Ppm1-Encoded Polyprenyl Monophosphomannose Synthase Activity Is Essential for Lipoglycan Synthesis and Survival in Mycobacteria

The biosynthesis of mycobacterial mannose-containing lipoglycans, such as lipomannan (LM) and the immunomodulator lipoarabinomanan (LAM), is carried out by the GT-C superfamily of glycosyltransferases that require polyprenylphosphate-based mannose (PPM) as a sugar donor. The essentiality of lipoglycan synthesis for growth makes the glycosyltransferase that synthesizes PPM, a potential drug target in Mycobacterium tuberculosis, the causative agent of tuberculosis. In M. tuberculosis, PPM has been shown to be synthesized by Ppm1 in enzymatic assays. However, genetic evidence for its essentiality and in vivo role in LM/LAM and PPM biosynthesis is lacking. In this study, we demonstrate that MSMEG3859, a Mycobacterium smegmatis gene encoding the homologue of the catalytic domain of M. tuberculosis Ppm1, is essential for survival. Depletion of MSMEG3859 in a conditional mutant of M. smegmatis resulted in the loss of higher order phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan. We were also able to demonstrate that two other M. tuberculosis genes encoding glycosyltransferases that either had been shown to possess PPM synthase activity (Rv3779), or were involved in synthesizing similar polyprenol-linked donors (ppgS), were unable to compensate for the loss of MSMEG3859 in the conditional mutant.


Introduction
Tuberculosis (TB) affects a third of mankind and causes 1.7 million fatalities annually [1]. The spread of TB has been facilitated in recent decades due to the susceptibility of HIVinfected individuals to Mycobacterium tuberculosis, the etiological agent of TB [2]. The problem has been compounded by the emergence of multi-and extensively-drug resistant M. tuberculosis strains [2]. Typically, the cell walls of the genus Mycobacterium contain mycolic acids (m), arabinogalactan (AG) and peptidoglycan (P), which are covalently linked to each other to form the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex [3]. A particular group of specialized glycophospholipids, phosphatidylmyo-inositol (PI) mannosides (PIMs) and lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are found in the outer leaflet of mAGP [4]. LM and LAM, which are based on a core PIM unit, possess an elongated a(1R6) linear, a(1R2) branched mannan, of approximately 30 mannopyranose (Manp) residues, and linked to its terminus to a branched D-arabinan domain of approximately 70 arabinofuranose (Araf) residues, assembled through several distinct structural motifs [4][5][6]. In mycobacteria, the large arabinan domain is capped to various degrees with short a(1R2) Manp chains in the case of M. tuberculosis [7], whereas in M. smegmatis, caps of inositol phosphate are present and termed PILAM [4], and M. chelonae possess non-capped LAM [8]. Both ManLAM and PILAM exhibit a broad range of immunomodulatory activities. For example ManLAM, which is predominantly found in the slow-growing pathogenic mycobacteria, inhibits a number of immune system effector functions, including interferonc-mediated activation of macrophages [4]. ManLAM also inhibits the production of the pro-inflammatory cytokines interleukin-12 [4] and tumor necrosis factor-a [9]. PILAM, which is characteristic of the fast-growing saprophytic mycobacteria, can induce a pro-inflammatory response in a Toll-like 2 receptor-dependent manner [10].
The current model of lipoglycan biosynthesis is supported by biochemical and genetic studies, and follows a linear pathway from PIRPIM 2 RLMRLAM [4]. PimA (Rv2610c) and PimB' (Rv2188), are a-mannopyranosyltransferases, belonging to the GT-B superfamily and utilize GDP-mannose [11,12], adding Manp residues at positions O-2 and O-6 of PI, respectively to produce PIM 2 , that can be subsequently acylated by Rv2611c, at the 6 position of the Manp residue to generate Ac 1 /Ac 2 PIM 2 [13]. RvD2-ORF1 from M. tuberculosis CDC1551, designated as PimC and an unknown glycosyltransferase (PimD), catalyzes the addition of a Manp residue from GDP-mannose to 6-OH of mannose at the non-reducing end of Ac 1 /Ac 2 PIM 2 to generate Ac 1 /Ac 2 PIM 3 [14] and Ac 1 /Ac 2 PIM 4 , respectively. At this key junction point in the biosynthetic pathway, polyprenylphosphate-based mannose (PPM) donors are employed by the GT-C superfamily for elongation and branching of Ac 1 /Ac 2 PIM 4 leading to Ac 1 /Ac 2 PIM 6 , LM and LAM [15].
The pathway splits into two branches from Ac 1 /Ac 2 PIM 4 : one leads to the formation of Ac 1 /Ac 2 PIM 6, whilst the other leads to the formation of LM and LAM. In the first branch, a(1R2) Manp is added to Ac 1 /Ac 2 PIM 4 to form Ac 1 /Ac 2 PIM 6 by two consecutive mannose additions catalyzed by PimE (Rv1159), a PPM-dependent a(1R2)-mannopyranosyltransferase and a second additional uncharacterized putative glycosyltransferase of the GT-C superfamily [16] . In the second branch, Ac 1 /Ac 2 PIM 4 is hypermannosylated by glycosyltransferases of the GT-C superfamily to generate LM. Recent studies have established MptB (Rv1459c) and MptA (Rv2174) to be involved in the synthesis of the mannan backbone, where MptB catalyzes the synthesis of the proximal end through the addition of 12-15 Manp residues to the backbone and MptA synthesizes the distal end of the a(1R6) mannan core of LM [17][18][19]. The a(1R6)-mannan core, synthesized by MptB and MptA, is further branched by MptC (Rv2181) which adds a(1R2)-Manp residues to the side chains of LM [20]. The transition from LM to LAM is catalyzed by EmbC (Rv3793) and involves the utilization of LM through initial priming with Araf units from the sugar donor decaprenylphosphate arabinan (DPA), by an unknown GT-C glycosyltransferase [21]. EmbC is then responsible for extension of the primed LM through the addition of 12-16 a(1R5)-Araf residues [22]. The addition of branch points is similar to AG, catalyzed by AftC [23]. The arabinan domain as found for AG synthesis is likely to be terminated by AftB (Rv3805c) [24]. The homologue of Rv1635c in M. tuberculosis CDC1551 has been shown to be involved in Man-LAM capping [6]. This enzyme was also shown to be PPMdependent and is now termed as CapA, which adds the first Manp residue onto the non-reducing arabinan termini of LAM [25]. In M. tuberculosis, MptC (Rv2181) a PPM-dependent a(1R2) mannosyltransferase has dual functionality in branching LM, and also for producing ManLAM that is fully functional via the capping of LAM with a(1R2)-Manp residues at the non-reducing end of LAM [4].
In vitro studies have shown that the key PPM sugar donor for the GT-C glycosyltransferases involved in LM/LAM biosynthesis is generated by Mt-Ppm1 (Rv2051c), a DPM-like synthase that uses GDP-Man and C 50 /C 35 -polyprenol phosphates as substrates [26]. Previous studies have identified subtle variations in the organization of the ppm1 locus in mycobacteria [26]. The well characterized Mt-ppm1 of M. tuberculosis encodes a large polypeptide consisting of two domains, Mt-ppm1/D1, which is membrane-anchored via six transmembrane segments, and Mt-ppm1/D2 which is sufficient for DPM synthase activity [26,27]. However, in Mycobacterium smegmatis, these two domains are encoded by two distinct ORFs MSMEG3860 (Domain 1, Ms-ppm2) and MSMEG3859 (Domain 2, Ms-ppm1) arranged in an operon (Figure 1), an arrangement also found in Mycobacterium leprae, Mycobacterium avium and the related Corynebacterium glutamicum [26,28]. Studies using bacterial two-hybrid systems have shown that MSMEG3859 and MSMEG3860 interact with each other [27]; while MSMEG3859 was sufficient for PPM synthase activity, this interaction with MSMEG3860 stabilized the synthase [26,27]. Similarly, in M. tuberculosis, a recombinant protein consisting only of the C-terminal domain (Mt-Ppm1/D2) was sufficient for generation of PPM activity [26]. However, whilst in vitro data has shown that Mt-Ppm1 (and MSMEG3859) has PPM synthase activity, there have been no genetic studies that demonstrate that Mt-Ppm1 is the sole PPM generating enzyme encoded by the M. tuberculosis genome. If this was the case, in view of the critical role of PPM for biosynthesis of LM/ LAM, Mt-ppm1 would be expected to be an essential gene and thus genetic studies would be possible only via the generation of a conditional knockout strain. The need to study the in vivo role of ppm1 by generation of a conditional mutant strain was further necessitated by conflicting reports that another membraneassociated glycosyltransferase, Rv3779 functions as a PPM synthase in M. tuberculosis [29], and as a glycosyltransferase that uses polyprenyl-P-D-GalNAc as a D-GalNp (or D-GalNAc) donor for transfer to 3,5-branched D-Araf residues of AG [30]. Additionally, the latter report also describes Rv3631 (ppgS) and Rv3632 as genes encoding a polyprenyl-P-D-GalN synthase and a small integral membrane protein respectively, analogous to Mt-Ppm1/D2 and Mt-Ppm1/D1. While PpgS is a GT-2 family glycosyltransferase involved in the generation of polyprenylphospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc, it could be envisaged as a secondary, potential PPM synthase.
In an effort to first confirm the in vivo role of Mt-Ppm1/D2 in LM/LAM biosynthesis we aimed to test the essentiality of PPM glycosyltransferase activity in M. smegmatis by using CESTET, a genetic tool for testing gene essentiality in M. smegmatis. As M. smegmatis does not encode homologues of ppgS or Rv3779, it also provided us with a valuable surrogate to probe the in vivo role and potential essentiality of MSMEG3859 in the absence of any potential functional redundancy caused by an alternative PPM synthase. Additionally, mutant or conditional mutant strains of M. smegmatis could subsequently be used as a host strain to test functional complementation of PPM synthase activity using recombinant ppgS or Rv3779.

Generation of the DMsPpm Conditional Mutant
The M. smegmatis conditional mutant DMsPpm was generated using CESTET [33]. Briefly, a merodiploid was first generated by introducing pTIC6a-MSMEG3859 by electroporation into M. smegmatis mc 2 155 [33]. The merodiploid strain mc 2 155::pTIC6a-MSMEG3859 was then subjected to specialized transduction as previously described [34] using a temperaturesensitive, recombinant phage phDMsPpm designed to replace MSMEG3859-MSMEG3860 with a hygromycin resistance marker. Transductants were selected at the non-permissive temperature of 37uC on selective plates containing 25 mg/ml kanamycin, 100 mg/ml hygromycin B and 50 ng/ml anhydrotetracycline (ATc). After confirmation of gene replacement by Southern blot, one such transductant was named DMsPpm and was selected for further analysis.

Conditional Depletion of DMsPpm Conditional Mutant
The DMsPpm conditional mutant was grown in Tryptic Soy Broth (TSB; Difco) containing 0.05% Tween 80, 25 mg/ml kanamycin, 100 mg/ml hygromycin B and 50 ng/ml ATc and subsequently passaged twice in medium without ATc. To visualize the effects of the conditional depletion of MSMEG3859 in DMsPpm on lipids and lipoglycans, the strains were grown to OD 0.8, labelled with 10 mCi/ml glucose D-[ 14 C(U)] (specific activity 250-360 mCi (9.25-13.3 GBq)/mmol; Perkin Elmer) and incubated at 37uC for 4 hours.

Extraction of Polar Lipids
Polar lipids and apolar lipids were extracted as described previously [35]. Briefly, cells from a 10 ml volume culture were washed once with 2 ml phosphate buffer saline (PBS) and treated with 2 ml CH 3 OH:0.3% NaCl (aq) (100:10, v/v) and 2 ml petroleum ether for 30 min. The suspension was centrifuged and the upper layer containing apolar lipids was separated. An additional 2 ml of petroleum ether was added, mixed and centrifuged as described above and the two upper apolar lipid fractions were combined and dried. For polar lipids, 2.3 ml of CHCl 3 :CH 3 OH:0.3% NaCl (aq) (90:100:30, v/v/v) was added to the cell pellet and mixed for 1 h. This mixture was centrifuged and the supernatant was separated. The remaining cell pellet was mixed with 750 ml of CHCl 3 :CH 3 OH:0.3% NaCl (aq) (50:100:40, v/v/v) for 30 min, centrifuged and the supernatant was combined to the previous fraction. After repeating this step, 1.3 ml of CHCl 3 and 1.3 ml of 0.3% NaCl (aq) was mixed with the pooled supernatant, centrifuged, and the lower layer containing the polar lipids was recovered and dried. The polar lipid extracts were dried and resuspended in CHCl 3 :CH 3 OH (2 : 1, v/v), and incorporation of glucose D-[ 14 C(U)] was quantified by liquid scintillation counting using 5 % of the lipid fractions in 5 ml EcoScint A (National Diagnostics). Equal counts of polar lipid extracts (50 000 cpm) were applied to Silica Gel 60 F 254 (Merck 5554)

Extraction and Purification of Lipoglycans
Lipoglycans were extracted as described previously [36]. Briefly, dried cells from a 10 ml volume culture were resuspended in water and refluxed five times with equal volume of 50% C 2 H 5 OH at 85uC, for 6 h intervals, followed by centrifugation and recovery of the supernatant. The combined supernatants were dried and subjected to hot phenol-H 2 O treatment at 65uC. The aqueous phase containing the crude lipoglycan fraction was dialyzed against water, dried and the incorporation of glucose D-[ 14 C(U)] was quantified by liquid scintillation counting using 5 % of the lipoglycan fractions in 5 ml EcoScint A (National Diagnostics). Equal counts (50 000 cpm) were loaded on a 15% SDS-PAGE gel and separated by electrophoresis. Lipoglycans were visualized by 48 h exposure on x-ray films by autoradiography (Kodak Biomax MR film).

PPM Synthase Assay
Membranes were prepared as described previously [37]. Briefly, cells were grown to mid-log phase, harvested, washed with PBS and stored at -20uC. Cells were washed and resuspended in buffer A containing 50 mM MOPS (adjusted to pH 7.9 with KOH), 5 mM b-mercaptoethanol and 10 mM MgCl 2(aq) at 4uC and subjected to sonication for a total time of 10 min using 60 s pulses and 90 s cooling intervals. The preparations were centrifuged at 270006g for 25 min at 4uC and the membranes were obtained by further centrifugation of the supernatant at 100,0006g for 1 h at 4uC. Membranes were resuspended in 1 ml of buffer A and concentration was determined using the BCA Protein Assay Reagent Kit (Thermo Scientific). Reaction mixtures for assessing [ 14 C]Man incorporation consisted of 6.25 mCi GDP[Man-14 C(U)] (262 mCi/mmol; Perkin Elmer), 100 mM ATP, 10 mM MgCl 2(aq) , 100 mM dithiothreitol, 20 mM NaF (aq) and membrane preparations corresponding to 50-400 mg protein in a final volume of 100 ml. Decaprenyl monophosphate was added to the reaction mixtures at a final concentration of 125 mM. The reaction mixtures were then incubated at 37uC for 30 min. The enzymic reactions were terminated by the addition CHCl 3 /CH 3 OH/ 0.8 M NaOH (aq) (10:10:3 by vol.) (6 ml/100 ml) followed by further incubation at 55uC for 20 min. The mixtures were then allowed to cool; 2.625 ml of CHCl 3 and 1.125 ml of water were added. The mixture was vortexed and centrifuged and the upper aqueous phase discarded. The organic phase was washed three times with 2 ml of CHCl 3 :CH 3 OH:H 2 O (3:47:48 by vol.), dried to yield an organic fraction containing PPMs. These were dried in a scintillation vial before scintillation counting using 5 ml of EcoScint A (National Diagnostics) [26].

Essentiality of MSMEG3859 in M. smegmatis
Due to its role in the biosynthesis of LM/LAM, Mt-ppm1 was considered to be an essential gene. We constructed a knockout phage phDMsPpm designed to replace MSMEG3859-MSMEG3860 in M. smegmatis with a hygromycin resistance cassette; we were however unable to generate a null mutant due to the failure to yield any transductants. In contrast, we were able to generate a MSMEG3859-MSMEG3860 null mutant by trans-ducing a merodiploid strain containing a second, inducible copy of MSMEG3859-MSMEG3860 by CESTET (Conditional Expression Specialized Transduction Essentiality Test) [33]; suggesting that one or both genes were essential in M. smegmatis. As MSMEG3859 was shown to be sufficient for the enzymatic generation of PPM in vitro, we reasoned that the PPM synthase-encoding MSMEG3859, but not the membrane segment-encoding MSMEG3860, was an essential gene. To address this, we used CESTET again to test the essentiality of MSMEG3859 in M. smegmatis. First, a merodiploid strain was constructed by introducing a second copy of MSMEG3859 cloned in pTIC6a, an integrative plasmid driving expression via a tetracycline inducible promoter [38]. Expression of this recombinant copy of MSMEG3859 can be induced by adding anhydrotetracycline (ATc) to the growth medium. Following transduction with knockout phage phDMsPpm, we were able to generate knockout mutants only when transductants were selected on plates containing hygromycin and ATc suggesting that MSMEG3859 was essential in M. smegmatis. One such conditional mutant, designated DMsPpm was used for further analysis. Subsequent passages of the DMsPpm mutant in medium without the inducer ATc resulted in loss of viability indicating that expression of the pTIC6a-driven copy of MSMEG3859 was required for cell growth, confirming the essentiality of MSMEG3859 (Figure 2).
The ability to generate an MSMEG3859-MSMEG3860 double mutant in a MSMEG3859 merodiploid strain indicated that while the PPM synthase-encoding MSMEG3859 was essential, MSMEG3860 (which encodes a six transmembrane sectioncontaining membrane-anchored protein) was not essential. This Ppm1 Is Essential in Mycobacteria PLOS ONE | www.plosone.org correlated with earlier reports that MSMEG3859 was sufficient for PPM synthase activity [26,27].

Loss of MSMEG3859 Results in Alteration of PIMs and Cessation of LM Biosynthesis
As Ac 1 /Ac 2 PIM 4 is at the branch point for the biosynthesis of higher PIMs (Ac 1 /Ac 2 PIM 6 ) and LM/LAM biosynthesis, the conditional DMsPpm mutant could be used to determine whether loss of MSMEG3859-encoded PPM activity affected the biosynthesis of these molecules. The mutant was grown for 36 hours in media in the presence, or absence, of ATc labelled with [ 14 C]glucose and subjected to lipid extractions. Cultures grown in ATccontaining media showed all PIM intermediates present. In contrast, cultures of the conditional mutant grown in the absence of ATc showed increasing amounts of PI accompanied by decreasing levels of Ac 1 /Ac 2 PIM 6 ( Figure 3). The loss of Ac 1 / Ac 2 PIM 6 and accumulation of PIM x precursors suggested that MSMEG3859 is required for the synthesis of higher order PIMs, particularly Ac 1 /Ac 2 PIM 6 . Conditional depletion of PPM synthase activity in the DMsPpm mutant should also affect the biosynthesis of lipoglycans and cultures grown in the absence of ATc did show diminished levels of [ 14 C]LM ( Figure 4). However, we did not see major differences in [ 14 [37]. These data suggest that MSMEG3859, which encodes the equivalent of M. tuberculosis Ppm1/D2, is solely responsible for the PPM synthase activity required for the generation of higher order PIMs and LM/LAM.  Effects of Loss of MSMEG3859 on Membrane-associated PPM Synthase Activity Membrane preparations from cultures of the DMsPpm mutant, grown in the presence and in the absence of ATc, were used to assay membrane-associated PPM synthase activity. A PPM synthase assay was used to analyse PPM activity in the membranes using GDP-[ 14 C] Man and polyprenol phosphate as substrate. This assay was used to assess the effects of depletion of MSMEG3859 function on the ability of the membrane preparations to catalyse the formation of PPM (C 50 -P-Man) donors via the incorporation of radioactive mannose into polyprenol substrate through pooled organic extracts containing PPMs [26]. While membrane preparations from cultures grown in the presence of ATc were able to catalyse incorporation of [ 14 C] Man into polyprenols, those from cultures grown in the absence of ATc displayed poor PPM synthase activity ( Figure 5). This co-relation between depleted MSMEG3859 function and low PPM synthase activity confirmed that MSMEG3859 was the key synthase required to catalyze the production of PPM donors in M. smegmatis.
Together, the data obtained from the depletion experiments with the conditional mutant highlight the potential of the MSMEG3859-encoded PPM synthase as a potential drug target that affects not only viability, but also the biosynthesis of a immunomodulatory mycobacterial lipoglycan.
Potential Ability of the Alternative M. tuberculosis PPM Synthases to Rescue Viability and Restore Wild-type Phenotype in the DMsPpm Mutant In contrast to M. smegmatis, M. tuberculosis encodes two other membrane-associated glycosyltransferases, Rv3779 and Rv3631 (PpgS), which have been suggested to have putative roles as PPM synthases in M. tuberculosis [30]. As mentioned above, homologues of neither are not present in M. smegmatis. The DMsPpm conditional mutant thus offered us an opportunity to assess the possible roles of Rv3779 and PpgS as alternative PPM synthases.
Each gene could be functionally analyzed in vivo by testing the ability of the DMsPpm conditional mutant transformed with a plasmid-borne copy of either Rv3779 or ppgS to rescue Ac 1 / Ac 2 PIM 6 biosynthesis when cultured in medium devoid of ATc. As expected, Ac 1 /Ac 2 PIM 6 biosynthesis was not affected in non-ATc cultures of DMsPpm containing plasmid clones of either MSMEG3859 ( Figure 6). We then tested the ability of the M. tuberculosis equivalent, Mt-ppm1/D2 to rescue the loss of MSMEG3859 function in the same manner and found Ac 1 / Ac 2 PIM 6 levels unaffected in the recombinant strains ( Figure 6) indicating that Mt-ppm1/D2 was functional in M. smegmatis. In contrast, however, Rv3779 was unable to rescue Ac 1 /Ac 2 PIM 6 biosynthesis in the conditional mutant when grown in the absence of ATc, leading to a loss of Ac 1 /Ac 2 PIM 6 and a noticeable accumulation of intermediary Ac 1 /Ac 2 PIM x s ( Figure 6). Thus while Rv3779 was shown to have PPM synthase activity in vitro [29], it surprisingly failed to substitute for loss of MSMEG3859 in the conditional DMsPpm mutant. Thus, in contrast to its in vitro activity, it is unlikely that Rv3779 functions in vivo as a PPM synthase. Instead, its predominant role in M. tuberculosis seems to be the utilisation of polyprenyl-P-D-GalNAc as a donor for the biosynthesis of galactosamine-modified AG [30].
As mentioned above, while PpgS is involved in the generation of polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-Gal-NAc) from polyprenyl-P and UDP-GalNAc, it could be envisaged as a secondary, potential PPM synthase. Its syntenic association with the small integral membrane protein Rv3662 is analogous to MSMEG3859-MSMEG3860, and Mt-Ppm1/D2-Mt-Ppm1/D1. However, ppgS also failed to compensate for the loss of MSMEG3859 in the conditional mutant ( Figure 6), and thus is unlikely to function as a PPM synthase in vivo.
Together, these results indicate that ppm1 is the gene encoding the sole PPM synthase in M. tuberculosis capable of generating the mannose donor for subsequent higher order PIMs and LM/LAM biosynthesis.