velocity profile of MtLAP (Figure 3A) obtained at 18.2 μM shows a major species, approximately 70% of the signal, that sediments at 11.2 S (S_20,w = 12.6 S). The s-value is directly related to the molar mass (M) and the Stokes radius (R_S) of the particle. The combination of the s-values with R_S = 5.9 nm estimated from calibrated size exclusion chromatography gives an estimate for the MtLAP complex of M = 323 kDa, which is close to the expected molecular mass for a hexamer (334.4 kDa). Considering a hexamer, the R_S value corresponds to a frictional ratio of 1.30, a value close to 1.25 that relates to globular proteins. It suggests that the MtLAP hexamer has a globular shape. Larger species were also detected but a well-defined peak was difficult to identify. The larger species most probably represents aggregates due to sample aging and also to the fact that MtLAP was prone to precipitate even in high ionic strength buffers. The smaller peaks correspond to only less than 8% of the signal and give estimated masses of 64 and 140 kDa, close to the expected mass of a monomer and a dimmer, respectively (Figure 3B). The presence of these smaller forms may be related to the high salt concentration in the buffer. These data clearly indicate that active MtLAP is an oligomeric protein formed by six monomers.

The activity of recombinant MtLAP was determined by measuring the fluorescence of AMC (7-amido-4-methylcoumarin) released by hydrolysis of the enzyme substrate Leu-AMC. However, in the initial assays, no MtLAP activity was observed when the enzyme was incubated with substrate in Tris-buffer in the absence of metal ions. As MtLAP belongs to the metallo aminopeptidase M17 family, that has a broad range of preferences for metal ions, we decided to test MtLAP metal dependence. The enzyme did show activity when the assay was conducted in the presence of metal ions including Co²⁺, Mg²⁺, Mn²⁺, Ni²⁺, and Zn²⁺. Conversely, Ca²⁺, Cu²⁺, and Fe²⁺ could not activate MtLAP. The maximum activity was observed in the presence of 1.5 mM Ni²⁺, followed by Mn²⁺, Co²⁺, Zn²⁺, and Mg²⁺ (Figure 4A). Regarding pH dependence, its optimal activity was observed at pH 7.5. Although at acidic pHs the enzyme rapidly loses activity and no substrate cleavage was observed at pH 5, the enzyme was resistant to alkaline environment displaying about 50 and 25% of its maximal activity at pH 9 and 10, respectively (Figure 4B). The activity of MtLAP was determined at different temperatures in the range of 10 – 100°C. At 37°C, the enzyme showed 70.78% of its maximal activity measured at 50°C (Figure 4C). To assess the enzyme substrate preference, MtLAP was incubated with aminopeptidase substrates (Figure 4D). Calculated K_m values are 69.4 ± 3.6 μM for Leu-AMC, 61.8 ± 4.4 μM and 344.8 ± 49.2 μM for Met-AMC and Pro-AMC, respectively (Supplementary Figure 1). The enzyme showed maximal activity on substrates with hydrophobic amino acids leucine and methionine. Low activity was observed with the non-polar cyclic amino acid proline based substrate. No cleavage was detected on substrates with charged side chains containing arginine and aspartic amino acid residues.

Enzyme hydrolytic activity was not sensitive to the classical serine (PMSF, TLCK or TPCK) and cysteine (E-64) protease inhibitors. The enzyme was not inhibited by leupeptin, an inhibitor of cysteine/serine/threonine proteases, nor by pepstatin A that inhibits aspartyl proteases. The chelating agents 1,10-phenanthroline and EDTA inactivated 86.5 and 68% of the enzyme activity, respectively. Furthermore, the enzymatic activity of MtLAP on Leu-AMC was completely inhibited by 30 μM bestatin (Figure 5A). Since bestatin is a potent inhibitor of LAPs, we assayed its inhibition on MtLAP activity in a range of 0.01–100 μM using Leu-AMC as a substrate (Figure 5B). As a result, the calculated IC₅₀ was 53.78 nM as shown in Figure 5B with 95% CI of 46.44–62.28 nM.

Since bestatin is efficient at inhibiting MtLAP activity, we asked if it could constrain M. tuberculosis growth. To address this question, we cultivated the bacteria in liquid media with different concentrations of bestatin and measured CFUs. At day seven of culture, a dose-dependent manner inhibition of M. tuberculosis growth was observed (Figure 6A). Bestatin shows a 50% growth inhibitory concentration value (GI50) of 174.2 μg/mL (95% CI 143.1–212.0 μg/mL). We next examined the effect of bestatin on M. tuberculosis survival inside macrophages. The bestatin treatment done after M. tuberculosis alveolar macrophages infection showed a significant dose-dependent reduction in colony counts. Compared to PBS infection control, 29.8% of the mycobacteria were recovered using 200 μg/mL of inhibitor and 46.4% of the bacteria were viable using bestatin at 100 μg/mL (Figure 6B). Those results suggest that the activity of LAP may be essential for M. tuberculosis survival and virulence.

Bestatin has been used before as a low molecular immunomodulator. It exhibits antitumoral and antimicrobial activities through the activation of host defense mechanisms and shows low toxicity even after a long-term administration (Ota, 1991). In several mouse-model studies, effective and safe bestatin doses ranged from 0.1 to10 mg/kg of body weight or in doses of 10–100 μg per animal (Bruley-Rosset et al., 1979; Carter et al., 2012; Reinhoudt et al., 2012; Lis et al., 2013). To test whether bestatin has any effect on tuberculosis progression, IFN-γ knockout mice were infected with 10⁴ CFU of M. tuberculosis H37Rv and treated with the inhibitor at 1 mg/kg/day intranasally. After completion of therapy, bestatin reduced bacterial burden (∼1.4 Log) in the lungs when compared to PBS administration and isoniazid corroborates the efficacy of treatment method (Figures 7A,B). Thus, bestatin inhibits up to 95.37% of M. tuberculosis growth in the mice lungs. In addition, treatment reduced lung lesions caused by infection (Figure 7C) from 9.09 mm² of lung lesion area in PBS group to 2.46 mm² in bestatin treated mice (Figure 7D). Taken together, our results indicate that bestatin is a promising drug and a model compound for anti-TB drugs.