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J Biol Chem, Vol. 274, Issue 44, 31625-31631, October 29, 1999
From the The biosynthesis of lipoarabinomannan (LAM), a
key mycobacterial lipoglycan that has been implicated in numerous
immunoregulatory functions, was examined utilizing
D-mannosamine (ManN) as a tool to identify
mannosyltransferase genes involved in LAM synthesis. Cell-free
reactions utilizing cellular membranes of mycobacteria as the enzyme
source indicated that ManN inhibited the synthesis of
phosphatidylinositol mannosides, early precursors to LAM. A selection
strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones
conferring conditional resistance to ManN, with the rationale that
overexpression of the gene(s) encoding a target of ManN would impart a
ManN-resistant phenotype under these conditions. This strategy led to
the identification of pimB, whose deduced amino acid
sequence shows similarity to mannosyltransferases and other
glycosyltransferases. Partially purified recombinant PimB protein
from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in
cell-free assays to show that PimB catalyzes the formation of
triacylphosphatidylinositol dimannoside from GDP-mannose and
triacylphosphatidylinositol monomannoside.
The emergence of multidrug-resistant strains of
Mycobacterium tuberculosis and the increased incidence of
tuberculosis, especially in developing countries, have made it clear
that there is a need for new chemotherapeutic agents (1). In this
regard, knowledge of the genetics and biochemistry of key biochemical
pathways in mycobacteria will provide a basis for the rational design
of new drugs (2). The mycobacterial cell wall (reviewed in Ref. 3) is
the site of action of many of the first-line antimycobacterial agents
(4), and it contains numerous components that are presumed to be
required for cell viability or survival in the host and are thus
attractive drug targets. One of these is lipoarabinomannan (LAM),1 a key lipoglycan of
the cell wall that is anchored in the mycobacterial plasma membrane
and/or the outer leaflet of the pseudo-outer membrane (3). LAM has been
implicated in various immunomodulatory effects, including the
down-regulation of cell-mediated immunity (5-9). In addition, mannose
"caps" on the LAM of M. tuberculosis (10) have been
shown to mediate attachment to macrophages and to aid in the initial
phagocytosis of this intracellular pathogen (11).
Although little is known about the biosynthesis of LAM, structural
similarities have suggested that the early precursors are phosphatidyl-myo-inositol (PI) and certain
phosphatidylinositol mannosides (PIMs) (12-14), and this hypothesis is
now supported by recent direct biosynthetic evidence (15, 16). The
proposed sequence of PI The PIM intermediates between PIM2 and larger LM-like
molecules are most likely PIM3-PIM5 on the
basis of their structures (14, 15), and these PIMs are a subset of the
so-called higher PIMs (PIM3-PIM6) that all
lack the branched configuration of LM (i.e. the
Further evidence that PIM2 is the biosynthetic precursor to
LAM comes from the location of the acyl functions elaborating these
molecules. The PIMs can vary in the extent of acylation, from the lyso
form with only C16:0 palmitate on the 1-position of the
glycerol to the multiply acylated forms. The latter are composed of the
predominant diacyl form containing C16:0 palmitate and
C19:0 tuberculostearate fatty acids esterified to the 1- and 2-positions of the glycerol, respectively, as well as the triacyl and tetraacyl forms that contain additional palmitate moieties on
specific Man residues (14). Several studies (20, 21) indicate that the
triacyl form2 of
PIM1 serves as the substrate for the subsequent
mannosylation step leading to triacyl-PIM2, and Khoo
et al. (14) have confirmed that it is the triacyl form of
PIM2 that is the predominant structural feature found
within LM and LAM.
The above structural and biosynthetic studies have established that
PIM2 is an intermediate in the LAM biosynthetic pathway and
provided the tools needed to initiate our analysis of the genetics of
LAM biosynthesis. In this report, we describe the biochemical and
genetic characterization of the pimB gene from M. tuberculosis. Evidence is presented that PimB is the
Bacterial Strains and Growth Conditions--
Mycobacterium
smegmatis strain mc2155 (22) was propagated in
Middlebrook 7H11 broth or agar medium (Difco). Escherichia
coli strains XL1-Blue and XL2-Blue (both from Stratagene) and TB1
(New England Biolabs, Inc.) were grown in LB broth or agar medium (Life Technologies, Inc.) that was supplemented with 20 mM
glucose for strain TB1. All cultures were incubated at 37 °C except
when noted, and all broth cultures were aerated by shaking. Media for
the propagation of recombinant E. coli or M. smegmatis strains contained 25 µg/ml kanamycin, 12.5 µg/ml
tetracycline, or 100 µg/ml ampicillin (all antibiotics were from Sigma).
M. smegmatis cells utilized in growth curve, whole-cell
labeling, or cell-free experiments were grown in glycerol alanine salts
medium (23). Unless otherwise noted, D-mannosamine (ManN) (ICN) was used at a concentration of 5 mg/ml, a concentration that was
empirically determined to completely inhibit the growth of freshly
diluted (A600 nm = 0.010-0.030) cells grown overnight in 7H11 broth. When necessary, cultures were centrifuged every 18-24 h, and fresh ManN and glycerol alanine salts medium were
added because ManN has been shown to be relatively unstable due to
nonenzymatic modifications of the amino sugar in vitro (24).
For growth assays with D-glucosamine (GlcN) and ManN, the
empirically determined concentration of 0.2 mg/ml GlcN was used in
addition to 5 mg/ml ManN. All assays were done in triplicate and
repeated at least three times.
Cell-free Assay for Mannolipid Synthesis Using
GDP-[14C]Man--
The incorporation of
GDP-[14C]Man into membrane lipids was assayed according
to Besra et al. (15) using membranes isolated as described
previously (25). Briefly, 15-20 g of cells were broken in a French
pressure cell and centrifuged at 27,000 × g, and the
supernatant was subjected to ultracentrifugation at 100,000 × g. The pelleted membrane fraction was suspended in Buffer A (50 mM MOPS and 10 mM MgCl2, pH
7.9); and the protein concentration, determined by the method of
Bradford (26), was adjusted to 20 mg/ml. Prior to the addition of 0.25 µCi of GDP-[14C]Man (321.4 mCi/mmol; NEN Life Science
Products), membranes (2 mg of protein) were preincubated for 10 min
with varying concentrations of ManN and, in some cases, amphomycin (100 µg/ml). Reactions were stopped by the consecutive additions of
H2O, CH3OH, and CHCl3 to give a
final ratio of 1:2:4 and extracted for 15 min. The lipids were
Folch-washed five times with
CHCl3/CH3OH/H2O (4:2:1) (27) and
dried before reconstituting in CHCl3/CH3OH
(2:1) for analysis by TLC. The total cpm of radiolabel incorporated
into the extracted lipids was measured by scintillation counting of
10% of the labeled material. Another 10% of the labeled material was
subjected to TLC analysis in
CHCl3/CH3OH/NH4OH/H2O
(65:25:0.5:3.6) (15) on aluminum-backed plates of Silica Gel 60 F254 (E. Merck, Darmstadt, Germany). Autoradiograms were
obtained by exposing thin-layer chromatograms to x-ray film at
Cell-free Assay for Mannolipid Synthesis Using
[14C]PIM1--
Triacyl-[14C]PIM1
was purified from a scaled-up cell-free reaction by TLC as described
above. The dry preparation was suspended by sonication in Buffer A
containing 100 mM CHAPS. Reaction mixtures contained 1000 cpm [14C]PIM1, 62.5 µM ATP, 10 µM GDP-Man, and either mycobacterial membranes (2 mg) or
partially purified recombinant protein (0.5 mg). The reactions were
incubated at 37 °C for 30 min, extracted with
CHCl3/CH3OH/H2O (4:2:1), and
analyzed by TLC autoradiography as described above. All assays were
done in triplicate and repeated at least three times.
Identification of Mannolipids--
The initial characterization
of the mannolipids affected by ManN was carried out by subjecting the
mannolipids to mild acid (in 0.5 N HCl) or mild alkali (in
0.1 N NaOH) hydrolysis as described previously (15). The
presence of Man was demonstrated by hydrolyzing the
[14C]Man-labeled lipids with 2.0 M
trifluoroacetic acid, followed by TLC analysis of the aqueous phase of
the Folch wash in comparison with sugar standards (28). For structural
analyses, nonradioactive mannolipids were synthesized in
vitro as described above using unlabeled GDP-Man and isolated by
preparative TLC using the radiolabeled mannolipids as markers.
Following autoradiography, the relevant regions of the TLC plate were
scraped off and extracted with CHCl3/CH3OH (2:1), and the organic phase of the Folch wash
(CHCl3/CH3OH/H2O (4:2:1)) was
collected and dried under nitrogen.
Mannolipids were analyzed by fast atom bombardment mass spectrometry
(FAB-MS) (14) either directly in negative ion mode or as
perdeuteroacetyl derivatives (100 µl of
pyridine-d6/acetic anhydride (1:1, v/v) for
2 h) in positive ion mode. Samples were redissolved in
CH3OH for loading onto the probe tip coated with triethanolamine or m-nitrobenzyl alcohol as matrix for
negative and positive ion modes, respectively. FAB mass spectra were
acquired on an Autospec orthogonal acceleration-time of flight mass
spectrometer (Micromass, Manchester, United Kingdom) fitted with a
cesium ion gun operating at 26 kV. Collision-induced desorption (CID)
MS-MS was performed by introducing argon gas to the collision cell to a
reading of ~1.2 × 10 Whole-cell Radiolabeling Experiments--
The effect of ManN on
PIM/LM/LAM synthesis in M. smegmatis was determined by the
whole-cell radiolabeling procedure described by Miku Selection of ManN-resistant Clones--
A genomic library of
M. tuberculosis strain H37Rv was screened for cosmid clones
conferring ManN resistance in the presence of exogenous GlcN (defined
as the ability of the bacteria to grow, as determined by growth curve
experiments, in the presence of 5 mg/ml ManN and 0.2 mg/ml GlcN). The
library (generated by Dr. Aimee E. Belanger) was constructed by cloning
35-40-kilobase pair partial Sau3AI fragments of chromosomal
DNA into the shuttle cosmid pYUB18 (30, 31). Following electroporation
of M. smegmatis (31), the cells were diluted to a low
A600 nm (0.010-0.030) and subjected to passage
in 7H11 broth prior to plating on 7H11 agar while maintaining
continuous selection with kanamycin, ManN, and GlcN. M. smegmatis transformed with pYUB18 alone was utilized as a control
during the selection procedure. To ensure that ManN resistance (in the
presence of GlcN) was conferred by the cosmid, putative ManN-resistant
cosmids were electroduced to E. coli (32) and purified with
QIAGEN columns, and the phenotype was retested after electroporating
into M. smegmatis.
DNA Sequencing, PCR, and Cloning Procedures--
DNA sequencing
was performed by Macromolecular Resources (Colorado State University)
using an ABI Prism 377 Automated DNA Sequencer. DNA sequence
comparisons were done by BLAST analysis (National Center for
Biotechnology Information). Alignments of deduced amino acid sequences
were performed using the MULTALIGN program. Standard PCR strategies
(33) with Vent DNA polymerase (New England Biolabs, Inc.) were used to
amplify the putative mannosyltransferase gene MTCY25D10.36 with primers
(5'-AATTATCCACGGGCATGCG-3' (sense primer for cloning into pYUB18),
5'-TGTGTGGCGTGCGCGTTGCGATC-3' (sense primer for cloning into pMV261),
5'-GTGTGTGGCGTGCGCGTTGCG-3' (sense primer for cloning into pMAL-c2),
and 5'-GGGGATCCTTGTCCAAGGC-3' (antisense primer)) derived from the
M. tuberculosis H37Rv genome sequence (34). PCR programs
consisted of an initial 4-min denaturation step (94 °C); followed by
30 cycles of 1) 94 °C for 30 s, 2) 66.6 °C for 30 s,
and 3) 72 °C for 1.5 min; and then a final elongation period at
72 °C for 7 min. PCR amplification of MTCY20G9.12 was facilitated
using Pfu DNA polymerase and a program identical to that
described above with primers 5'-GCAGGCATCGTGTCCCTAAGC-3' (sense
primer for cloning into pYUB18) and 5'-CGCTCAGGATGGTGTTGATCTTG-3' (antisense primer). Amplified genes were then cloned into
pYUB18 (31), pMV261 (35), or pMAL-c2 (New England Biolabs, Inc.) for
further analysis.
Partial Purification of Active Recombinant PimB--
Active
recombinant PimB protein was partially purified using the pMAL fusion
protein system (New England Biolabs, Inc.) according to the
manufacturer's instructions. The pimB gene was amplified by
PCR and cloned into the XmnI site of pMAL-c2 to generate
pMAL:pimB2, encoding a maltose-binding protein (MalE)-PimB fusion
protein. E. coli TB1(pMAL:pimB2) was grown at 37 °C to
A600 ~ 0.5 and then induced by the addition of
0.3 mM isopropyl- Effect of Mannosamine on in Vitro PIM Synthesis--
ManN
(2-deoxy-2-amino-D-mannose) inhibits the synthesis of
glycosylphosphatidylinositol anchors in Trypanosoma by chain
termination, forming ManN-Man-GlcN-PI that cannot be further
mannosylated at the 2-position of ManN (36). This mode of action
suggested that ManN might be a useful tool to study PIM biosynthesis in
mycobacteria, and so it was applied to the cell-free system of Besra
et al. (15) that permits mannolipid synthesis in the
presence of enzymatically active membranes from M. smegmatis. Dose-response curves showed decreasing incorporation of
radiolabel from GDP-[14C]Man into total mannolipids with
increasing ManN concentration, and TLC autoradiography (Fig.
1A), followed by scintillation
counting of the relevant bands, indicated that ManN inhibited the
synthesis of two mannolipids (designated mannolipid-1 and -2) as well
as the higher PIMs (Fig. 1B). ManN did not inhibit the
synthesis of the PPMs (mannosyl-1-phosphoryldecaprenol
(decaprenolphosphorylmannose (DPM), C50-P-Man) or
mannosyl-1-phosphorylheptaprenol (heptaprenolphosphorylmannose (HPM),
C35-P-Man)), which are the lipid carriers of Man that are involved in certain aspects of LAM biosynthesis (15). Unlike the
situation in trypanosomes, there was no evidence for the incorporation of ManN into any of these products when [14C]ManN was
used in whole-cell radiolabeling experiments (see below).
Mannolipid-1 and -2 were mild acid-stable and mild alkali-labile,
indicating that they are members of the PIM family as opposed to PPMs.
Both were also shown to contain the hexose Man by TLC analysis of the
trifluoroacetic acid-hydrolyzed sugars in comparison with sugar
standards. The chemical identities of mannolipid-1 and -2 were further
determined by mass spectrometry. Direct FAB-MS analysis (Fig.
2) of the isolated mannolipid-1 in
negative ion mode afforded a major [M
The results of the cell-free experiments and structural analyses of
mannolipid-1 and -2 were consistent with the model that ManN inhibits
the synthesis of PIM1 and PIM2. Specifically,
the hypothesis was that ManN was affecting the mannosyltransferase activity or activities responsible for the addition of Man to either PI
(to form PIM1) or PIM1 (to form
PIM2). This model was also consistent with the observed
decrease in the in vitro synthesis of the higher PIMs since
they are derived from PIM1/PIM2.
Feasibility of Cloning the Putative Mannosyltransferase Gene(s) by
Selection for ManN Resistance--
In conjunction with the above
cell-free assays, whole-cell radiolabeling experiments were conducted
by growing M. smegmatis with
D-[14C]glucose in the presence or absence of
an empirically determined, growth inhibitory concentration of 5 mg/ml
ManN. Treatment with ManN decreased the incorporation of radiolabel
into PIM2, but not PIM1, as compared with the
untreated controls and had no effect on the incorporation of radiolabel
into LM/LAM as judged by SDS-PAGE followed by autoradiography (data not
shown). The difference between the cell-free and whole-cell results
regarding the effect of ManN on PIM1 synthesis may reflect
the availability of PI precursors in purified membrane preparations
versus whole cells. In agreement with the cell-free results,
ManN did not inhibit the synthesis of the PPMs (DPM/HPM). These studies
indicated that it might be feasible to clone the gene(s) for the
ManN-sensitive mannosyltransferase, whose activity could be studied in
the cell-free assays, by screening a M. tuberculosis genomic
library in M. smegmatis for clones that confer a
ManN-resistant phenotype by virtue of overexpression of the ManN target
on a multicopy plasmid. Preliminary experiments indicated the need to
add exogenous GlcN (0.2 mg/ml) along with ManN to avoid cloning another
ManN target that resides in the GlcN synthase pathway rather than the
LAM biosynthetic
pathway.3
Cloning of Putative Mannosyltransferase Genes by Selection for ManN
Resistance in the Presence of GlcN--
The above strategy was used to
isolate two different cosmid clones (pMLS45 and pMLS47) from the H37Rv
genomic library by screening M. smegmatis transformants for
ManN resistance in the presence of GlcN. The availability of the
complete M. tuberculosis genome sequence (34) facilitated
the identification of the genes of interest: the genomic regions
present in the cosmids were delineated by sequencing the cosmid insert
junctions, and then those regions were subjected to BLAST analysis.
Each cosmid was found to contain one putative mannosyltransferase. That
on pMLS45 corresponded to gene MTCY20G9.12, whereas that on pMLS47
corresponded to gene MTCY25D10.36. Each of these genes was individually
cloned into pYUB18 via PCR to generate pMLS46 and pMLS48, respectively,
and both imparted the phenotype associated with their parental cosmids, namely ManN resistance in the presence of GlcN. Gene MTCY25D10.36 was
selected for this study, whereas gene MTCY20G9.12 (which appears to be
involved in polyprenolmannose synthesis) is the subject of a
separate investigation.4
The 1134-base pair MTCY25D10.36 gene, designated pimB, is
predicted to encode a 41.2-kDa protein that is 378 amino acids in length. BLAST analysis of this amino acid sequence revealed similarity to several mannosyltransferases and other glycosyltransferases, including MTH173 (GenBankTM/EBI accession number AE000805)
and MTH450 (AE000829) from Methanobacterium sp., which are
related to RfbU (a mannosyltransferase involved in lipopolysaccharide
biosynthesis), and GPI-3 (GenBankTM/EBI accession number
P32363) from Saccharomyces cerevisiae, which is believed to
be involved in the transfer of a glycosyl residue to PI. PimB contains
the sequence EXFCXXXXE (amino acids 282-290),
which differs by one amino acid from that
(EXFGXXXXE) present in several bacterial
Evidence That PimB Catalyzes the Formation of PIM2 from
GDP-Man and PIM1--
The function of PimB was examined by
overexpressing the pimB gene in both M. smegmatis
and E. coli and assaying the membrane fraction or partially
purified protein, respectively, in the cell-free system. For expression
in M. smegmatis, pimB was cloned downstream of
the hsp60 heat shock promoter in vector pMV261 (35) to generate pMV36Short. Membranes from heat-shocked (42 °C) cells (35) were assayed in the cell-free system, and representative results are shown
in Fig. 4A. The incorporation
of radiolabel from GDP-[14C]Man into PIM2 was
27% with the vector control as compared with 49% with pMV36Short (the
difference of 22% incorporation represents a 1.8-fold increase in
relative activity), whereas there was no significant change in the
amount of PIM1 synthesized. As expected, there was also no
apparent change in the incorporation of [14C]Man into the
DPM/HPM populations. Since the cell-free assay supports a number of
mannosyltransferase activities (15), there was concern that the major
products (notably the PPMs) might obscure any other results on the
chromatogram. Therefore, amphomycin (which inhibits polyprenol
phosphate-requiring transferases) was added to inhibit the synthesis of
DPM/HPM (15) and thus restrict product formation. Under these
conditions, there was again no significant change in the incorporation
of radiolabel from GDP-[14C]Man into PIM1
compared with that obtained with the control, and all of the observed
mannosyltransferase activity was associated with PIM2
synthesis (Fig. 4B). The difference between the control and
the pimB construct remained close to 1.8-fold, although the overall efficiency of the reactions increased (55 and 96%
incorporation, respectively, for a difference of 41%).
These results showed that PimB was associated with the addition of Man
to PI in the formation of PIM2, but it could not be discerned if it was involved in the first or second Man transfer because any newly synthesized PIM1 might be chased directly
into PIM2. Since Takayama and Goldman (20) provided
convincing evidence that two distinct mannosyltransferase activities
are required for the mannosylation of PI to form PIM2, it
was highly unlikely that PimB was involved in both reactions, and so
the cell-free assays were repeated using TLC-purified
triacyl-[14C]PIM1 as the sole source of
radiolabel. Initial attempts to use the M. smegmatis
membranes in these assays indicated that the reaction was inefficient
(~10% maximal incorporation of radiolabel) for three reasons. First,
and probably most important, any endogenous, unlabeled
triacyl-PIM1 in the membrane will compete with
triacyl-[14C]PIM1. A second problem arises
because triacyl-[14C]PIM1 is purified by
preparative TLC, and the residual silica was found to inhibit the
reaction. Finally, the in vitro synthesized radiolabel is of
low specific activity. The major problem was addressed by partially
purifying the active recombinant enzyme from E. coli. The
pMAL fusion protein system was used to generate a fusion protein
between maltose-binding protein (MalE) and PimB, which could then be
purified via an amylose column. As shown in Fig.
5A, SDS-PAGE analysis of the
column eluate showed that a major band of ~90 kDa and a minor band of
~48 kDa are visible by silver staining. The 90-kDa band is close to
the predicted size of the MalE-PimB fusion protein (82 kDa), and both
the 90- and 48-kDa bands were recognized by anti-MalE antibodies in
Western blot analysis, indicating that the latter is probably a
breakdown product of the fusion. The intact fusion protein was not
active in the cell-free assay; however, once it was digested with
Factor Xa (to precisely cleave off MalE and thus produce a mixture of PimB and MalE as well as Factor Xa), it catalyzed the transfer of Man
from GDP-Man to triacyl-[14C]PIM1 to form
triacyl-[14C]PIM2 (representative results are
shown in Fig. 5B). In this case, the incorporation of
radiolabel was 26%. Further purification of PimB in an active form has
not yet been achieved, but this work is currently in progress. Although
this reaction was sensitive to ManN (Fig. 5B), only 56%
inhibition was observed (the incorporation of radiolabel in the
presence of ManN was 12%). This suggests that ManN does not bind
directly or efficiently to the active site of the enzyme, but
interferes indirectly with the enzyme's access to GDP-Man, and this
conclusion is supported by the high concentration of ManN (5 mg/ml)
that is required for inhibition. It should be emphasized that the
reaction mediated by recombinant PimB occurs in the complete absence of
any mycobacterial membrane, thus addressing any concerns that may arise
regarding the validity of using recombinant M. smegmatis
membrane preparations to study the activity of a M. tuberculosis protein.
The biochemical analysis of pimB indicates that the
gene product is involved in the utilization of GDP-Man as the donor for Man in the LAM biosynthetic pathway. Specifically, the cell-free results with partially purified recombinant PimB provide evidence that
pimB encodes the
The analysis of glycosyltransferases from eukaryotic sources indicates
that the majority of the enzymes have a single transmembrane region
with a C-terminal catalytic domain, and they are oriented with the
catalytic domain within a membrane-bound compartment (40). As noted
earlier, a single transmembrane region is predicted for PimB, and the
preferred orientation would place the amino terminus outside the cell.
Assuming that the glycosyltransferase domain of PimB resides in the C
terminus, it would be on the cytoplasmic side of the membrane. This
would be consistent with PimB being an enzyme that utilizes GDP-Man, a
cytoplasmic component.
BLAST analysis of the M. tuberculosis genome sequence data
base shows similarity between PimB (Rv0557) and seven predicted M. tuberculosis proteins. Rv0486 (the mannosyltransferase
encoded by MTCY20G9.12),4 Rv3032, Rv2610c, and Rv3709c all
contain the sequence EXFGXXXXE, whereas Rv2188c,
Rv0225, and Rv1212c contain variant sequences (EX(L/W)GXXXXE). This similarity to PimB suggests
that these proteins are potential candidates for the other
mannosyltransferases that must be involved in LAM biosynthesis,
although it should be noted that the EXFGXXXXE
motif is also found in bacterial glycosyltransferases other than
mannosyltransferases (37).
The total number of mannosyltransferases that are needed for LAM
biosynthesis is a matter of speculation, depending on how the
The mannosyltransferases involved in the PIM/LM/LAM biosynthetic
pathway are potential targets for the rational design of novel
chemotherapeutic agents against M. tuberculosis. The
evidence that PIMs are early precursors to LM and LAM is well
established (14, 15), and drugs that inhibit PIM synthesis could have an effect on the organism's ability to produce LAM. Although the importance of LAM in retaining the structural integrity of the cell
wall can only be speculated, its potent immunomodulatory effects show
that LAM clearly plays a role in the pathogenesis of mycobacterial
disease, and so agents that disrupt its biogenesis will undoubtedly
affect the ability of M. tuberculosis to survive within the
host. This identification of PimB provides groundwork for determining
if this particular enzyme and this biosynthetic pathway are viable drug
targets in M. tuberculosis.
We thank Dean Crick for helpful suggestions
regarding the cell-free assay.
*
This work was supported in part by National Cooperative Drug
Discovery Group Opportunistic Infections of AIDS Grant AI-38087 (to
P. J. B., Program Primary Investigator) and Grants AI-35220 (to
G. S. B.), AI-37139 (to D. C.), and AI-01185 (to J. M. I.) from
the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF061562. (The original submission was with the gene
designation mtfB; however, the name was changed to
pimB to more accurately reflect the function of the gene
product and to distinguish this gene from other mtfB genes
that are denoted as being methyltransferases or mannosyltransferases).
¶
Supported by National Science Council Grant 88-2113-M-001-003
(Taiwan, Republic of China).
**
To whom correspondence should be addressed. Tel.: 970-491-7543;
Fax: 970-491-1815; E-mail: jinamine@cvmbs.colostate.edu.
2
Variations in nomenclature exist in the
literature. For example, triacyl-PIM1 has been called
"monoacyl-PIM1" to highlight only the additional acyl
function on Man (15).
3
M. L. Schaeffer and J. M. Inamine,
manuscript in preparation.
4
S. Gurcha and G. S. Besra, manuscript in preparation.
The abbreviations used are:
LAM, lipoarabinomannan;
PI, phosphatidyl-myo-inositol;
PIM, phosphatidylinositol mannoside;
LM, lipomannan;
PIM1, phosphatidylinositol monomannoside;
PIM2, phosphatidylinositol dimannoside;
PPM, polyprenolmannose;
ManN, D-mannosamine;
GlcN, D-glucosamine;
MOPS, 4-morpholinepropanesulfonic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
FAB-MS, fast atom bombardment mass spectrometry;
CID, collision-induced
desorption;
MS-MS, tandem mass spectrometry;
PAGE, polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
DPM, decaprenolphosphorylmannose;
HPM, heptaprenolphosphorylmannose.
The pimB Gene of Mycobacterium
tuberculosis Encodes a Mannosyltransferase Involved in
Lipoarabinomannan Biosynthesis*
,
,,,, and**
Mycobacteria Research Laboratories,
Department of Microbiology, Colorado State University, Fort Collins,
Colorado 80523, the § Institute of Biological Chemistry,
Academia Sinica, Taipe 115, Taiwan, Republic of China, and the
School of Microbiological, Immunological, and Virological
Sciences, Medical School, University of Newcastle upon Tyne,
Newcastle upon Tyne NE2 4HH, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PIM lipomannan (LM) LAM (15) builds
on work that started over 30 years ago with the PIMs (17-19) and that led to the identification of two distinct mannosyltransferase activities (20) that transfer Man from GDP-Man to the 2-position of the
myo-inositol ring of PI to form phosphatidylinositol
monomannoside (PIM1) and then to the 6-position to form
phosphatidylinositol dimannoside (PIM2) (14, 15). From
here, it has been proposed that PIM2 is further
glycosylated with Man to form LM, which is characterized by a linear
(16)-linked mannan backbone punctuated by (12)-linked
mannopyranose side chains, and LM is further glycosylated with arabinan
to form LAM (13).
(12)-linked Man side chains) (14). PIM6 appears to be
a terminal product because it contains linear (12)-linked Man,
which is not found in LM or LAM (14). Besra et al. (15) have
used a cell-free assay to show that PIM2 (or
PIM3) may be extended by the addition of Man residues from
an alkali-stable polyprenol-based mannolipid (polyprenolmannose (PPM))
(15) to form "linear LM" containing only (16)-linked Man.
These authors also suggest that this linear LM is then further
mannosylated to form mature, branched LM (15). If this is the case,
then PIM5 would also have to be a terminal product because
it contains (12)-linked Man (14).
-D-mannose-(16)-phosphatidyl-myo-inositol-monomannoside transferase responsible for the formation of triacyl-PIM2
from GDP-Man and triacyl-PIM1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C for 4-5 days. Relevant portions of the TLC plate were
scraped off, dissolved in CHCl3/CH3OH, and analyzed by scintillation counting. All assays were done in triplicate and repeated at least three times.
6 millibars on the time of
flight ion gauge. The source accelerating voltage was at 8 kV, and the
laboratory frame collision energy was maintained at 800 eV, with a
push-out frequency of 56 kHz for orthogonal sampling. A 1-s integration
time per spectrum was chosen for the time of flight analyzer with a
0.1-s interscan delay. Individual spectra were summed for data processing.
ová
et al. (28). Briefly, cultures (100 ml) were grown to
mid-log phase (A600 nm = 0.20-0.40) prior to
the addition of 1 µCi/ml D-[14C]glucose
(296 mCi/mmol; NEN Life Science Products) in the presence or absence of
5 mg/ml ManN and then incubated for 8 h. In certain experiments,
the radiolabel was [14C]ManN (55 mCi/mmol; ICN). Cells
were then harvested and washed, and the cell pellets were delipidated
with CHCl3/CH3OH (2:1) to provide the
extractable lipids containing the PIMs (29) that were analyzed by TLC
autoradiography as described above. The delipidated cell pellet was
then subjected to 50% aqueous ethanol reflux, followed by partitioning
between hot phenol and water (12). The aqueous phase, containing the
LM/LAM population, was analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE) on 13.5% gels (28). Gels were either stained
with periodic acid and silver nitrate or blotted onto nitrocellulose
and autoradiographed.
-D-thiogalactopyranoside with incubation at room temperature for 10-12 h. The cells were harvested, resuspended in Buffer A, and frozen at 20 °C overnight. Cells were broken in a French pressure cell and centrifuged at 27,000 × g. The supernatant was loaded onto an amylose
resin column, and the fusion protein eluted with Buffer A and maltose
was concentrated 8-fold. The eluate was analyzed by SDS-PAGE on a 10%
gel, followed by Western blotting using a ProTran membrane (Schleicher
& Schüll) with anti-MalE antibodies (New England Biolabs, Inc.).
Aliquots (~500 µg of protein) either were used directly in the
cell-free assay with [14C]PIM1 as the
substrate or were first incubated at room temperature for 12-16 h with
Factor Xa to cleave MalE from PimB.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
Effect of ManN on the in vitro
incorporation of [14C]Man into mannolipids.
M. smegmatis membranes (2 mg of protein) were preincubated
with ManN for 10 min prior to the addition of
GDP-[14C]Man. Reactions were incubated at 37 °C for
1 h and stopped with the consecutive additions of H2O,
CHCl3, and CH3OH. Lipids were Folch-washed
several times, and 10% of the labeled material was applied to TLC
plates and developed with
CHCl3/CH3OH/NH4OH/H2O
(65:25:0.5:3.6, v/v). A, TLC autoradiograph of reactions
carried out in the presence of ManN at the following concentrations: 0 mg/ml (lane 1), 1 mg/ml (lane 2), 5 mg/ml
(lane 3), and 20 mg/ml (lane 4). B,
table of percent inhibition of the incorporation of
[14C]Man into mannolipid-1 and -2 and higher PIMs
determined by scintillation counting of the bands from the TLC plate
shown in A.
H]
molecular ion at m/z 1251, whereas mannolipid-2 gave a
molecular ion at m/z 1413, corresponding to a difference of
one hexose increment from that of mannolipid-1. Based on previous
studies (14) and the TLC mobility in comparison with standards, the
molecular ion signals afforded by mannolipid-1 and -2 can be assigned
as triacylated PIM1 (with a total of three fatty acyl
chains, namely two C16:0 fatty acyl chains and one
C19:0 tuberculostearate) and PIM2 (also triacylated with C16:0, C16:0, and
C19:0), respectively. Supporting evidence was provided by
the fragment ions observed directly (Fig. 2, A and
B) as well as those from CID-MS-MS (Fig.
3, A and B). In the
former, the key fragment ion at m/z 689 corresponds to the
phosphoglycerol moiety diacylated with C16:0 and
C19:0. Both fatty acyl functions afforded strong
carboxylate ions in the CID daughter spectra at m/z 255 and
297. Loss of one or both fatty acyl functions by CID generated a number
of other fragment ions as indicated in Fig. 3. An additional
C16:0 substituent on Man was confirmed by several
complementary fragment ions observed. In direct FAB-MS analysis (Fig.
2), loss of a C16-Man from PIM1 gave the ions
at m/z 851 and 879, whereas similar loss from
PIM2 afforded the ions at m/z 1013 and 1041. Significantly, both PIM1 and PIM2 can lose a
single C16 function (m/z 1013/995 and 1175/1157, respectively), whereas only PIM2 can lose a single Man
(m/z 1251) without also losing a C16. Direct
loss of a Man residue from PIM1 to give the expected
m/z 1089 ion was not observed, and this is consistent with
the additional C16 being carried on the single Man residue
on PIM1. Further evidence for the assigned composition was
derived from FAB-MS analysis of the perdeuteroacetyl derivatives, in
which mannolipid-1 and -2 afforded molecular ions at m/z
1590 and 1887, corresponding to [M + Na]+ of triacylated
PIM1 and PIM2, respectively, with similar fatty acyl content (data not shown).
View larger version (38K):
[in a new window]
Fig. 2.
FAB-MS analysis of mannolipid-1
(A) and -2 (B) in negative ion
mode. Key fragment ions afforded are illustrated by the schematic
drawings. Stereochemistry of the implicated triacylated
PIM1 and PIM2 was inferred from previous
studies (14). The deduced structures were further supported by
composition analysis and negative ion CID-MS-MS (see Fig. 3), which
gave additional corroborative daughter ions. The fragment ions
generated by the analysis in direct or MS-MS modes are not identical.
Ino, myo-inositol.
View larger version (33K):
[in a new window]
Fig. 3.
FAB-CID-MS-MS analysis of the parent [M H] ions at
m/z 1251 and 1413 for mannolipid-1
(A) and -2 (B), respectively.
The key daughter ions generated from m/z 1251 are shown in
the schematic drawing. Ions corresponding to fragments containing only
the phosphodiacylglycerol were similarly observed for both parent ions,
whereas those carrying the mannosylated inositol moiety were shifted
162 mass units higher in the daughter spectrum of m/z 1413. Ino, myo-inositol.
-mannosyltransferases (37). Analysis of PimB using TMPred or
MacVector (Oxford Molecular Group) predicted a single transmembrane
region from amino acids 224 to 239, suggesting that PimB may be a
membrane protein or membrane-associated protein. This is consistent
with the observed co-localization of the associated mannosyltransferase
activity (see below) with the membrane fraction.
View larger version (39K):
[in a new window]
Fig. 4.
Effect of overexpressing pimB
on the in vitro incorporation of
[14C]Man into mannolipids. Cell-free reactions
containing membranes (2 mg of protein) and GDP-[14C]Man
were incubated at 37 °C for 1 h and stopped with the
consecutive additions of H2O, CHCl3, and
CH3OH. Lipids were Folch-washed several times, and 10% of
the labeled material was applied to TLC plates and developed with
CHCl3/CH3OH/NH4OH/H2O
(65:25:0.5:3.6, v/v). Membranes were prepared from M. smegmatis transformed with pMV261 (vector control)
(lanes 1) and pMV36Short (pMV261 containing
pimB expressed from the hsp60 promoter)
(lanes 2). A and B, TLC
autoradiograph of reactions carried out in the absence and presence of
amphomycin, respectively. The indicated increases in activity are based
on the differences in the percent incorporation of
[14C]Man into PIM2 between lanes 1 and 2 determined by scintillation counting of the indicated
bands.
View larger version (31K):
[in a new window]
Fig. 5.
Partial purification of recombinant PimB from
E. coli (A) and its use in the
cell-free synthesis of triacyl-PIM2 from
triacyl-PIM1 (B). A,
SDS-10% PAGE, followed by silver staining, of a 10-kDa ladder (Life
Technologies, Inc.) (lane 1) and intact MalE-PimB fusion
protein eluted from the amylose column (lane 2);
B, TLC autoradiograph of unreacted
triacyl-[14C]PIM1 (lane
1), triacyl-PIM2 generated with cleaved
MalE-PimB fusion protein and
triacyl-[14C]PIM1 (lane 2), and
same as lane 2 but in the presence of 5 mg/ml ManN
(lane 3). These results are from one TLC plate, but samples
between lanes 1 and 2 were spliced out.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-mannose-(16)-phosphatidyl-myo-inositol-monomannoside transferase that mediates the transfer of Man from GDP-Man to triacyl-PIM1 to form triacyl-PIM2. Sequence
analysis of PimB originally suggested that the gene encoded a
glycosyltransferase based on the presence of the deduced amino acid
sequence EXFCXXXXE, which differs by one amino
acid (C instead of G) from the sequence found in bacterial
-mannosyltransferases (37), as well as similarity to other
glycosyltransferases and certain enzymes that transfer glycosyl
residues to PI.
(16)-linked mannan backbone and (12)-linked Man side chains are assembled. However, one can predict that numerous enzymes are
needed for the formation of the different linkages as well as the
different acceptor and donor specificities and the possibility that
certain Man residues might be added sequentially while others might be
derived from intermediates built up on lipid carriers. One should also
expect there to be a few species-specific enzyme requirements given
that, beyond the structurally conserved PI/PIMs, the mature LM/LAMs
from different mycobacteria species have been found to differ in the
extent of glycosylation and modification (38). For example, the
synthesis of the Man caps on LAM will certainly involve enzymes that
are distinct from those required to form the mannan core. Other
unrelated biosynthetic pathways in M. tuberculosis will also
need specific mannosyltransferases, most notably those involved in
producing the mannosyl portions of the glycoproteins (39).
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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