The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

Abstract

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.

INTRODUCTION

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance (MDR) underlines the need for the discovery of novel antibiotics (1). This is particularly true for tuberculosis (TB), for which 3.7% of new cases and 20% of previously treated cases are estimated to have MDR-TB. In several countries in Eastern Europe and central Asia, these numbers rise to 9 to 32% and more than 50%, respectively, making the situation particularly worrisome. In addition, in the case of TB, which claimed 1.3 million lives in 2012, the treatment of the least-complicated, drug-sensitive cases is lengthy and uncomfortable, and new drugs are urgently needed to shorten the regimen and make it more acceptable to the patients (2).

The cell envelope of Mycobacterium tuberculosis represents a highly attractive target for the discovery of novel TB drugs. Supporting this notion, several key medicines used in the current TB therapy are inhibitors of essential enzymes participating in cell envelope biosynthesis. The first-line drug ethambutol has been shown to target at least three unique integral membrane-associated arabinofuranosyltransferases (EmbA, EmbB, and EmbC) involved in the biosynthesis of arabinogalactan (AG) and lipoarabinomannan (LAM) (3, 4). The inhibition of InhA by isoniazid (INH) in M. tuberculosis leads to the specific depletion of mycolic acids from the bacterial cell wall (5, 6). Interestingly, promising new compounds, which recently emerged from different screening programs, also target various steps in cell envelope metabolism (reviewed in reference 7). These are exemplified by (i) the inhibitors of MmpL3 (8–12), the trehalose monomycolate transporter required for the proper localization of mycolic acids, and (ii) the inhibitors of the decaprenyl-phosphoribose 2′-oxidoreductase DprE1 (11, 13–15), involved in the biosynthesis of mycobacterial AG and LAM. Specifically, DprE1 inhibitors block the biosynthetic pathway of the arabinan precursor and, therefore, the buildup of a covalently linked complex of mycolic acids, AG, and peptidoglycan, which form the so-called cell wall core (16). This unique envelope structure is responsible for many specific features of the tubercle bacilli, including resistance against commonly used antibiotics or the ability to withstand the immune system during infection (17). In addition, a variety of noncovalently linked glycoconjugates have been found to display significant immunomodulatory properties. A prominent group of these molecules comprises the family of phosphatidyl-myo-inositol mannosides (PIMs) and their highly glycosylated counterparts, lipomannan (LM) and LAM (reviewed in reference 18; 16, 19, 20). Several recent reports also demonstrated the structural relevance of PIM, LM, and LAM, underscoring their role in the physiology of mycobacteria (21–23).

PIMs are unique glycolipids anchored through a phosphatidyl-myo-inositol (PI) moiety to the inner and outer membranes of the cell envelope of all Mycobacterium species. PIMs can contain one to six mannose residues and up to four acyl chains, with tri- and tetra-acylated phosphatidyl-myo-inositol dimannoside (PIM₂) and phosphatidyl-myo-inositol hexamannoside (PIM₆) being the predominant species (Fig. 1) (19, 24, 25). PIM₂ and its acylated versions, Ac₁PIM₂ and Ac₂PIM₂, are considered both metabolic end products and intermediates in the biosynthesis of Ac₁PIM₆, Ac₂PIM₆, LM, and LAM. The biosynthesis of PIM is initiated by the phosphatidyl-myo-inositol mannosyltransferase PimA, which catalyzes the transfer of mannose from GDP-Man to the 2 position of the myo-inositol ring of PI, giving rise to phosphatidyl-myo-inositol monomannoside (PIM₁) (20, 26). PimB′ catalyzes the second step by transferring a mannosyl residue from GDP-Man to position 6 of the myo-inositol ring of PIM₁ (26–28). The acyltransferase AcylT1 (Rv2611c in M. tuberculosis H37Rv) can acylate PIM₁ and PIM₂ at the 6 position of the mannose ring transferred by PimA to form Ac₁PIM₁ and Ac₁PIM₂, respectively (29). Ac₁PIM₂ can be further acylated at position 3 of the myo-inositol ring to form Ac₂PIM₂. This acyltransferase as well as most of the mannosyltransferases that catalyze the formation of higher PIMs still remains to be identified (19, 30, 31). Notably, four enzymes participating in the early steps of the PIM pathway, the phosphatidyl-myo-inositol synthase PgsA1, PimA, PimB′, and AcylT, whose orthologs in M. tuberculosis are Rv2612c, Rv2610c, Rv2188c, and Rv2611c, respectively, were found to be essential in vitro in Mycobacterium smegmatis (26, 32, 33). From a drug discovery perspective, the essential character of PIM biosynthetic enzymes and their restriction to mycobacteria and a few other actinomycetes emphasize the interest in them as novel targets for TB drug discovery.

FIG 1.

PIM biosynthetic pathway in mycobacteria. The major PIM species have two (PIM₂) or six (PIM₆) mannose residues with different degrees of acylation. PIM₂ species account for ∼4.5% of the total phospholipids, whereas the abundance of PIM₆ species is ∼10 to 30% the level of PIM₂ species (20). Two pathways have been proposed for the biosynthesis of Ac₁PIM₂ in the cytoplasmic phase of the plasma membrane. Recent analysis suggests that the sequence PI → PIM₁ → PIM₂ → Ac₁PIM₂ is favored. According to this model, the mannosyltransferase PimA acts first, followed by PimB′ and then the acyltransferase AcylT1 (26). R₁, R₂, and R₃ represent fatty acyl chains. In Mycobacterium bovis BCG, major acyl forms contain two palmitic acid residues (C₁₆) and one tuberculostearic acid residue (10-methyloctadecanoate, C₁₉) (62, 63). PimC, a GDP-Man-dependent enzyme, has been proposed to catalyze the third step of the PIM pathway. However, homologs of this enzyme are missing in other mycobacterial strains. It is believed that either PIM₂, Ac₁PIM₂, or Ac₁PIM₃ is flipped from the cytoplasmic side to the periplasmic leaflet of the plasma membrane (19). The mannosyltransferases involved in the biosynthesis of Ac₁PIM₃ to Ac₁PIM₆ are dependent on polyprenol-phosphate-mannose (PPM) rather than GDP-Man as the mannose donor. PimE is the only PPM-dependent mannosyltransferase involved in PIM biosynthesis identified to date, transferring a mannose residue to Ac₁PIM₄ to form Ac₁PIM₅ (31). n.d., not determined.

Here, we have focused on PimA, an enzyme that belongs to the emerging family of membrane-associated glycosyltransferases B (GT-B) (34, 35). Interestingly, a significant number of its members have been found to participate in the biosynthesis of essential glycoconjugates in bacteria, including major human pathogens such as Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae (36, 37). The crystal structure of PimA in the presence of GDP or GDP-Man was disclosed in 2007 and represented the very first structure of a glycosyltransferase found to be involved in mycobacterial cell envelope biosynthesis (38). Overall, the availability of both structural and strong mechanistic information about PimA (19, 39, 40), along with its essentiality for in vitro growth in mycobacteria (33, 41) and a lack of a human homolog, makes PimA an appealing drug target (42). Our goal in this study was to investigate the essentiality of M. tuberculosis PimA in a mouse model of TB in order to evaluate its potential for drug discovery purposes.

MATERIALS AND METHODS

Bacterial strains and media.

M. tuberculosis H37Rv and its derivative, TB38, were grown at 37°C in Middlebrook 7H9 (liquid medium) or 7H10 (solid medium) (Difco) supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), 0.2% (vol/vol) glycerol (Sigma-Aldrich), and 10% ADN (2% glucose, 5% bovine serum albumin, 0.85% NaCl). TB38 is an H37Rv derivative with pFRA42 integrated in its genome at the unique mycobacteriophage L5 attB site. The pFRA42 integrative vector contains the tetR gene, the gene encoding the Pip repressor under the control of P_furA102-tetO, and the Pip-dependent P_ptr promoter upstream of a promoterless lacZ gene (43).

For cloning procedures, Escherichia coli strain HB101 was grown in Luria-Bertani medium (LB) (44). Hygromycin (Roche) was used at a final concentration of 100 μg/ml (solid medium) or 50 μg/ml (liquid medium) for M. tuberculosis and at a final concentration of 200 μg/ml for E. coli. Streptomycin and kanamycin (Sigma-Aldrich) were used at a concentration of 20 μg/ml.

Construction of the M. tuberculosis pimA conditional mutant.

The first 768 bp of M. tuberculosis pimA were amplified using primers RP917 (5′-ATGCATCGGATCGGCATGATTTGTC-3′) and RP918 (5′-ATCGATTCGTCCACCTGACCCAGAA-3′) and cloned downstream of the ptr promoter region in the suicide plasmid pFRA50 (43) to obtain pMAR4. Subsequently, 2 μg of pMAR4 was used to transform M. tuberculosis H37Rv in order to replace the promoter of pimA with the Pip-controlled promoter P_ptr by insertional duplication. Selection of recombinants was performed on 7H10 agar plates containing hygromycin. Integration of the plasmid was confirmed by PCR. The resultant mutant strain was finally transformed with the integrative plasmid pFRA42B (containing the TetR-Pip off system) to obtain TB99. Since pimA might be cotranscribed with its downstream gene (rv2609c), the latter gene was provided in trans on pFRA94 (a pMV261-derived plasmid), which expresses rv2609c from P_hsp60. The final pimA conditional knockdown (cKD) strain was named TB101.

Analysis of lipid composition of pimA cKD strain TB101.

M. tuberculosis TB101 was grown at 37°C with shaking in Middlebrook 7H9 supplemented with 200 ng/ml anhydrotetracycline (ATc). After 48 h, the culture was diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in fresh medium with ATc at 50 ng/ml and further incubated for 48 h. A culture of M. tuberculosis TB101 grown without ATc served as a control. Cells were harvested by centrifugation, and pellets were heat inactivated at 100°C for 1 h and used for lipid extraction. About 10 mg of cell pellet was incubated at room temperature (RT) for 12 h in 2 ml CHCl₃-CH₃OH (1:2) with constant stirring. The organic extract was separated from the mixture by centrifugation (1,000 × g, 5 min, RT), and the pellet was reextracted under the same conditions with 2 ml CHCl₃-CH₃OH (2:1). The two organic extracts were combined, dried under a stream of nitrogen at RT, and subjected to a biphasic Folch wash (45) with 2.1 ml of CHCl₃-CH₃OH-H₂O (4:2:1). Centrifugation (1,000 × g, 5 min, RT) of the mixture resulted in the formation of two phases. The bottom, organic phase was removed, dried under a stream of nitrogen at RT, and redissolved at a ratio of 4 μl CHCl₃-CH₃OH (2:1) per 1 mg of cell dry weight. Five microliters of each sample was applied on 60 F₂₅₄ silica gel plates (Merck), and lipid components were separated by thin-layer chromatography (TLC) using CHCl₃-CH₃OH-concentrated NH₄OH-H₂O (65:25:0.5:4) as a mobile phase. The complete lipid profiles on the TLC plates were obtained by staining with 10% (wt/vol) CuSO₄ in 8% (vol/vol) H₃PO₄; glycolipids were visualized using α-naphthol staining (46).

Generation of polyclonal anti-PimA antibodies.

Recombinant PimA from M. smegmatis (MsPimA) was purified as previously described (40). The purified enzyme was diluted to 100 μg in 500 μl of 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4, and emulsified with 500 μl of complete Freund adjuvant (Sigma-Aldrich). Five subcutaneous injections of 0.2 ml per site were performed on a rabbit. Three successive subcutaneous injections of 100 μg of MsPimA emulsified with incomplete adjuvant were carried out at 15-day intervals. A week after the last injection, serum was collected and evaluated for anti-PimA antibodies. The animal was bled 15 days after the last injection. The anti-PimA antibodies were able to recognize both M. smegmatis and M. tuberculosis enzymes in cell extracts at a 1:3,000 dilution. Rabbit experiments were approved by the Committee of Ethics for Animal Welfare of the University of the Basque Country.

Protein extraction and Western blotting.

TB101 was grown in Middlebrook 7H9 with hygromycin, streptomycin, kanamycin, and ATc (200 ng/ml) starting from an OD₅₄₀ of 0.3, and after 24 and 48 h of incubation at 37°C in rolling bottles, samples were collected by centrifugation (3,500 rpm at 4°C for 5 min). TB101 was also grown under the same conditions but without ATc, and cells were collected after 48 h and after 7 days of incubation (control samples). Pellets were washed with 1 volume of a solution containing 10 mM Tris-HCl, pH 9.5, 5 mM EDTA, and 1× complete EDTA-free protease inhibitor cocktail (Roche) and finally resuspended in 600 μl of the same solution at 4°C. Thereafter, samples were transferred into a clean tube containing 0.5 ml of 0.1 mm zirconia/silica beads (BioSpec Products). The samples were homogenized with three 30-s pulses in a Mini-Beadbeater apparatus (BioSpec Products), followed by refrigeration on ice. Finally, samples were centrifuged at 13,000 rpm for 3 min at 4°C. The supernatants were filtered through 0.22-μm-pore-size filters with low protein-binding activity (Millipore) and further centrifuged at 13,000 rpm for 10 min at 4°C. Protein samples were quantified by a Bradford assay (Bio-Rad), as previously described (47). Samples collected at 6, 12, and 24 h were concentrated using Microcon centrifugal filter devices (cutoff, 30 kDa; Millipore). Protein samples were boiled and separated by SDS-PAGE, as follows: 22.5 μg of protein lysates was run on 10% polyacrylamide gels (44) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) by Western blotting. PimA was visualized by immunoblotting using antibodies directed against the enzyme (1:3,000), while a monoclonal antibody (dilution, 1:200; BEI Resources) was used to detect GroEL. A goat anti-rabbit IgG–horseradish peroxidase conjugate (dilution, 1:3,000; Cell Signaling Technology) or a goat anti-mouse IgG–horseradish peroxidase conjugate (dilution, 1:2,000; Santa Cruz Biotechnology) was used as a secondary antibody. A LiteAblot Turbo extrasensitive chemiluminescent substrate kit (EuroClone) was used to develop the signal. Image acquisitions were performed using a Versadoc imaging system (Bio-Rad) and Quantity One (version 4.2.3) software (Bio-Rad).

RNA extraction and quantitative reverse transcription-PCR.

RNA extraction and quantitative reverse transcription-PCR were performed as previously described using the primers shown in Table S1 in the supplemental material (48). sigA mRNA was used as internal invariant control for data normalization (49). RNA samples that had not been reverse transcribed were included in all experiments to exclude significant DNA contamination. For each sample, melting curves were performed to confirm the purity of the products.

Infection of macrophages.

THP-1 monocytes (American Type Culture Collection) were grown in suspension at 37°C in 5% CO₂ in bicarbonate-buffered RPMI (Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Gibco), 50 μmol/liter β-mercaptoethanol, and 50 μg/ml gentamicin to a density of about 0.5 × 10⁶ cells/ml. Differentiation of monocytes into macrophages was obtained by plating the cells in 96-well plates at a density of 7.5 × 10⁴ cells/well in the presence of 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich). After 24 h, PMA was removed and cells were infected with M. tuberculosis H37Rv or TB101 at a multiplicity of infection of 1:20 (number of CFU/cell) for 90 min, as previously described (50). After infection, extracellular bacteria were removed by washing with phosphate-buffered saline (PBS) and fresh medium with or without 200 ng/ml ATc was added. The medium was replaced every 48 h. At different time points, macrophages were lysed and extracts were plated to determine the viable counts (50).

Aerosol infection in mice.

The AstraZeneca Animal Ethics Committee, registered with the Government of India (registration no. CPCSEA 99/5), approved all animal experimental protocols and usage. BALB/c mice (age, 8 to 10 weeks) were infected via low-dose aerosol with about 10² CFU of either M. tuberculosis H37Rv or TB101. One group of mice in each category was fed a normal diet, while the rest were fed mouse chow containing 2,000 ppm doxycycline (Research Diets). Among these, one group each of M. tuberculosis H37Rv- and TB101-infected mice was fed doxycycline-containing mouse chow from the day of infection, whereas 2 more groups of animals infected with TB101 were fed doxycycline-containing mouse chow from 2 weeks (acute phase) and 4 weeks (chronic phase) postinfection. To compare the rate of bacterial elimination in the acute phase, another control group of mice infected with TB101 was treated with 10 mg/kg of body weight/day of INH by gavage starting from day 3 postinfection and continuing until week 4. Four mice per group were sacrificed at day 0, week 2, week 4, week 8, week 12, and week 16 postinfection. For CFU enumeration, appropriate dilutions of lung homogenates were plated on Middlebrook 7H11 plates for M. tuberculosis H37Rv and Middlebrook 7H11 plates containing 50 μg/ml hygromycin and 20 μg/ml each of kanamycin and streptomycin for TB101.

i.v. infection in mice.

Female C57BL/6 mice (age, 5 to 6 weeks) were obtained from Charles River Laboratories. For infections, logarithmic-phase TB101 bacteria were suspended in PBS-Tween 80 and delivered intravenously (i.v.) in the lateral tail vein (10⁸ CFU per mouse). One group of mice served as a control and was fed normal mouse chow, whereas the other received doxycycline-containing food (Research Diets) from the day before infection. Mice were sacrificed at 4 and 8 weeks postinfection. Lung and spleen homogenates were plated on Middlebrook 7H10 plates with the appropriate antibiotics for evaluation of the number of CFU. Experimental procedures involving animals were approved by Swiss cantonal and federal authorities (authorization number 2218).

RESULTS

Silencing of pimA in axenic cultures results in bacterial death.

In order to study the role of PimA in M. tuberculosis physiology, we constructed an M. tuberculosis conditional mutant where the pimA promoter was replaced by the repressible promoter P_ptr in a strain carrying the TetR-Pip off repressible system previously developed in our laboratory (TB38). In the resulting strain, named TB101, expression of PimA was expected to be downregulated by the addition of anhydrotetracycline (ATc) to the culture medium, thus leading to its depletion.

As shown in Fig. 2, when the pimA conditional mutant TB101 was plated on Middlebrook 7H10 containing 500 ng/ml ATc, growth was inhibited, confirming the predicted essentiality of the gene in vitro (41, 51). As a control, the parental strain TB38 was unaffected by the presence of the drug at this concentration (data not shown). A similar experiment was performed in liquid medium: in this case, TB101 was grown in Middlebrook 7H9 in the presence of increasing concentrations of ATc (0, 50, 100, 200 ng/ml). After 4 days, no difference among the different cultures was detected (data not shown). Each culture was then diluted to an OD₅₄₀ of 0.06 in Middlebrook 7H9 containing the same ATc concentration used in the first culture. As depicted in Fig. 3A, growth of the TB101 strain was inhibited in a clear dose-dependent manner after this passage in fresh ATc-containing medium. To determine whether PimA depletion resulted in bacteriostasis or in bacterial death, TB101 was grown in liquid medium containing 200 ng/ml ATc until growth stopped. At different time points, an aliquot of the culture was diluted and plated to determine the viable counts. As shown in Fig. 3B, the number of CFU decreased by ∼10,000-fold in 6 days, demonstrating that depletion of PimA is indeed bactericidal. Interestingly, the drop in the number of CFU preceded the stop of the optical density increase by about 24 h, suggesting that at this point cells are still able to go through one or two rounds of division but generate unviable daughter cells.

FIG 2.

pimA is an essential gene. Five microliters of a suspension of TB101 was spotted at the indicated dilutions on Middlebrook 7H10 plates with or without 500 ng/ml ATc.

FIG 3.

(A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

To correlate PimA depletion with growth arrest, the conditional mutant TB101 and its parental strain, TB38, were grown for 24 h with or without 500 ng/ml ATc. Under this condition, both strains grew at the same rate, irrespective of the presence of ATc (Fig. 4 and 5). The cultures with ATc were then diluted in fresh medium with or without 500 ng/ml ATc. TB38 was able to grow in these subcultures regardless of the presence of ATc (Fig. 5). On the contrary, TB101 could grow only when it was subcultured in the absence of ATc. When incubated with ATc, it grew at a lower rate, and growth stopped completely after 72 h at an OD₅₄₀ of 0.43 (Fig. 4). Samples from the different cultures were collected and proteins were extracted for Western blot analyses. As is visible in Fig. 4 (see also Fig. S1 in the supplemental material), during the first culture in the presence of ATc, the amount of PimA in TB101 after 24 h of incubation became barely detectable, while it was clearly identified in the culture without ATc. After dilution in medium without ATc, the amount of protein was poorly detectable after 48 h, but then the amount increased and became evident after 72 h of incubation, suggesting that in the absence of ATc, production of PimA was restored, allowing growth. However, in the culture with ATc, PimA was undetectable after both 72 and 96 h of incubation (Fig. 4; see also Fig. S1 in the supplemental material), indicating that under these conditions, depletion of PimA correlated with growth arrest. When the same experiment was performed with TB38, the protein remained well detectable for the entire course of the experiment, as expected, independently of the presence of ATc (Fig. 5; see also Fig. S2 in the supplemental material). The level of the control protein GroEL remained stable in all samples for the entire course of the experiment (see Fig. S1 and S2 in the supplemental material).

FIG 4.

Growth curves of the pimA conditional mutant TB101 with or without 500 ng/ml ATc. The two upper panels represent the first cultures, while the two lower panels represent the second cultures started from a dilution of the first culture grown in the presence of ATc (dashed arrows). (Insets) PimA levels at specific time points (arrows) (details from the Western blot are shown in Fig. S1 in the supplemental material).

FIG 5.

Growth curves of TB101 parental strain TB38 with or without 500 ng/ml ATc. The two upper panels represent the first cultures, while the two lower panels represent the second cultures started from a dilution of the first culture grown in the presence of ATc (dashed arrows). (Insets) PimA levels at specific time points (arrows) (details from the Western blot are shown in Fig. S2 in the supplemental material).

We also performed a quantitative reverse transcription-PCR experiment with RNA extracted from the same cultures described above. The relative amounts of pimA mRNA detected in TB101 were compared to those detected in the parental strain TB38 grown for 24 h in the absence of ATc, representing the physiological level of pimA mRNA (Fig. 6). The level of pimA mRNA detected in the conditional mutant TB101 grown in the absence of ATc was about 6-fold higher than that detected in TB38, demonstrating that P_ptr is stronger than the physiological pimA promoter, thus resulting in overexpression of PimA in the conditional mutant. During the first culture in the presence of ATc, the level of pimA mRNA decreased and was about 5-fold (6 h) and 16-fold (24 h) lower than that found in TB38. During the second culture with ATc, the level of pimA mRNA dropped to a level 32-fold (72 h) and 1,000-fold (96 h) lower than that detected in TB38. These data indicate that the level of pimA mRNA was dramatically reduced in the mutant strain incubated with ATc. More importantly, the pimA transcript was found to be strongly silenced when its level was compared with the level in the wild type in those cultures that underwent growth arrest. In addition, it is worth noting that the level of pimA mRNA in the parental strain TB38 was not affected by the presence of ATc in the medium.

FIG 6.

Changes in pimA mRNA levels at different time points upon exposure to 500 ng/ml ATc. Samples were collected during the experiment whose results are presented in Fig. 4 and 5. Values are expressed as the ratio between the number of cDNA copies detected in samples obtained from bacteria exposed to ATc and the number of cDNA copies detected in samples from TB38 not exposed to ATc. The values were normalized to the level of sigA cDNA, which represented the internal invariant control.

Finally, we also compared the mRNA levels specific for pimA and for the two flanking genes before and after incubation with ATc. As shown in Fig. 7, while the level of pimA-specific mRNA decreased more than 100-fold after incubation with ATc for 48 h, the mRNA levels of the flanking genes were not affected by the treatment. Therefore, the phenotype of TB101 correlates with silencing of the pimA gene and the corresponding protein depletion.

FIG 7.

Changes in mRNA levels after growth in ATc (48 h). The values are expressed as the ratio between the number of cDNA copies detected in samples obtained from the ATc-treated culture and the number of cDNA copies detected in samples obtained from untreated bacteria. The values were normalized to the sigA mRNA level.

Production of PIM is greatly reduced in PimA-depleted mycobacteria.

The effect of pimA gene silencing on PIM biosynthesis was examined by TLC analysis of the lipids extracted from M. tuberculosis TB101 cells grown with or without ATc for 48 h. As shown in Fig. 8, a clear reduction in the levels of PIM was detected in the cells grown in the presence of ATc compared to the control cells. This was particularly evident in the case of Ac₁PIM₂ and Ac₂PIM₂, which are considered end products of the pathway. Moreover, the levels of PI significantly increased in cells grown in the presence of ATc. In contrast, the amounts of the whole range of unrelated lipids remained unchanged. Altogether, this profile is consistent with (i) the biochemical reaction catalyzed by PimA, which transfers a mannose residue from GDP-Man to PI, leading to the synthesis of PIM₁, and (ii) the fact that Ac₁PIM₂, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA followed by PimB′ (18, 23). It is evident that the depletion of PimA caused a block in the biosynthesis of PIM, resulting in severe changes in the composition of the mycobacterial cell envelope, which correlates with the loss of viability observed.

FIG 8.

Loss of PIMs in the PimA-depleted strain. Lipids were extracted from M. tuberculosis TB101 grown in the absence (−) and presence (+) of ATc, separated by TLC in the solvent CHCl₃-CH₃OH-concentrated NH₄OH-H₂O (65:25:0.5:4), and visualized with 10% (wt/vol) CuSO₄ in 8% (vol/vol) H₃PO₄ for the complete lipid profiles (A) and α-naphthol for glycolipids (B).

pimA is essential for growth in macrophages.

To determine whether PimA was required during intracellular replication, we infected THP-1-derived macrophages with the pimA conditional mutant TB101 or with the parental strain TB38 and incubated cells in the presence or absence of ATc. TB38 efficiently multiplied under both conditions, indicating that the presence of the drug and of the TetR and Pip regulators did not affect its virulence (Fig. 9A). Conversely, Fig. 9B shows that TB101 was able to divide intracellularly in the absence of ATc, whereas the presence of the inducer prevented survival and the number of viable bacteria decreased rapidly, thus validating that PimA is essential during infection.

FIG 9.

Growth curves of TB101 and of the parental strain TB38 in THP-1-derived macrophages. The pimA conditional mutant TB101 and the parental strain TB38 were used to infect THP-1-derived macrophages at a multiplicity of infection of 1:20 (bacteria/macrophage). While TB38 was able to divide regardless of the presence of ATc in the cell culture medium (A), TB101 was able to replicate intracellularly only in the absence of ATc (B). Triangles, no ATc; squares, addition of 200 ng/ml ATc to the culture medium.

pimA is essential for growth during the acute and chronic phases of infection.

In order to assess the essentiality of pimA in vivo, BALB/c mice were aerosol infected with the conditional knockdown strain TB101. Silencing of pimA was achieved by feeding the animals doxycycline-containing mouse chow either starting on the day of infection or starting 2 or 4 weeks postinfection. Two control groups of mice did not receive doxycycline in food, thereby allowing regular expression of pimA from the P_ptr promoter. Both groups were fed normal mouse chow; however, one of the groups was treated with 10 mg/kg/day of INH. The results reported in Fig. 10 show that the lungs of mice receiving doxycycline from the beginning of the experiment were completely cleared by week 4, underlining the essential function carried out by PimA in vivo. The rate of clearance of the bacterial burden was similar to that obtained in the other control group infected with TB101 and treated with 10 mg/kg/day INH from day 3 postinfection. In addition, when doxycycline was administered at later time points, a significant decrease in the number of CFU was soon observed and the lungs were completely cleared by week 16, strongly suggesting the essential nature of PimA even in the chronic phase of infection.

FIG 10.

Validation of pimA essentiality in the mouse model of infection. The graph illustrates the bacterial loads in the lungs of BALB/c mice infected with TB101 and fed doxycycline (Doxy)-containing chow either starting on the day of infection or starting at weeks 2 or 4 postinfection. Results represent the mean value and standard deviation for four mice per group and time point.

Further support for the previously described results was obtained by intravenous infection of C57BL/6 mice with TB101. As shown in Fig. 11, the bacterial burden in both spleens and lungs strongly decreased in animals fed doxycycline-containing chow for 8 weeks compared to that in the control groups. In particular, the infection level was reduced by approximately 2 log units in the lungs and by 1.2 log units in the spleen. Altogether, these findings clearly highlight the essential role played by PimA during both the acute and chronic phases of infection.

FIG 11.

Intravenous infection of C57BL/6 mice. Graphs show the bacterial loads in lungs (A) and spleens (B) of mice infected intravenously with TB101 and fed doxycycline-containing chow from the day before infection. Results represent the mean value and standard deviation for four mice per group and time point. Filled symbols, control group without doxycycline; empty symbols, group to which doxycycline was provided in the mouse food.

DISCUSSION

The aim of this work was to validate the mannosyltransferase PimA as a potential drug target in M. tuberculosis by demonstrating its essentiality. By exploiting a repressible promoter system based on the TetR and Pip regulators (43), we showed that this protein plays a vital role in M. tuberculosis physiology both in vitro and in vivo.

The pimA gene (rv2610c) is part of a cluster potentially organized as a single transcriptional unit (23). The upstream genes encode the acyltransferase Rv2611c (rv2611c) and the phosphatidyl-myo-inositol synthase PsgA (rv2612c and pgsA), whereas the downstream open reading frame (ORF) codes for a putative GDP-mannose hydrolase (rv2609c). Although rv2609c was predicted to be nonessential (51), we decided to provide this ORF to the TB101 strain in trans on a replicative plasmid in order to counteract potential polar effects caused by the single recombination event in the pimA gene. The phenotype that we have described and analyzed was therefore due to the lack of the pimA gene product only.

Addition of different amounts of ATc to in vitro cultures had a dose-dependent impact on M. tuberculosis growth that became apparent after 1 dilution in fresh medium with ATc. Western blot analyses demonstrated that, in spite of the reduction of the PimA level in the first culture with ATc, the protein could still be detected (Fig. 4), and it disappeared only after dilution in fresh medium with ATc. Undetectable PimA levels correlated well with growth arrest, thus confirming that the enzyme is required for bacterial multiplication (Fig. 4). The finding that the basal level of PimA in the conditional mutant was about 6-fold higher than that in the wild-type parental strain (Fig. 6) suggests that transcriptional silencing of the gene under investigation was probably not sufficient per se to generate an effect and protein turnover had to take place before a phenotype could be observed. It is possible that the conditional mutant accumulates both PimA and PIMs that allow the cells to undergo a few rounds of replication even after the pimA mRNA is depleted to a level lower than that found in wild-type cells. Similar findings have already been reported for other conditional mutants, which needed several rounds of subculturing before a phenotype was evident (52, 53) or which were still able to undergo some cycles of cell replication after the depleted essential protein became undetectable by Western blotting (54).

The drastic decrease in bacterial viability strongly confirmed that PimA is required for survival and correlated with the depletion of the target, as proved by Western blotting. Indeed, the protein was undetectable 48 h after addition of ATc, which tallied with the decrease in the number of CFU. Finally, our data also prove the lack of redundancy for the function carried out by PimA in M. tuberculosis. Biochemical analysis provided further support with the demonstration that downregulation of pimA led to the reduced production of Ac₁PIM₂ and Ac₂PIM₂ and, consequently, the accumulation of the precursor PI, while it did not affect the remaining pool of lipids.

A dramatic decrease in the number of CFU was detected upon doxycycline treatment of the infected animals. The effect was evident in two different mouse strains (BALB/c and C57BL/6) and when two distinct infection protocols (aerosol and i.v.) were used. Silencing of PimA might have accelerated the process of lung clearance by provoking a pleiotropic effect, which possibly involved an increased permeability of the bacteria to doxycycline and a more favorable interplay with the host immunity. We observed increased bacterial clearance between weeks 12 and 16 postinfection in aerosol-infected animals, irrespective of whether the doxycycline treatment was initiated at day 14 or 28. The bacterial burden was gradually reduced after the doxycycline treatment initiation at day 14 and day 28, but a sharp decline was observed only beyond 12 weeks of infection, suggesting that this is probably due to the immune response and not related to the kinetics of pimA downregulation.

While both the wild-type and TB101 strains were able to infect mice to similar extents, the pimA conditional mutant did not multiply as efficiently as H37Rv and a difference between the two strains of approximately 1 log unit was noted in the chronic phase. We can hypothesize that the levels of pimA expression from the ptr promoter were not optimal and affected the ability of the strain to expand in vivo. Interestingly, silencing of pimA at the onset of the chronic phase (i.e., at week 4) resulted in a reduction of the bacterial burden in the lungs, thus demonstrating the essentiality of the target in the chronic phase as well. PimA therefore represents another candidate target whose depletion causes severe effects, similar to what has been reported for icl, the proteasome, pckA, or pptT (55–58).

Overall, the data presented here highlight the importance of PimA for M. tuberculosis growth, survival, and persistence in vivo and thereby make this enzyme a potential candidate for in vitro target-based drug screening approaches. In that sense, recent reports using such screens provided clear evidence that they can identify inhibitors which act on a target when tested against mycobacteria. One example is the structure-guided discovery and development of phenyl-diketo acids (PDKA) as inhibitors of Mycobacterium tuberculosis malate synthase (GlcB) (59). In another example, the M. tuberculosis dihydrofolate reductase (DHFR) was subjected to high-throughput screening (HTS), which identified a quinazoline-containing enzyme inhibitor displaying potency against in vitro-grown wild-type M. tuberculosis (60). In addition, modulating the expression of the target by means of a conditional mutant could find application in target-based whole-cell screening assays for the identification of compounds targeting PimA or other cellular processes that are part of the same pathway as PimA. This approach was successfully applied with a pantothenate synthase (panC) conditional mutant, where the authors identified hits with increased activity against the anhydrotetracycline-treated, PanC-depleted bacteria (61). Encouraged by the findings of our rigorous validation, we are currently conducting similar screens against PimA.