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The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal α-[1→2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death.
One of the key steps in the pathogenesis of Mycobacterium tuberculosis (M.tb) infection is the phagocytosis and replication of the tubercle bacillus within human macrophages. In this process, M.tb has been shown to engage macrophage complement, mannose, scavenger, and toll-like receptors (reviewed in Schlesinger et al. (2008)). Studies indicate that entry via the mannose receptor (MR) provides a safe portal for the bacillus by bypassing the oxidative response and by modulating calcium responses, phagosome–lysosome fusion, and cytokine responses (Astarie-Dequeker et al. 1999; Kang et al. 2005). Therefore, M.tb surface-exposed molecules like phosphatidyl-myo-inositol mannosides (PIMs) and mannose-capped lipoarabinomannan (ManLAM), which contain terminal mannose residues are of particular interest due to their interaction with the host MR (Schlesinger et al. 1994, 1996; Kang et al. 2005; Torrelles et al. 2006). M.tb ManLAM has been shown to regulate host cell trafficking, signaling, and nitric oxide production (reviewed in Schlesinger et al. (2008)). M.tb PIMs have been shown to mediate host cell receptor-mediated interactions, where lower-order PIMs interact with complement receptor 3 and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) (Villeneuve et al. 2005; Torrelles et al. 2006) and higher order PIMs interact with the MR on human macrophages reducing phagosome–lysosome fusion (Torrelles et al. 2006). Based on their importance in regulating macrophage immune responses, we are generating M.tb gene disruption mutants involving key enzymes in the biosynthesis of PIMs and ManLAM to determine their impact on the production of these lipoglycans and effects on M.tb–human macrophage interactions.
Although ManLAM, lipomannan (LM), and PIMs share a common biosynthetic pathway (Besra et al. 1997), the inherent complexity of this pathway adds difficulty to the search for key enzymes involved in their biosynthesis. The implied biosynthetic pathway of PI→PIM1→PIM2→PIM3→ PIM4→linear-LM→LM→ManLAM is somewhat supported by biochemical and genetic studies. For the PIM biosynthesis pathway, PgsA has been described as the enzyme responsible for the synthesis of the PI, which serves as a membrane anchor for PIMs, LM, and ManLAM (Jackson et al. 2000). PimA (Rv2610c) was later described to add the first mannosyl residue to the PI anchor to form diacylated PIM1 (PIM1) (Kordulakova et al. 2002). PIM1 is further acylated by Rv2611c (Kordulakova et al. 2003) and mannosylated by a reaction catalyzed by PimB (Rv0557) resulting in the formation of Ac1PIM2 (Schaeffer et al. 1999). PimC (RvD2-ORF1) has been shown to add a third mannose to Ac1PIM2 to form Ac1PIM3 (Kremer et al. 2002). The mycobacterial mannosyltransferase PimE (Rv1159) has been described to catalyze the formation of higher order PIMs, specifically Ac1PIM5 (Morita et al. 2006). Recently, a gene has been identified involving the regulation of higher order PIM synthesis versus LM and/or LAM production (Kovacevic et al. 2006) suggesting independent biosynthetic pathways for these lipoglycans.
In the present study, we focus on M.tb Rv0557 (pimB). As noted above, Schaeffer et al. (1999), using in vitro cell-free reactions with M. smegmatis membranes, have shown that PimB encodes a functional GDP-mannose (GDP-Man)-dependent phosphatidyl-myo-inositol monomannoside transferase that catalyzes the addition of a second mannose residue from GDP-Man to the inositol moiety of Ac1PIM1 to generate Ac1PIM2. Ac1PIM2 is thought to constitute the anchor portion of LAM; therefore, PimB is proposed to be a key enzyme for the production of LM and LAM (Schaeffer et al. 1999). Our results show that the inactivation of pimB in a virulent strain of M.tb by allelic-exchange mutagenesis does not alter growth of the mutant strain in culture relative to the control strain indicating that pimB is not essential for M.tb growth. In addition, although the inactivation of pimB produced an alteration in colony morphotype, it does not abolish the presence of PIMs, LM, and ManLAM in the cell wall, pointing to the complexity and likely redundancy that exist in the steps of the LAM biosynthetic pathway. Although there were no qualitative differences in structure, biochemical analyses of LM and ManLAM revealed a reduction in their cell wall content by ~68% and ~57%, respectively, in the pimB mutant when compared to M.tb Erdman wild type, whereas there was no reduction in either lower order or higher order PIMs. The gene inactivation of pimB did not affect growth of the mutant strain within human macrophages; however, unexpectedly the pimB mutant strain exhibited an enhanced killing of primary human macrophages in in vitro cultures.
To further define the role of M.tb surface mannose-containing glycoconjugates in bacteria–macrophage interactions, we inactivated the gene encoding PimB, a proposed key mannosyltransferase which is thought to be essential in the biosynthesis of ManLAM. The pimB knock-out strain (pimB-KO) was achieved after electroporation of a suicide vector containing a kanamycin resistance (aph) cassette inserted into the center of a PCR-amplified 2.7 Kb segment of M.tb DNA encompassing the pimB ORF and flanking DNA segments. Allelic replacement and homologous recombination of pimB were confirmed by PCR (Figure (Figure1A)1A) and Southern blot analyses (Figure (Figure1B),1B), respectively. The pimB mutation was complemented by construction of a single insertion of the wild-type gene into the attB locus (pimB-Compl). A strain over-expressing pimB (pimB-Overexp) was produced after a multicopy plasmid containing pimB was transformed into M.tb Erdman.
The absence of transcript in the pimB-KO and its presence in the complemented and over-expressing strains was assessed by RT-PCR (Figure (Figure1C).1C). RNA was isolated from log phase bacteria, reverse transcribed, and PCR performed on cDNA targeting transcripts for pimB and controls hspX and aroB. The pimB transcript was not detected in the pimB-KO demonstrating that insertion of the aph Kanr cassette had abrogated gene transcription. The pimB transcript was present in the M.tb Erdman and pimB-Compl strains. Although this assay is nonquantitative, the pimB transcript in the pimB-Overexp strain appears to be present at higher levels when compared to the wild-type M.tb Erdman strain (Figure (Figure11C).
Several studies show a direct correlation between changes in colony morphology and alterations in the mycobacterial cell wall (reviewed in Barry (2001)), and altered morphology was observed for the pimB mutant when compared to controls (Figure (Figure2).2). The pimB-KO strain displayed a smoother colony morphotype with less ruffling and shallower surface grooves. The presence of multiple copies of pimB in the pimB-Overexp strain created a particularly dense, rough, and elevated colony morphotype (data not shown). These variations in colony morphology suggested the possibility of alterations in the cell wall architecture that are directly related to the level of PimB produced.
To examine for alterations in components of the mycobacterial cell wall that are related to pimB expression, we performed several biochemical analyses. First, neutral sugar analysis of whole cells was performed using gas chromatography (GC) following hydrolysis and conversion of carbohydrates to alditol acetates. There was an overall reduction in the content of arabinose (~4.2-fold), mannose (~4.8-fold), and myo-inositol (~4.5-fold) in the pimB-KO strain (Table (TableI)I) suggesting a reduction in mannosylated cell wall components due to the absence of PimB. Our analysis of the rhamnose and galactose content in whole cells suggested that the reduction observed in arabinose may result primarily by a decrease in ManLAM or possibly in the related arabinomannan. The carbohydrate profile returned to control Erdman strain levels in the complemented strain. The carbohydrate content in the pimB-Overexp strain resembled the content in the control strain (data not shown).
Other modifications in the cell wall components were analyzed in total lipid extracts by 1D thin layer chromatography (TLC). Results did not show significant qualitative differences between strains (data not shown). However, neutral sugar analysis of the crude lipid extracts (Table (TableI,I, normalized by protein content) showed that the pimB-KO strain had a reduction in mannosylated glycolipids as indicated by its decrease in the myo-inositol and mannose content. The strain over-expressing pimB did not have an alteration in any of these carbohydrates when compared to the control Erdman strain (data not shown).
As pimB has been described to function in the production of Ac1PIM2 (Schaeffer et al. 1999), the anchor domain of LM and ManLAM, PIM profiles were determined for the mutant and wild-type Erdman strains (Figure (Figure3).3). PIMs were extracted and analyzed by 2D TLC and results showed that the PIM profiles were similar for all strains; most notably Ac1PIM2 was present in the pimB-KO strain. Purification by preparative TLC (Figure (Figure4A)4A) and characterization by MALDI-MS (matrix-assisted laser desorption-time of flight mass spectrometry) (Figure (Figure4B)4B) confirmed that the PimB-KO strain had all of the predicted PIM types. The signal m/z 1459.92 (band B, Figure Figure4B)4B) can be assigned as the sodiated molecular ion [M−H+2Na]+ of Ac1PIM2 with palmitic (C16:0) and tuberculostearic (TBST) acids on the C-1 and C-2 position of the glycerol and an additional C16:0 either on the C-6 position of the α(1→2)-linked mannose or on the C-3 of the myo-inositol (Khoo et al. 1995). The observation that the molecular ion of band A at m/z 1740.39 is a tuberculosterate anion (m/z 297) minus a hydroxyl group (–OH, m/z 17) higher than m/z 1459.92 of band B suggested that band A corresponded to the sodiated molecular ion of Ac2PIM2 [M−H+2Na]+ with its additional fatty acid being TBST. Additionally, no differences in PIM acylation patterns and in the relative amounts of higher order PIMs were observed among all strains studied.
Characterization of LM and ManLAM was performed on M.tb lysates from the different bacterial strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) followed by periodic acid silver (PAS) staining showed that LM and ManLAM were present in all strains with no discernible differences in the size distribution of these lipoglycans (Figure (Figure5A).5A). The quantities of LM and ManLAM did not appear to differ between the wild-type Erdman strain and pimB-KO strain when the samples were analyzed by gel electrophoresis at 20 μg protein equivalents or higher. However, as the protein equivalents were serially reduced, SDS–PAGE of the extracts showed a consistent, quantitative decrease in LM and ManLAM in the pimB-KO strain with respect to the control strain (Figure (Figure5A).5A). Densitometry analysis (Figure (Figure5C)5C) of the gels at 6.25 μg protein equivalents confirmed this result, where the pimB-KO strain had a 67.5 ± 6.0% and 56.8 ± 1.0% reduction in its LM and ManLAM content, respectively. This decrease was also verified by Western blot using the anti-LAM mAb CS-35 (data not shown). These data support the reduction in the LM and ManLAM content observed by neutral sugar analysis of these soluble carbohydrate extracts (Table (TableI,I, lysates) where a 1.8-fold decrease in total arabinose and a 1.6-fold decrease in total mannose were observed only for the pimB-KO strain. This decrease was restored to control Erdman strain levels in the complemented strain. The presence of equal myo-inositol amounts among lysates in Table TableII may be explained by traces of soluble and/or micellar PIMs and phosphatidyl-myo-inositol (PI) observed in these samples.
M.tb surface components are known to interact with specific host cell receptors; therefore, we tested whether the decrease in LM and ManLAM observed in the pimB-KO strain affected the association of M.tb with primary human macrophages. Human monocyte-derived macrophage (MDM) monolayers were incubated for 2 h with M.tb strains in the presence of autologous serum (as a measure of opsonic binding), no serum (as a measure of nonopsonic binding), and serum plus mannose-BSA (to study specific bacterial association with the MR). No consistent differences in the number of bacteria associated with macrophages for the M.tb Erdman and pimB-KO strains were observed under all three conditions. The number of bacteria per cell in the Erdman versus pimB-KO strain with serum was 9.4 ± 0.9 and 10.6 ± 0.3 (mean ± SD, n = 2) respectively, with no serum 1.8 ± 1.2 and 4.0 ± 0.5, and with serum plus mannose-BSA 3.4 ± 0.5 and 3.9 ± 0.5. Both the Erdman and pimB-KO strains showed the greatest level of macrophage association in the presence of serum, and a marked reduction in the number of bacteria per macrophage in either the absence of serum or the addition of soluble mannan.
Several studies have shown that alterations in the M.tb cell wall result in a decrease in bacterial growth within macrophages in vitro (reviewed in Barry (2001)). However, we observed no differences in bacterial growth in MDM cultures that were incubated with control M.tb Erdman, pimB-KO, or pimB-Compl strains in the presence of autologous serum (the condition in which the greatest level of association was observed) up to 40 h postinfection (data not shown). Colony morphotype and PCR analysis (for strain genotype) from randomly selected colonies on colony forming unit (CFU) plates showed no alterations related to passage through macrophage culture. Antibiotic selection in the M.tb medium had no effect on subsequent macrophage infection and/or growth, as the pimB-KO strain cultured on 7H11 agar plates with and without Kan produced similar results in MDM cultures. In summary, these results indicate that the inactivation of pimB does not affect phagocytosis and intracellular growth of M.tb in in vitro macrophage cultures despite reducing the cell wall content of LM and ManLAM.
Despite no effects on macrophage uptake and intracellular growth, striking differences were observed in the phenotype of macrophages infected with the pimB-KO strain when compared to the control M.tb Erdman strain. MDMs infected with M.tb Erdman displayed characteristic homotypic adhesion over time (Figure (Figure6A,6A, arrow) (Desjardin et al. 2002). As the time following initial infection increased, macrophage aggregates enlarged in size and number ultimately detaching from the surface of the tissue culture plate. MDMs present in smaller aggregates or as single cells on the monolayer excluded trypan blue; thus, they were viable. Larger clumps were found mainly as mixtures of trypan blue-excluding and -staining cells, with the majority of the detached cells staining with trypan blue, and therefore considered dead. Timing of macrophage aggregation, detachment, and death varied amongst individual macrophage donors and according to the multiplicity of infection (MOI); however, the appearance of the macrophage monolayers and progression in cytopathology observed were comparable in all cases. In contrast, infection with the pimB-KO strain resulted in an altered macrophage phenotype with the level of homotypic adhesion greatly reduced at all time points. Macrophages also appeared to have contracted cytoplasm and monolayers became less dense at a faster rate than those observed following control M.tb Erdman infection for a given MOI (Figure (Figure6B).6B). Sham-infected human macrophages displayed a round appearance without any changes observed during the course of manipulation or incubation (Figure (Figure66C).
Given the marked differences in appearance of the macrophage monolayers following infection with pimB-KO and the control Erdman strain, and the apparent more rapid loss in cell monolayer following infection with pimB-KO, nuclei were isolated from monolayer and supernatant fractions and counted to allow for a more accurate quantification of macrophage cell numbers (Nakagawara and Nathan 1983). Results from macrophages derived from three independent donors showed a significantly greater loss of viable macrophages (reduction in nuclei) only after infection with pimB-KO (Figure (Figure7A).7A). The loss in viability occurred despite no significant differences in CFUs following the 2 h infection period compared to the control M.tb Erdman strain (Figure (Figure7B),7B), indicating that the more rapid monolayer destruction produced by pimB-KO was not simply a reflection of a faster growth rate and/or greater numbers of bacilli per macrophage. Assessment of macrophage viability using the pimB over-expressing strain showed results comparable to those obtained with the control M.tb Erdman strain (data not shown).
The specific number of α-mannosyltransferases involved in PIM, LM, and ManLAM biosynthesis is still unknown. In this study, we focus on the mannosyltransferase PimB, a proposed key enzyme in the biosynthesis of PIMs, LM, and ManLAM (Schaeffer et al. 1999). Although the function of PimB was clearly demonstrated in in vitro studies using M. smegmatis membranes and partially purified recombinant PimB protein from E. coli, here we demonstrate that the inactivation of pimB in a virulent M.tb strain leads to a quantitative decrease rather than abolishment of the production of LM and ManLAM.
Our results show that there are likely to be other mannosyltransferases that are functional in the absence of pimB. Analysis of the M.tb H37Rv genome shows that there are four ORFs homologous to pimB (Rv0486c, Rv2160c, Rv2188c, and Rv3032) with ~25% and 40% amino acid identity and similarity, respectively, and Rv2188c has recently been described to be important in the conversion of Ac1PIM1 to Ac1PIM2 in Corynebacterium (Lea-Smith et al. 2008). All of these pimB homologs contain the conserved glycosyltransferase EX7E domain. Although Rv0486 has been recently described to catalyze the first step of mycothiol biosynthesis (mshA) (Newton et al. 2003), it is possible that in vivo one or more of these homologs are able to serve in a similar fashion to pimB, thus allowing for the biosynthesis of PIMs, LM, and ManLAM in the pimB-KO strain.
This is consistent with work by Kremer et al. (2002), where the inactivation of pimC, which catalyses the formation of Ac1PIM3 in M. bovis BCG, did not affect the production of higher order PIMs, LM, and ManLAM when compared to WT M. bovis BCG, supporting the concept of gene redundancy or alternative pathways in ManLAM biosynthesis. It is also possible that pimB has an additional primary activity in vivo different from that previously described in vitro. For example, M.tb pimB expressed in Corynebacterium has been recently shown to participate exclusively in the biosynthesis of a diacylglycerol lipid modified with a Manα(1→4)GlcA head group (Tatituri et al. 2007), whereas M.tb Rv2188c was found to be the gene that participated in the conversion of Ac1PIM1 to Ac1PIM2 (Lea-Smith et al. 2008). These studies together with our results and the fact that the M. leprae genome does not contain a pimB ortholog (Cole et al. 2001) yet produces PIMs, LM, and ManLAM (Torrelles et al. 2004), demonstrate the high redundancy and alternative biosynthetic pathways that must exist in mycobacteria to build these essential mannosylated lipoglycans. Different acceptor and donor specificities (e.g., degree of acylation in the substrates) may play an important role at some biosynthetic steps (Figure (Figure8)8) (Berg et al. 2007).
In the current study, the reduction of the ManLAM/LM content in the pimB-KO strain correlates with a change in bacterial colony morphology and the macrophage response to its infection. However, this reduction does not lead to a decrease in bacterial association with the human macrophage MR. This can be explained by the fact that no differences were observed in the relative amounts of higher order PIMs, which together with ManLAM serve as major ligands for the MR as well as for the dendritic cell C-type lectin DC-SIGN (Villeneuve et al. 2005; Torrelles et al. 2006). It is also possible that the residual amount of ManLAM exposed on the surface of the pimB-KO strain was enough to allow for detectable recognition by the MR within the sensitivity limits of the in vitro assay.
From our study, the most striking phenotype associated with the pimB-KO is the reduction in macrophage homotypic adhesion in conjunction with faster death, features that were reversed in macrophages infected with the complemented strain. The level of macrophage death was quantifiable at early time points after infection, when the monolayer was still largely intact. However, the entire monolayer was lysed more rapidly following infection with the pimB-KO without any large aggregations of detached macrophages, as is typically observed following infection with wild-type M.tb Erdman. This increased rate of death in the mutant strain was not due to a higher initial infection of macrophages, measured as either bacteria per cell or numbers of cells infected, or to a greater increase in the numbers of intracellular bacteria over time. Eventually, both pimB-KO and the parent Erdman strain kill all of the macrophages in monolayer culture; however, the progress toward that outcome was substantially altered in the pimB-KO. How or if the reduction in the abundance of LM and ManLAM affects the macrophage response is unknown. One possibility is that a reduction in the presence of LM and ManLAM displayed on the cell wall surface may enhance exposure to the host of other normally hidden cytotoxic or inflammatory-producing glycolipids (e.g., trehalose dimycolate (TDM), sulfolipids, phenolic glycoplids). In the case of TDM, however, its total content (quantified by TLC) or surface exposure (assayed by whole cell ELISA using an anti-M.tb TDM mAb) showed not significant differences between all strains in this study (data not shown).
Several M.tb isogenic mutants have been described that show an attenuated phenotype via a reduction of growth in macrophages and/or macrophage-like cell lines (Kaushal et al. 2002; Geiman et al. 2004). Although an increased rate of macrophage death does not necessarily equate to increased virulence, to our knowledge, the pimB-KO is the first genetically defined M.tb strain that results in a faster rate of macrophage death without altering its intracellular growth. Several studies provide evidence that M.tb and its derived cell wall components can regulate apoptosis (and other cell death pathways) in macrophages, with the most virulent M.tb strains having been shown to have a lower apoptotic index. This is the case for the virulent M.tb strain H37Rv, which induced lower levels of apoptotic death in primary alveolar macrophages than its attenuated M.tb H37Ra counterpart (Keane et al. 2000). M.tb ManLAM has been shown to be involved in the inhibition of the apoptosis process (Rojas et al. 2000) via activation of a pro-survival pathway (Maiti et al. 2001; Dao et al. 2004). Thus, it is possible that the pimB-KO strain is attenuated compared to the parent M.tb Erdman strain with enhanced cell death being an ultimate advantage to the host.
We conclude that pimB plays a role in defining the nature of the M.tb cell wall without being essential in the biosynthesis of PIMs, LM, and ManLAM. Undefined mannosyltransferases can act in PimB's behalf or there are unknown alternative steps in the biosynthesis of these lipoglycans. Genetic analyses of M.tb strains deficient in cell wall mannosylation are likely to unravel new mannosyltransferases involved in this process and will enhance our understanding of the interaction between the mannosylated cell wall components of M.tb and the host immune response during infection.
All chemical reagents were of the highest grade from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. CS-35 and CS-40 murine mAbs were kindly provided by both the “TB Vaccine Testing and Research Materials” contract (NOI-AI-75320) and “Leprosy Research Support” contract (NO1-AI-25469). Rabbit anti-M.tb TDM was kindly provided by Dr. Robert L. Hunter (University of Texas).
A 2.7 Kb fragment of the M.tb genome, encompassing the ORF of pimB with 835 bp 5′- and 667 bp 3′-flanking sequences, was amplified from genomic M.tb DNA by PCR using the oligonucleotide primers 5′-ATGACAACGGCGGCGGCATCTTCGA-3′ and 5′-GGGCTGGCACCCGCTAAGTCTTCCT-3′. PCR was performed in 50 μL reaction mixtures (1× Hi Fidelity buffer, 2 mM MgCl2, 0.2 μM each primer, 200 μM each dNTPs, 1 U Platinum Taq Polymerase (Invitrogen, Carlsbad, CA)) and cycling parameters of 95°C for 2 min, (94°C for 30 s, 65°C for 30 s, 68°C for 4 min) ×40 cycles, 72°C for 15 min. The amplified fragment was cloned using the Perfectly Blunt kit into vector pT7Blue (Novagen, Gibbstown, NJ) to make the construct pBlue3K. The gene encoding kanamycin resistance (aph, isolated from Puc4K, Amersham Pharmacia Biotech, Piscataway, NJ) was inserted into a unique PshAI site located in the center of the cloned M.tb fragment to make pBlue3Kaph. The disrupted pimB construct was excised from pBlue3Kaph with XbaI and EcoRv and ligated into the suicide vector pSM270, containing a streptomycin resistance gene for positive selection and sacB for negative selection (kindly provided by Dr. Issar Smith, New York Public Health Research Institute) to make the plasmid pS/pimB::aph. Accuracy of all plasmid constructs was confirmed by DNA sequencing with an Applied Biosystems Model 373A stretch fluorescent automated sequencer at the University of Iowa DNA Facility.
Electrocompetent M.tb strain Erdman bacteria were prepared from mid-log phase cultures as previously described (Pelicic et al. 1996), and 100 μL (~109 CFU) were transformed at 37°C with 2 μg plasmid suicide vector pSpimB::aph in a 0.1 cm gap cuvette using a BTX electroporator at the settings 1.7 kV, 25 μF, and 200 Ω. Bacteria were incubated for 15 h in 7H9 broth with 10% oleic acid-albumin-dextrose-catalase (OADC) enrich supplement at 37°C with 5% CO2, before plating on 7H11 agar, 10% OADC, 0.5% glycerol, 0.1% Tween 80 containing 25 μg/mL streptomycin, and 20 μg/mL kanamycin (Kan) for selection of transformants possessing a single crossover. Recombination of the pSpimB::aph plasmid into the correct region of the M.tb genome was confirmed by Southern blot. The merodiploid strain was grown to the mid-log phase in 7H9 broth, OADC, 0.1% Tween 80 without antibiotic selection and plated on 7H11 agar containing 5% sucrose and 25 μg/mL Kan for selection of double crossover. Several M.tb colonies grew and displayed the phenotype of Kanr, sucrose resistance, and streptomycin sensitivity. Allelic exchange was confirmed by PCR and Southern blot analyses and the M.tb strain with the pimB mutated designated pimB-KO.
To obtain the complemented strain (pimB-Compl), the Kanr gene was removed by restriction digest with NheI and SpeI from the attP/integrase vector pMV306 (kindly provided by Dr. Ken Stover Pathogenesis, Corp., Seattle, WA (Stover et al. 1991)) and replaced with a hygromycin (Hyg) resistance gene and promoter, obtained by HindIII and SmaI restriction digests of the vector pSMT3 (Herrmann et al. 1996) to make the vector pMV306hr. Plasmid pBlue1.5K was digested with PstI and SpeI to remove the entire ORF of pimB with 443 bp of 5′-flanking sequence and subcloned into pMV306hr. This construct was introduced by electroporation into pimB-KO as described above with subsequent selection on 7H11agar + OADC with 50 μg/mL Hyg. Integration of the plasmid into the genome was confirmed by PCR and Southern blot. For the over-expression of pimB, the PstI/SpeI fragment from pBlue1.5K (described above) was subcloned into the mycobacterial shuttle vector pNBV1-NL (kindly provided by Dr. Gunter Harth, UCLA, Los Angeles, CA, a modification of pNBV1 (Howard et al. 1995)), electroporated into M.tb and recombinants were selected by plating on 7H11 agar + OADC with 50 μg/mL Hyg. This plasmid was present at approximately 10 copies per bacillus as estimated by relative amounts of mycobacterial genomic and plasmid DNA observed by semiquantitative gel electrophoresis. The pimB over-expressing strain was designated pimB-Overexp.
Genomic DNA was isolated from cultures grown in 7H9 broth, OADC, 0.1% Tween 80 to late log phase (OD580 = ~1.00). Cultures were centrifuged (3000 × g, 7 min, 24°C), the bacterial pellet resuspended in 5 mL water, with the addition of 500 μL packed volume sterile glass beads (100 μM, acid washed, Sigma) and 5 mL phenol: CHCl3 (pH 8) (Amresco, Solon, OH). Samples were mixed by vortex for 2 min, and aqueous phase was removed following centrifugation (4500 × g, 10 min, 24°C). The aqueous layer was extracted twice more with 5 mL CHCl3:isoamyl alcohol (24:1), treated with 100 μL RNase A at 37°C for 30 min, followed by 500 μL ProCipitate (CPG, Lincoln Park, NJ) and 100 μL Cleanascite (CPG). DNA was precipitated by adding 1/10 volume of 3 M sodium acetate, 2.5 volumes of 95% ethanol, and overnight incubation at −20°C.
RNA was isolated by a combination of mechanical and organic lysis as described (Desjardin 2000). Briefly, RNA was treated with DNase I (Ambion, Austin, TX) and reverse transcribed with AMV reverse transcriptase or without enzyme as a control for the presence of DNA contamination. PCR was performed in 50 μL reaction mixtures (1× Hi Fidelity buffer, 2 mM MgCl2, 0.2 μM each primer, 200 μM each dNTPs, 1 U Platinum Taq Polymerase (GIBCO BRL Life Technologies, Rockville, MD)) and cycling parameters of 95°C for 5 min, 40 cycles of 94°C for 1 min, 65°C for 1 min, and 70°C for 1 min to end with 72°C for 10 min. Primers 5′-CGCCGTCCACAGCGACAATG-3′ and 5′-CGACGCCAGCGCTTCCTGCA-3′ amplified a 360 bp fragment of pimB. The 235 bp of hspX transcript, encoding alpha crystallin homolog, and 166 bp of aroB transcript, encoding dehydroquinate synthase, were amplified as described (Desjardin et al. 2001) and served as control targets for RNA isolation and the RT reaction.
Extraction and analysis of PIMs were performed as described (Besra 1998). Briefly, M.tb cultures were harvested by centrifugation and washed with PBS. The bacterial pellets (100 mg of protein by the BCA method) were extracted using CH3OH/0.3% NaCl (2 mL, 100:10, v/v) and 1 mL of petroleum ether. Following centrifugation, the upper petroleum ether layer was removed and the polar lipids further extracted from the residue. The methanolic-saline fraction was heated at 65°C for 5 min and mixed with 2.3 mL of CHCl3:CH3OH:0.3% NaCl (9:10:3, v/v/v). After centrifugation, the supernatant was retained and the residue further extracted by adding 0.75 mL of CHCl3/CH3OH /0.3% NaCl (5:10:4, v/v/v). The solvent extracts were then combined with 1.3 mL of CHCl3 and 1.3 mL of 0.3% NaCl. The lower organic phase obtained after centrifugation was evaporated to dryness and the polar lipids resuspended in CHCl3:CH3OH:H2O (10:10:3, v/v/v). Equivalent amounts of material (based in 50 μg of protein) were loaded onto silica gel plates (Silica gel 60 F254; Merck, Darmstadt, Germany). Two-dimensional TLC was performed using CHCl3:CH3OH:H2O (60:30:6, v/v/v) in the first dimension and CHCl3/CH3COOH/CH3OH/H2O (40:25:3:6, v/v/v/v) in the second dimension. PIMs were revealed following charring with α-naphthol or 10% H2SO4 in ethanol at 120°C for 5 min. PIM purification for further biochemical analysis was performed by preparative TLC and confirmed by TLC as above described.
M.tb bacilli were scraped from 7H11 + OADC agar plates and delipidated using CHCl3:CH3OH (2:1, v/v) followed by CHCl3:CH3OH:H2O (10:10:3, v/v/v) for 48 h at room temperature with constant shaking. The residual biomass was suspended in PBS and disrupted using a FP-120 bead beater (Q-Biogene) applying a total of seven cycles (1 min-on/1 min-off). Protein quantification of disrupted cells was performed following the BCA method (Thermo Fisher Scientific, Rockford, IL) before treatment with proteinase K (1 mg/mL, Invitrogen), at 37°C for 16 h. The suspension was pelleted by centrifugation at 27,000 × g for 10 min, and the supernatant dialyzed against MQ-H2O using a 3500 MWCO membrane (Thermo Fisher Scientific). The retentate containing ManLAM/LM/PIMs mixture was analyzed by 15% SDS–PAGE followed by PAS staining of the gel (Torrelles et al. 2004). Densitometry analysis was performed using Image Acquisition and Analysis Labworks software version 4.6 (UVP Bioimaging system, UVP Inc, Upland, CA). Immunoblotting with mAb CS-35 was performed essentially as previously described (Prinzis et al. 1993). MAb CS-35 was generated against M. leprae and has been shown to cross-react with ManLAM from M.tb as well as other mycobacteria.
Whole cells (normalized by 1×107 bacterial equivalents), crude lipid extract, total PIMs, and ManLAM/LM fractions (all fractions normalized based on 500 μg of protein using the BCA method) were converted to alditol acetates using scyllo-inositol as an internal standard and analyzed by GC as previously described (McNeil et al. 1989). GC of alditol acetates was performed on a ThermoQuest Trace Gas Chromatograph 2000 connected to a GCQ/Polaris MS detector (ThermoQuest, Austin, TX) at an initial temperature of 50°C for 1 min, increasing to 170°C at 30°C/min and finally to 270°C at 5°C/min. All experiments were performed in duplicate using three independent lysates derived from different cell batches.
Analyses by MALDI-TOF were carried out on a Bruker Daltonic Reflex III (Bruker Daltonics Inc., Billerica, MA) mass spectrometer using the DE-reflectron mode. Ionization was affected by irradiation with pulsed UV light (337 nm) from a N2 laser. PIMs were analyzed by the instrument operating at 22.5 kV in the positive ion mode using an extraction delay time set at 200 ns. Typically, spectra from 100 to 250 laser shots were summed to obtain the final spectrum. Up to 10 μg/μL of PIM sample in CHCl3:CH3OH:H2O (10:10:3, v/v/v) solution and 5 μL of the matrix solution (saturated α-cyano-4-hydroxycinnamic acid (Bruker Daltonik) in 50% acetonitrile/0.1% aqueous trifluorocetic acid (50:50)) were deposited in a stainless steel target, mixed with a micropipet, and allowed to air-dry, forming a co-crystalline sample/matrix complex. The measurements were externally calibrated at two points with PIMs.
Bacilli were grown for 11 days on 7H11 with 10% OADC agar plates. Immediately prior to macrophage infection, bacteria were scraped from plates into a tube containing two 4 mm glass beads and 1 mL of RPMI1640 (Difco/BD Franklin Lakes, NJ), mixed by pulse vortex five times (1 s each), and allowed to settle for 30 min (Schlesinger et al. 1990). The top 600 μL was carefully removed to a fresh tube without disturbing the pellet. Bacteria were centrifuged at 500 × g for 1 min and the top 300 μL were transferred to a fresh tube. This solution contained 1 × 108 bacilli per ml with fewer than 10% bacterial clumps as observed by microscopy. Bacterial viability and concentration were determined by plating serial dilutions of the bacterial solution on 7H11 + 10% OADC agar and calculating CFU. Viability was consistently ~90%, and the measurement of CFU was consistent with the numbers of bacteria determined by Petroff–Hauser counting.
Human macrophages were obtained from human peripheral blood mononuclear cells (PBMC) as previously described (Schlesinger 1993). Briefly, heparinized blood was obtained by venipuncture from purified-protein derivative negative donors using an approved IRB protocol, mononuclear cells separated on a Ficoll cushion, and PBMCs cultured for 5 days in Teflon wells containing RPMI 1640 plus 20% autologous serum. MDMs were adhered to glass coverslips for 2 h, nonadherent cells were removed by washing, and MDMs were cultured overnight in RH (RPMI 1640 + 20 mM HEPES) plus 20% autologous serum at 37°C with 5%CO2. MDMs on coverslips were incubated with M.tb strains at a MOI of 10–20:1 for 2 h in the presence or absence of 2.5% autologous serum or in the presence of serum and 0.2 mg/mL mannose-BSA (EY Laboratories, San Mateo, CA). After washing, coverslips were fixed for 10 min in PBS containing 10% formaldehyde, washed three times with PBS, dried, and bacilli stained with auramine-rhodamine (Schlesinger et al. 1990). M.tb associated with MDMs was determined by counting the number of M.tb per MDM in 100 consecutive macrophages on triplicate coverslips in each test group using phase-contrast and fluorescence microscopy.
MDM monolayers were prepared as above and cultured an additional 7 days in RH plus 20% autologous serum at 37°C with 5%CO2 (Olakanmi et al. 2000). Monolayers were washed three times with RPMI and macrophages incubated with M.tb in RH plus 2.5% autologous serum at a MOI of 1:1 for 2 h. Nonadherent bacteria were removed by washing, and monolayers were cultured in RH and 1% autologous serum until time of harvest (from 2 to 90 h postinfection). At designated time points, CFU were enumerated separately for liquid medium (i.e., RH supernatant) and monolayer-associated bacteria. The RH supernatant from individual wells was removed to 1.5 mL polypropylene tubes after mild agitation by pipetting of the well contents and pelleted by centrifugation at 5000 × g for 10 min at 4°C. This pellet containing bacteria from the RH supernatant fraction was resuspended in a 100 μL NP40 lysis buffer (10 mM Tris, pH 8, 5 mM MgCl2, 10 mM NaCl, 60 mM KCl, 0.1 mM EDTA, 250 mM sucrose, 0.5% NP-40) by a combination of vortex mixing and seven strokes of a mini-pestle dounce. This method results in the recovery of >99% of bacteria from the RH supernatant fraction.
Macrophage monolayers were lysed by the addition of 500 μL NP-40 lysis buffer per well and removal from the plastic well surface with a cell scraper. The lysate was removed to a 1.5 mL polypropylene tube, dounced seven times with a mini-pestle, and mixed by vortex. Separate serial dilutions of both mycobacteria from supernatant (described above) and MDM monolayer lysate fractions were made in 7H9 broth, 10% OADC, 0.1% Tween 80, and aliquots plated on 7H11 + 10% OADC agar. Cultures were incubated at 37°C with 5%CO2 and CFU counted from 2 to 4 weeks postplating. CFU measurements of supernatant fractions were consistently <10% of the number associated with the monolayer. Data reported in the text represent the sum of supernatant and monolayer CFU enumeration.
The number of nuclei present in macrophage cultures was determined by removing 10 μL of the supernatant and monolayer fractions in the NP-40 lysis buffer, adding 9 μL 10% formaldehyde in PBS and 1 μL 0.4% trypan blue and counting on a hemocytometer. Few nuclei were observed in the supernatant fraction under control conditions and >90% were associated with the monolayer lysate.
Statistical analyses were performed using GraphPad Prism version 4.0.
The National Institutes of Health (AI33004, AI52458); the Veteran's Administration (to L.S.S.); and the University of Iowa CIFRE Award (to L.E.D.). GSB acknowledges support in the form of a Personal Research Chair from Mr. James Bardrick, a Royal Society Wolfson Research Merit Award, former Lister Institute-Jenner Research Fellow; the Medical Research Council; and The Wellcome Trust.
We thank Drs. Abul K. Azad and Evelyn Guirado for their review and assistance with the manuscript. We also thank the Campus Chemical Instrument Center at The Ohio State University for technical support.