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Disruption of the serine/threonine protein kinase H
affects phthiocerol dimycocerosates synthesis in
Mycobacterium tuberculosis
Anaximandro Go
´mez-Velasco,
1
Horacio Bach,
1
Amrita K. Rana,
2
Liam R. Cox,
3
Apoorva Bhatt,
2
Gurdyal S. Besra
2
and Yossef Av-Gay
1
Correspondence
Yossef Av-Gay
yossi@mail.ubc.ca
Received 10 July 2012
Revised 15 December 2012
Accepted 8 February 2013
1
Department of Medicine, Division of Infectious Diseases, Faculty of Medicine, University of British
Columbia, Vancouver, BC, Canada
2
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
3
School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for
interaction of the pathogen with its human host. Emerging evidence suggests that the
biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs).
Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted
deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates
(PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed
that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH
strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan
(LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus,
we provide experimental evidence that PknH contributes to the production and synthesis of M.
tuberculosis cell-wall components.
INTRODUCTION
Mycobacterium tuberculosis possesses a complex cell wall
characterized by the presence of a high content and diverse
array of lipids (Minnikin, 1982). Phthiocerol dimycocer-
osates (PDIMs) are a family of surface-exposed polyketide
lipids which constitute the most-abundant free lipids
found in the cell wall. These non-polar complex lipids
are composed of a mixture of long chain b-diols (C
33
–C
41
)
termed the phthiocerols, which, in turn, are esterified by
two multimethyl-branched fatty acids, termed mycocerosic
acids (Daffe
´& Laneelle, 1988; Minnikin et al., 2002;
Onwueme et al., 2005). Depending on chemical modifica-
tions, the PDIMs are classified into series: the phthiocerol
series A have a 3-methoxy group, the phthiocerol series B
have a 2-methoxy group, whereas the series C, the
phthiodiolone family, have a 2- or 3-keto group (Fig. 1a).
Biosynthesis, transport and translocation of PDIMs to the
surface of the bacterium are well studied. Biosynthesis of
PDIMs is initiated by activation and transfer of C
12
–C
18
fatty acids by FadD26 (Trivedi et al., 2004). The activated
fatty acids are transferred to the type I polyketide synthases
(PKSs) PpsA–PpsE, which elongate straight-chain fatty
acids until the final phthiocerol backbone is synthesized
(Azad et al., 1997; Trivedi et al., 2005). In parallel, an
iterative type PKS, the Mas protein, produces mycocerosic
acids, which, in turn, are transferred to the b-diol backbone
of the phthiocerol by the acyltransferase PapA5 (Azad et al.,
1996; Onwueme et al., 2004; Trivedi et al., 2005). Release of
the elongated phthiocerol moiety from PpsE is carried out
by the type II thioesterase TesA (Alibaud et al., 2011).
Transport and translocation of PDIMs to the cell wall is
carried out by either MmpL7 or DrrABC (Camacho et al.,
1999, 2001; Cox et al., 1999).
Protein phosphorylation, carried out by protein kinases, is
the principal mechanism by which extracellular envir-
onmental stimuli are translated into adaptive gene
expression. The M. tuberculosis genome encodes 11
serine/threonine protein kinases (STPKs), shown to be
involved in the regulation of pathogenesis, cell division and
cell-wall synthesis (Av-Gay & Everett, 2000; Chao et al.,
2010a; Cole et al., 1998). Recent studies have shown the
involvement of two STPKs, PknB and PknD in the
production of PDIMs. PknB was shown to phosphorylate
PapA5 on threonine residues (Gupta et al., 2009), whereas
Abbreviations: ACN, acetonitrile; FT-ICR, Fourier transform ion cyclotron
resonance; iTRAQ, isobaric tags for relative and absolute quantitation;
LAM, lipoarabinomannan; LM, lipomannan; MBP, maltose-binding
protein; m/z, mass to charge; PDIM, phthiocerol dimycocerosate; PKS,
polyketide synthase; scFv, single-chain, fragment-variable; STPK,
serine/threonine protein kinase.
A set of supplementary methods, five supplementary figures and a
supplementary table are available with the online version of this paper.
Microbiology (2013), 159, 726–736 DOI 10.1099/mic.0.062067-0
726 062067 G2013 SGM Printed in Great Britain
PknD may phosphorylate MmpL7 (Pe
´rez et al., 2006).
These studies provide experimental evidence that PDIMs
are regulated by STPKs.
We have previously shown that deletion of pknH leads to
improved survival of the mutated strain in a BALB/c
mouse model of infection, indicating that PknH is needed
for in vivo bacterial growth (Papavinasasundaram et al.,
2005). More recently, we found that PknH is linked to the
M. tuberculosis dormancy regulon by phosphorylating the
control enzyme DosR (Chao et al., 2010b). Previous studies
have demonstrated that PknH is able to phosphorylate
enzymes participating in cell-wall biosynthesis. In vitro
kinase assays revealed that PknH phosphorylated EmbR,
and this interaction may play a role in the transcription of
the embCAB operon that encodes arabinosyltransferases
(Molle et al., 2003; Sharma et al., 2006; Zheng et al., 2007).
Overexpression of PknH in Mycobacterium smegmatis
activated EmbR, which induced the transcription of the
embCAB operon, leading to a higher lipoarabinomannan
(LAM) / lipomannan ( LM) ratio (Sharma et al., 2006). M.
tuberculosis PknH also phosphorylates DacB1, an enzyme
that in Bacillus subtilis is a sporulation-specific protein
involved in cell-wall biosynthesis (Zheng et al., 2007).
Together these results suggest that PknH plays an
important role in the regulation of M. tuberculosis growth
by controlling cell-wall compound synthesis and/or
transport. Rationalizing that M. tuberculosis cell-wall
components contribute to its virulence and that a DpknH
deletion mutant strain has been shown to be hypervirulent
(Papavinasasundaram et al., 2005), we undertook a
detailed cell-wall lipid analysis to investigate whether their
biosyntheses were affected by PknH. In this study we show
by radiolabelling, MS (lipidomics) and immunostaining
analyses that PDIM production and the ratio of LAM to
LM are specifically affected by knocking out the pknH gene.
METHODS
Bacterial strains and growth conditions. M. tuberculosis H37Rv,
DpknH and DpknH ::pknH strains were used in this study
(Papavinasasundaram et al., 2005). Starter cultures were initiated
from glycerol stocks using 10 ml 7H9 Middlebrook (Becton and
Dickinson) broth supplemented with 10 % (v/v) oleic acid/albumin/
dextrose/catalase (OADC), and 0.05 % (v/v) Tween-80 at 37 uC.
Actively growing bacterial cells were used to start 500 ml cultures for
rolling conditions (850 cm
3
roller bottles; Greiner Bio-one, catalogue
no. 680060, 1.25 r.p.m.) and were incubated at 37 uC. 7H11 agar
medium (B&D) was prepared according to the manufacturer’s
instructions and was supplemented with 10 % (v/v) OADC.
Antibiotics were supplemented as required: 25 mg kanamycin ml
21
and 50 mg hygromycin ml
21
.
Extraction of apolar and polar lipids. For cell-wall lipid analysis,
mycobacterial strains were grown at early exponential phase (OD
600
~1, measured by Optizen POP spectrophotometer, path length
10 mm). Cells were harvested by centrifugation, washed three times
with PBS (100 mM K
2
HPO
4
, 10 mM NaCl, pH 7.4) and were
autoclaved at 121 uC. Autoclaved cells were then lyophilized prior to
extraction and purification of apolar and polar lipids as described
previously (Besra, 1998; Dobson et al., 1985). Each lipid extract
(100 mg) was loaded onto TLC plates (silica gel 60F
254
, Merck) and
was separated using 2D-TLC and solvent systems A–E (Besra, 1998;
Dobson et al., 1985). To visualize PDIMs, lipids were separated using
a mixture of petroleum ether/ethyl acetate (98 : 2, v/v), three times in
H37Rv
(a) (b)
β-diol moiety
(c)
MK
TAG
D2
D1
III II I
ΔpknH ΔpknH::pknH
Mycocerosic acids
Fig. 1. (a) PDIM structure. The long chain b-diol is esterified by multimethyl-branched fatty acids (mycocerosic phthioceranic
acids). R
1
is CH
2
-CH
3
for phthiocerol series A and C or CH
3
for phthiocerol series B; R
2
is OCH
3
for the phthiocerol family or
is O for the phthiodiolone family (series C). (b) Colony morphology. Colonies of H37Rv wild-type, DpknH and DpknH ::pknH
strains were grown on 7H10 agar plates supplemented with OADC. Colonies were obtained by inoculating 10 ml aliquots from
cultures at OD
600
0.010. Plates were sealed and incubated at 37 6C for 3 weeks. Bars, 1 mm. (c) Apolar lipid profile of the
DpknH mutant strain. Apolar lipid profile 2D-TLC reveals that PDIMs were not produced in the DpknH strain (circled), while the
wild-type and the complemented strains produced similar amounts. PDIMs (I, phthiocerol series A; II, phthiocerol series B; III,
series C in phthiodiolone family); MK, menaquinones; TAG, triacylglycerols.
Phthiocerol dimycocerosates synthesis regulation
http://mic.sgmjournals.org 727
the first direction, while a mixture of petroleum ether/acetone (98 : 2,
v/v) was used in the second direction. TLC plates were developed by
staining with 5 % ethanolic phosphomolybdic acid, followed by
charring at 100 uC.
For radiolabelling experiments [1-
14
C]-propionate 3.7610
10
Bq ml
21
[specific activity 54 mCi mmol
21
(1.998 GBq mmol
21
); American
Radiolabeled Chemicals]and [1,2-
14
C]-acetate 3.7610
10
Bq ml
21
[specific activity 57 mCi mmol
21
(2.109 GBq mmol
21
)]were added
at different time points (12 and 24 h, 5 and 10 days) to 10 ml
mycobacterial cultures at 37 uC. PDIMs were extracted, their activities
were measured in a scintillation counter (Beckman) and they were
purified as above using preparative TLC. Equal radioactivity was
loaded onto 2D-TLC. Autoradiograms were visualized using a
Phosphorimager SI (Molecular Dynamics). For lipid quantification,
spots were scraped from TLC plates and subjected to scintillation
counting. For statistical analysis three independent biological
replicates were used.
Fourier transform ion cyclotron resonance (FT-ICR) MS
analysis. Mycobacterial strains were grown as described above and
total lipids were extracted using the Bligh–Dyer method (Bligh &
Dyer, 1959). To avoid interference with the results Tween-80 used as a
supplement in the culture was removed as described previously by
Jain et al. (2007). Briefly, cell extracts were resuspended in a hexane/
water mixture (50 : 50, v/v), mixed thoroughly and centrifuged at
3500 gfor 5 min. The organic layer was extracted with water (five
times). For lipidomic analysis, total lipids were resuspended in a
chloroform/methanol mixture (2 : 1, v/v) and injected into an Apex-
Oe 12-Tesla Hybrid quadrupole-FT-ICR machine (Bruker Daltonics),
which was equipped with an Apollo electrospray ionization (ESI) ion
source. Samples were infused into the MS instrument at a flow rate of
2ml min
21
and were ionized with ESI. Mass spectra were acquired
within a mass to charge (m/z) ratio range of 250–300 in either positive
or negative mode, with broadband detection and using a data
acquisition size of 1024 kilobytes per second. Each spectrum was
accumulated from 100 scans. Total abundance of lipid species was
calculated by summing the peak intensities as measured by FT-ICR, as
reported previously by Jain et al. (2007).
Production of single-chain, fragment-variable (scFv) antibod-
ies. Purified PDIMs were kindly provided by Dr Jean-Marc Reyrat
(INSERM-UMR, France). scFv antibodies against purified PDIMs were
selected as described previously (Bach et al., 2001). Briefly, 1.7 mg
purified PDIMs was dissolved in 100 ml chloroform and then 100 ml
0.1 M 2-(N-morpholino)ethanesulfonic acid, pH 4.5, was added to the
dissolved PDIMs. The sample was sonicated and mixed with 30 ml
10 nM stock solution of hydrazine. The mixture was incubated at
65 uC for 10 min. After cooling down the reaction to room
temperature, the mixture was conjugated to 4 mg BSA using 1-ethyl-
3-[3-dimethylaminopropyl]carbodiimide hydrochloride (Pierce),
according to the manufacturer’s instructions. The Tomlinson I scFv
antibodies library was kindly supplied by Geneservice, Cambridge,
UK. The scFv antibodies were screened according to the instructions
supplied with the library. PDIMs coupled to BSA were used as
antigens for screening. Selected antibodies were subcloned into
pMAL-C5X (NEB) and were produced as recombinant proteins
fused to a maltose-binding protein (MBP) as described previously
(Bach et al., 2001).
Immunostaining and fluorescence microscopy. Bacteria were
labelled with rhodamine (10 mg ml
21
) for 1 h at 37 uC with gentle
rocking. Labelled bacteria were washed three times with PBS and
three times with double-distilled water and were immobilized on
coverslips by flaming. Coverslips containing bacterial cells were
incubated with scFv antibodies at room temperature for 30 min, and
unbound antibodies were washed away with PBS for 10 min (three
times). Coverslips were further incubated with an anti-MBP antibody
(1 : 1000 dilution) mixed with goat FITC-conjugated anti-mouse IgG
secondary antibody (1 : 1000 dilution) at room temperature for
20 min. Coverslips were washed again with PBS for 10 min (three
times) and mounted on glass slides containing FluorSave
(Calbiochem). Samples were analysed by fluorescence microscopy as
described previously by Sendide et al. (2004).
THP-1 cell infection and immunofluorescence microscopy. The
human monocyte cell line THP-1 (American Type Culture
Collection) was cultured in RPMI 1640 (Sigma) supplemented with
1% L-glutamine, 100 mg streptomycin ml
21
, 100 U penicillin, 0.1 %
fungizone (Invitrogen) and 10 % FCS (Sigma). THP-1 cells were
seeded on coverslips at 5610
6
cells per well in 2 cm
2
24-well tissue
culture plates and were differentiated by the addition of phorbol 12-
myristate 13-acetate (20 ng ml
21
) and they were incubated for 20 h
in a humidified atmosphere (5 % CO
2
). Before infection, bacteria
were labelled with rhodamine (10 mg ml
21
) and were incubated with
shaking for 30 min at 37 uC. After labelling, bacterial cells were
washed with incomplete RPMI (supplemented only with FCS and L-
glutamine) three times and were opsonized for 30 min at 37 uC with
10 % human serum. THP-1 cell monolayers were infected with
bacteria at an m.o.i. of 10 : 1 (THP-1/bacteria) and were incubated at
37 uC and 5 % CO
2
for 4 h. Non-internalized bacteria were removed
by several washes with incomplete RPMI. Tissue cultures were further
incubated at 37 uC and 5 % CO
2
for 24 h.
Infected macrophages were fixed with 4 % p-formaldehyde for 30 min
at room temperature and then permeabilized with saponin for
30 min. Coverslips were incubated with scFv antibodies at room
temperature for 30 min, and any unbound antibody was washed away
three times with PBS for 10 min. Coverslips were further incubated
with anti-MBP (1 : 1000 dilution) and goat anti-mouse IgG-FITC
conjugate secondary antibody (1 : 1000 dilution) at room temperature
for 20 min. Coverslips were washed again three times with PBS for
10 min and were mounted on glass slides containing FluorSave
(Calbiochem). Samples were analysed as described previously by
Sendide et al. (2004).
Isobaric tags for relative and absolute quantitation (iTRAQ)
analysis. Proteomic analysis was performed as previously reported
(Chao et al., 2010b). Strains were grown in rolling cultures, harvested,
washed and treated with 3 mM NaNO
2
. Total proteins were
extracted, digested with trypsin at 37 uC overnight and then labelled
with iTRAQ reagents. Tryptic-labelled peptides were separated using
a polysulfethyl A [10064.6 mm, 5 mM, 300 A
˚(30 nm)]strong cation
exchange column (Poly LC) in the first dimension. The column was
allowed to equilibrate for 20 min in buffer A [10 mM KH
2
PO
4
,
pH 2.7 and 25 % acetonitrile (ACN)]before a gradient was applied;
0–35 % buffer B (10 mM KH
2
PO
4
, pH 2.7, 25 % ACN, 0.5 M KCl)
for 30 min. The flow rate was set at 0.5 ml min
21
. Tagged peptides
were analysed by LC-MS/MS using an integrated Famos autosampler,
Switchos II switching pump and Ultimate micropump system (LC
Packings) with a hybrid Quadrupole-time of flight (TOF) LC-MS/MS
mass spectrometer (QStar Pulsar i), equipped with a nanoelectrospray
ionization source (Proxeon) and fitted with 10 mm fused-silica
emitter tip (New Objective). The second dimensional chromato-
graphic separation was carried out using a 75 mm615 cm C
18
PepMap Nano LC column [3mm, 100 A
˚(10 nm); LC Packings]and a
300 mM65mm C
18
PepMap 2 Guard column [5mm, 100 A
˚
(10 nm); LC Packings]. The mobile phase (solvent A) consisted of
water/ACN (98 : 2, v:v) with 0.05 % formic acid for sample injection
and equilibration on the guard column at a flow rate of 100 ml min
21
.
A linear gradient was created upon switching the tapping column
inline by mixing with solvent B, which consisted of ACN/water (98 :2,
v:v) with 0.05 % formic acid, and the flow rate was reduced to 200 nl
min
21
for high resolution chromatography and introduction into the
A. Go
´mez-Velasco and others
728 Microbiology 159
mass spectrometer. MS data were acquired automatically using
Analyst QS 1.0 software Service Pack 8 (ABI MDS SCIEX). An
information-dependent acquisition method, consisting of a 1 s TOF
MS survey scan of mass range 400–1200 atomic mass units (amu) and
two 2.5 s product ion scans of mass range 100–1500 amu, was
followed. The two most-intense peaks over 20 counts, with charge
state 2–5, were selected for fragmentation, and a 6 amu window was
used to prevent the peaks from the sample isotopic cluster from
fragmenting again. MS/MS was put on an exclude list for 180 s.
Curtain gas was at 23 uC, nitrogen was used as the collision gas and
ionization tip voltage was 2700 V.
MS data analysis. Data were obtained and analysed from two
independent experiments. The identification and quantification
of the proteins were performed using ProteinPilot 2.0.1 (Applied
Biosystems/MDS Sciex). The Paragon algorithm integrated in the
ProteinPilot software was used for peptide identification and was
further processed by Pro Group algorithm for peptide identification
and isoform-specific quantification, and the iTRAQ peak data were
normalized for loading error by auto-biased corrections calculated
using the ProteinPilot software. ProteinPilot software calculates an
unused score of 2 for a peptide with a 99 % identity confidence and an
unused score of 1.3 for a peptide with 95 % confidence level. An
unused score of .2 indicates that a minimum of two peptides, one
peptide with .95 % confidence plus at least one other peptide with
less than 95 % confidence, were used exclusively for the identification
of that protein. With a protein group of highly homologous proteins
(identical peptides), peptides are arbitrarily assigned to one protein
for which an unused score and iTRAQ ratio is determined. The
percentage of protein covered by identified sequences at a 95%
confidence level [% Cov(95)]is calculated by dividing the number of
amino acids of peptides identified with 95 % confidence by the total
number of amino acids in the protein. Relative quantification was
performed on MS/MS scans and denotes the ratio of the areas under
the peaks at 115 Da and 114 Da (untreated DpknH/WT), and 117 Da
and 116 Da (nitrite-treated DpknH/WT).
Statistical analysis. Statistical significance was determined with the
unpaired two-tailed Student’s test with GraphPad Prism Version 5.
P¡0.05 was considered statistically significant.
RESULTS
The DpknH mutant produces low levels of PDIMs
M. tuberculosis virulence has been associated with a cording
appearance and this is likely to be related to cell-wall
components. In our previous study, we observed that the
DpknH strain is hypervirulent in a mouse model
(Papavinasasundaram et al., 2005). As an initial analysis,
we first characterized the colony morphology of all strains.
An analysis of the parental wild-type and DpknH strains
shows morphological differences in colony formations (Fig.
1b). While the cording appearance was similar for both the
wild-type and the complemented strains, the DpknH shows
a more ruffled shape (Fig. 1b). This appearance may have
implications for the overall cell wall physical structure and
therefore for M. tuberculosis physiology.
We next examined whether the deletion of pknH altered
specific cell-wall components. For this purpose, we used a
range of 2D-TLC solvent systems designed to systematically
profile a wide range of mycobacterial lipids (Besra, 1998;
Dobson et al., 1985). Our screening demonstrated that the
DpknH mutant failed to produce observable levels of
PDIMs (Fig. 1c). 2D-TLC analyses of other cell-wall lipids
show no apparent differences between the wild type and
the DpknH strains (Fig. S1, available with the online
version of this paper). We ruled out the possibility of polar
effects associated with knock-outs of PDIM biosynthetic
genes (Domenech & Reed, 2009) by showing that the
complemented strain re-establishes the wild-type pheno-
type (Fig. 1c).
To further confirm that preparative 2D-TLC demonstrates
the absence of PDIMs, we labelled cell cultures with [1-C
14
]-
propionate and [1,2-
14
C]-acetate and monitored their
production at different time points (12 and 24 h, 5 and
10 days) (Figs. 2 and S2). Apolar lipids were further analysed
by 2D-TLC. This sensitive technique showed that PDIMs
were not fully abolished, but rather that their levels were
reduced in the DpknH strain compared with those of the
wild-type and the complemented strains (Figs 2 and S2). The
growth of all strains was similar (data not shown), as
reported previously (Papavinasasundaram et al., 2005);
therefore, the difference in PDIM biosynthesis was normal-
ized by loading equal radioactivity (c.p.m.). Thus, the spots
corresponding to PDIMs (series A/B and C, I/II and III in
figures, respectively) from parental, DpknH and comple-
mented strains were quantified by scintillation counting. For
both radiolabelled carbon sources, the general trend
observed was a reduction in PDIM production (Figs 2 and
S2). The lipid profile of apolar lipids extracted from cells
labelled with acetate remained similar at all time points
(Fig. S2). Interestingly, the profile of apolar lipids from
propionate-labelled cells remained similar at 12 and 24 h
(Fig. 2a, b), but altered at 5 (Fig. 2c, d) and 10 days (Fig. 2e,
f). Thus, radiolabelled-culture analysis, with both [1,2-
14
C]-
acetate and [1-C
14
]-propionate, revealed a reduction of
PDIM synthesis in the DpknH mutant as well as an alteration
in the level and profile of other unidentified lipids.
Structural analysis of total lipids from the DpknH
strain
Total lipids were extracted by the method described by
Bligh & Dyer (1959) and were then subjected to FT-ICR
MS analysis, shown previously to be a comprehensive
analytical method to analyse complex lipids from M.
tuberculosis (Jain et al., 2007). The intensity of a series of
molecular ions, corresponding to PDIM masses (C
86
–C
100
)
in the m/zrange 1300–1600, showed that these PDIMs
species were more abundant in the parental wild-type
strain than in the DpknH mutant (Fig. 3a). Comparison of
the relative abundances of PDIM A–B and PDIM C lipid
groups revealed significant reduction of both in the mutant
strain (Fig. 3b, c). Complete absence of ion species in the
1390–1449 m/zregion (DIM A/B) was observed in the
mutant, while in the wild-type these peaks were more
abundant, suggesting a higher production of these cell
components (Fig. 3a and Table S1). The complemented
Phthiocerol dimycocerosates synthesis regulation
http://mic.sgmjournals.org 729
strain partially restored the production of these same ion
molecular species (Fig. 3a). We also observed that the ratio
of total PDIM A/B to total DIM C lipids was 1.75 in the
wild-type, which is similar to results in previous reports
(Sartain et al., 2011). Thus, this sensitive MS technique,
together with the radiolabelled-lipid profile, confirmed that
PDIMs are produced at lower levels in the DpknH strain
compared with those of its parental strain.
Immunofluorescent detection of PDIMs
Both the MS- and TLC-based methods do not distinguish
between cytosolic and cell-wall-bound lipids. In order to
verify whether the observed PDIM levels reflect the relative
abundance on the cell wall, we generated synthetic
antibodies (scFv) against PDIMS and used them to monitor
PDIM production using fluorescence microscopy in the
examined strains. As a negative control, we incubated the
antibodies with M. smegmatis, a mycobacterial strain known
to be unable to produce PDIMs. Analysis of wild-type and
DpknH ::pknH bacterial cells showed high-intensity label-
ling and 100 % co-localization for scFv antibodies against
PDIMs in wild-type M. tuberculosis (Fig. 4a). A weak
fluorescence signal was detected in the DpknH strain,
corresponding to 18% of the relative fluorescence compared
with that the wild-type strain (Fig. 4b). Interestingly, higher
fluorescence in the complemented strain was observed,
suggesting that the overproduction of PDIMs may be related
to uncontrolled expression of pknH.
We further examined PDIM production during in vivo
analysis by infecting differentiated THP-1 cells with all
strains, followed by immunostaining and microscopic
analyses. As in our previous analysis, the wild-type and
the DpknH ::pknH strains were strongly labelled on their
surfaces with anti-PDIM antibodies (Fig. S3a), whereas in
the DpknH strain only a portion of the fluorescence signal
was detected (Fig. S3b).
12 h
PDIMs
III
1
2
I/II
PDIMs
III
I/II
5 days
(a) (b)
30 000
20 000
40 000 ***
*
***
*
*
*
10 000
3 000
2 000
4 000
1 000
0
(d)
30 000
20 000
25 000
35 000
15 000
10 000
5 000
0
6
5
4
3
2
1
III
I/II
7
6
5
4
3
2
1
III
I/II
7
(f)
40 000
20 000
30 000
Unknown lipids (c.p.m.)
50 000
10 000
5 000
0
6
5
4
3
2
1
III
I/II
7
(c)
10 days
(e)
H37Rv ΔpknH ΔpknH::pknH
D2
D1
7
6
5
4
3
1
2
7
6
54
3
PDIMs
III
I/II 1
2
7
65
4
3
Unknown lipids (c.p.m.) Unknown lipids (c.p.m.)
Fig. 2. Production of PDIMs using propionate as a carbon source. Mycobacterial strains grown in 7H9 medium supplemented
with OADC were labelled with [1-
14
C]-propionate and were further incubated at 37 6C to different time points. Apolar lipids
were extracted as described in Methods. Equal amounts of lipid samples (100 000 c.p.m.) were loaded onto each TLC plate
and plates were resolved as described in Methods. The observed general trend was the lower production of PDIMs in the
mutant strain. Apolar lipid profiles for 12 and 24 h (a and b, respectively) were similar. However, levels and lipid profiles of
unknown lipids changed at 5 days (c, d) and 10 days (e, f). Lipids were visualized by exposure to PhosphoImager SI. I, II and III,
as described above. For lipid quantification spots from TLC plates, as shown in each 2D-TLC, were scraped and subjected to
liquid scintillation counting. (b, d, f) Quantification of unknown lipids 1–7. Data are the means±SEM from three independent
biological experiments. *P¡0.05, ***P¡0.001, significant differences compared with wild-type samples by Student’s t-test.
White bars, H37Rv; grey bars, DpknH; black bars, DpknH ::pknH.
A. Go
´mez-Velasco and others
730 Microbiology 159
H37Rv
ΔpknH
ΔpknH::pknH
(a)
6
4
2
8
0
6
4
Relative abundance (10–8)
Relative abundance (10–8)
2
8
0
6
4
2
8
015251500147514501425
14001375135013251300 1550
1302.396725
1305.872197
1305.869337
Intensity –10
1310.381135
1319.387024
1324.407725
1346.920608
1354.909849
1368.930211
1376.919556
1406.935084
1420.942166
1463.922941
1475.838823
1501.878624
1545.893172
1559.867795
1559.927213
1545.901202
1503.894895
1501.886591
1487.930194
1481.917490
1471.956524
1462.419903
1460.415322
1448.412990
1433.374687
1420.373318
1406.362522
1391.340996
1384.924616
1376.924555
1354.405696
1341.403086
1332.902949
1327.382892
1318.884633
1310.382896
1302.396186
1529.92
1519.48
1503.89
1498.56
1491.45
1487.93
1485.91
1476.43
1465.22
1462.42
1455.51
1448.41
1439.47
m/z
m/z
1434.39
1427.47
1420.37
1406.36
1404.34
1398.43
1391.33
1385.43
1378.32
1362.29
1348.28
1318.88
1305.87
1545.9
1559.92
1470.52
1443.96
1430.42
1416.4
1412.45
1371.29
1354.4
1346.27
1341.4
1332.39
1327.38
1324.41
1310.38
1302.39
1481.92
1513.95
1305.367081
1324.414742
1346.272131
1348.282030
1363.409046
1368.432027
1371.913817
1398.932389
1454.048574
1469.932299
1476.431517
1491.456919
1498.062218
1513.951398
1519.868413
1529.925227 1529.919917
1519.868504
1513.947287
1503.892422
1496.407211
1491.822925
1487.922237
1481.912796
1471.955040
1465.947629
1459.886275
1454.036386
1449.403949
1443.960285
1432.368826
1398.931752
1390.946676
1385.423854
1371.903817
1362.902562
1348.20608
1341.399153
1332.395429
1327.380797
1302.395050
1310.383051
1318.884139
1324.414431
1327.382552
1332.399458
1341.403371
1348.282483
1346.271726
1362.299208
1385.431732
1398.433937
1404.342668
1406.362517
1412.457346
1420.373353
1430.425136 1434.390004
1448.412158
1455.510257
1470.526368
1476.434033
1487.932221
1498.561664
1519.485131
1559.927057
1545.900240
1529.927543
1513.952988
1503.897428
1491.457197
1462.420250
1439.476653
1427.474544
1416.400350
1391.339340
1378.326211
1371.415852
1354.406713
(b)
9
8
7
6
5
4
3
2
1
10
0
(c)
6
5
4
3
2
1
0
Fig. 3. (a) PDIM region of FT-ICR mass spectra. Total lipids were extracted from mycobacterial strains by the Bligh–Dyer
method. A series of molecular ions corresponding to PDIM masses m/z1300–1600 were observed. Based on the relative
intensity, PDIMs were more abundant in the wild-type and complemented strains compared with the DpknH strain. Absence of
ion species in the region between 1390 and 1449 m/z, corresponding mainly to dimycocerosates A and B, was observed in the
DpknH strain. Relative abundance of ion species corresponding to (b) dimycocerosates A/B and (c) dimycocerosates C.
Abundance is shown as measured by FT-ICR. Black bars, H37Rv; white bars, DpknH; grey bars, DpknH ::pknH. Error bars
represent SEM.
Phthiocerol dimycocerosates synthesis regulation
http://mic.sgmjournals.org 731
Proteins involved in PDIM synthesis are
differentially regulated in the DpknH strain
We have previously analysed the proteome of the DpknH
strain and compared it with that of the parental H37Rv
strain. For this purpose, we used iTRAQ labelling of
cultures grown under rolling conditions with or without
the inducer nitric oxide in the form of 3 mM acidified
nitrite (Chao et al., 2010b). This proteomic approach has
identified the role of PknH in the control of the
mycobacterial dormancy regulon (Chao et al., 2010b).
For the current study, we specifically examined the iTRAQ
experiment data, focusing on proteins encoded by the
PDIM biosynthetic pathway. We detected that, without
induction, the levels of six out of the seven proteins
participating in PDIM biosynthesis were similar when the
mutant was compared with its parental strain.
Interestingly, PpsE levels were higher in the DpknH strain
compared with those in the parental strain (Table 1). PpsE
levels remained high even in the presence of nitric oxide
induction, a treatment which significantly induced the
expression of only one biosynthetic protein, PpsD. Thus,
the iTRAQ experiment reveals that protein levels of
selected PDIM pathways are affected by PknH, which is
is triggered by nitric oxide.
Alteration of LAM/LM ratio in the DpknH strain
Previous in vitro and in vivo studies have shown the
interaction of PknH and EmbR (Molle et al., 2003; Sharma
et al., 2006; Zheng et al., 2007). Overexpression of PknH in
M. smegmatis activates EmbR, which induces the transcrip-
tion of the embCAB operon that encodes arabinosyltrans-
ferases, leading to a higher LAM/LM ratio (Sharma et al.,
2006). Therefore, we extended our study to M. tuberculosis
to further analyse the content of these lipoglycans in the
DpknH strain compared with those of the parental and
complemented strains. LAM and LM were extracted,
purified and analysed on 15 % SDS-PAGE, as shown in
Fig. S4(a). The ratio of LAM to LM was significantly
different between the wild-type and DpknH strains. This
ratio was twofold higher in the mutant strain (Fig. S4b). The
complemented strain showed a similar LAM/LM ratio to the
parental strain (Fig. S4b). Although results agree with those
from previously reported M. smegmatis studies (Sharma
et al., 2006), GC/MS analysis did not show any significant
difference in arabinose and mannose content (Fig. S5).
Nevertheless, we observed that the wild-type strain pro-
duced higher amounts of galactose and glucose compared
with those of the mutant strain (Fig. S5).
DISCUSSION
Despite extensive knowledge of mycobacterial cell-wall
biosynthesis, regulation of cell-wall components is still an
emerging area of study. In vitro kinase studies suggest that
STPKs are important regulatory enzymes in M. tuberculosis
cell-wall biosynthesis (Chao et al., 2010a). However, few
studies have addressed in vivo interactions between STPKs
and the biosynthetic enzymes of the cell wall.
We have shown previously that infection of BALB/c mice
with a pknH knock-out strain resulted in a hypervirulent
phenotype. These results suggest that PknH acts as an in
vivo growth regulator (Papavinasasundaram et al., 2005).
In the present study, we carried out cell-wall lipid analyses
to determine whether deletion of pknH affected cell-wall
lipid biosynthesis. Strikingly, 2D-TLC analysis showed that
PDIMs were not produced in the DpknH strain. However,
when their production was evaluated using more sensitive
techniques, such as radiolabelled 2D-TLC, MS and
immunostaining, we found that although several species
of PDIMs were produced, their overall levels were lower in
the mutant compared with those in its parental wild-type
Fig. 4. (a) Immunofluorescence microscopy analysis. Bacteria were
labelled with rhodamine, and scFv antibodies against PDIMs were
used as primary antibodies. Anti-MBP antibody coupled to goat
anti-mouse IgG-FITC was used as the secondary antibody. The
merged images are shown in the panels on the right. M. smegmatis
was used as a negative control. (b) Immunofluorescence detection.
Data represent the means±SD of green fluorescence intensity
(labelled PDIMs) in arbitrary units (a.u.), which corresponds to
labelled PDIMs, per cellular area; n550 single bacterial cells.
*P¡0.05.
A. Go
´mez-Velasco and others
732 Microbiology 159
or complemented strains. Additionally, we also observed
the accumulation of unknown lipids in the DpknH strain.
The extracellular localization of PDIMs suggests an
important role in cell-wall integrity and pathogenicity. M.
tuberculosis strains unable to produce PDIMs or transport
PDIMs to the cell wall are attenuated in animal models
(Camacho et al., 1999; Cox et al., 1999; Kirksey et al., 2011;
Rousseau et al., 2004; Yu et al., 2012). However, a
discrepancy has been found in two studies in which the
H37Rv strain was used to assess the function of different
genes (Ioerger et al., 2010). These strains retained their
virulence in mice despite harbouring a frameshift mutation
in mas (Ioerger et al., 2010), a gene encoding an enzyme
that catalyses the synthesis of mycocerosic acids. These
strains might have compensating mutations that retain
virulence despite the loss of PDIMs (Ioerger et al., 2010). It
is still unknown how PDIMs mediate virulence, but a
recent study has shown that PDIMs may facilitate a
receptor-dependent phagocytosis and provide protection
against phagosome acidification (Astarie-Dequeker et al.,
2009), although a precise molecular mechanism has not
been defined.
Even though we found that PDIMs were produced at low
levels in the mutant strain, we cannot exclude the
possibility that biosynthesis of other cell-wall components
or other signalling pathways might have been affected by
disruption of the pknH gene. It is well known that PknH
has effects on the transcription of the embCAB operon via
phosphorylation of EmbR (Molle et al., 2003; Zheng et al.,
2007). Furthermore, the overexpression of pknH in M.
smegmatis results in a high LAM/LM ratio (Sharma et al.,
2006). Both LAM and LM act as ligands for host-cell
receptors and contribute to the pathogenesis of M.
tuberculosis, since they are located on its cell surface. It
has been hypothesized that M. tuberculosis adapts to its
human host by mimicking the glycoforms of mammalian
mannoproteins (Torrelles & Schlesinger, 2010). Thus, the
amount and nature of the mannose exposed on the surface
might be major determinants for the phagocytosis and host
response to M. tuberculosis (Torrelles & Schlesinger, 2010).
Bacilli strains with reduced mannose are considered
hypervirulent, whereas strains with abundant mannose
on their surface have become more host adapted (Torrelles
& Schlesinger, 2010). The latter strains may be highly
successful in establishing an infection, potentially leading
to a latent infection (Torrelles & Schlesinger, 2010). In line
with this hypothesis, an unbalanced LAM/ LM ratio might
partially explain the hypervirulence found in the DpknH
strain. Indeed, our lipoglycan analysis revealed that the
LAM/LM ratio was twofold higher in the DpknH strain
compared with that in the wild-type, and this might be due
to the transcriptional effect of the embCAB operon via
EmbR phosphorylation. In fact, deletion of pknH from M.
tuberculosis results in decreased transcription of embB and
embC in cultures treated with sublethal doses of ethambu-
tol (Papavinasasundaram et al., 2005).
We also observed different production levels of unknown
lipids (Fig. 2), which can also affect the course of M.
tuberculosis pathogenesis. Diverse studies have demon-
strated that correct structure and balance of cell-wall
synthesis components have a marked effect on the
pathogenesis of M. tuberculosis. For instance, a mutant
lacking the mmaA4 gene, which encodes the methyltrans-
ferase MmaA4 required for synthesis of keto- and
methoxy-mycolic acids, displayed enhanced production
of IL12p40, an important cytokine that controls intracel-
lular infection (Dao et al., 2008). Furthermore, the same
study found that trehalose dimycolate (TDM), a modified
mycolic acid linked with trehalose, derived from the
DmmaA4 mutant also stimulated IL12p40 (Dao et al.,
2008). The authors suggested that the different biological
activities observed for the TDM wild-type and DmmaA4
are based on the chemical and structural differences
conferred by the functional groups of their mycolates
(Dao et al., 2008). Similarly, certain M. tuberculosis clinical
isolates belonging to the W-Beijing family produce phenol
glycolipids (PGLs) that are hypervirulent in murine disease
models (Reed et al., 2004). However, most M. tuberculosis
strains, including the H37Rv strain, are devoid of PGLs. It
has been proposed that M. tuberculosis strains are natural
mutants deficient in PGLs due to a frameshift in the pks15/
1gene (Constant et al., 2002). Absence of this lipid in
mutants lacking the pks15/1 gene abrogates cytokine-
repressing activity and leads to attenuation of virulence
with extended survival in mouse infection studies
(Constant et al., 2002).
The current study leads us to propose that PknH positively
regulates PDIM biosynthesis, as in the absence of pknH M.
tuberculosis produces low levels of this class of lipids.
Furthermore, our study also suggests that regulation of
Table 1. iTRAQ analysis of PDIM biosynthetic proteins levels in DpknH/pknH with or without treatment with 3 mM acidified nitrite
Protein ORF Untreated ratio Treated ratio (no. of peptides)
PpsA Rv2931 1.05 0.93 (1)
PpsD Rv2934 0.97 1.48 (1)
PpsE Rv2935 2.36 1.93 (6)
PapA5 Rv2939 1.16 1.16 (1)
Mas Rv2940c 1.20 0.89 (8)
FadD28 Rv2941 1.05 0.94 (7)
Ketoreductase Rv2951c 1.06 1.11 (2)
Phthiocerol dimycocerosates synthesis regulation
http://mic.sgmjournals.org 733
PDIM-biosynthetic proteins is fine-tuned rather than
controlled through a strict on/off mechanism as proposed
previously (Veyron-Churlet et al., 2009). Recent studies
have shown that phosphorylation of enzymes involved in
mycolic acid biosynthesis results in a variety of biochemical
outcomes. The enzymes involved in the FAS-II system
during mycolic acid synthesis, such as malonyl-
CoA : : AcpM transacylase (mtFabD) and the b-ketoacyl-
AcpM synthases KasA and KasB, have been shown to
undergo in vitro phosphorylation by different STPKs
(Molle et al., 2006). Interestingly, although KasA and
KasB are similar enzymes that catalyse the condensation of
acyl-AcpM and malonyl-AcpM (Kremer et al., 2002;
Schaeffer et al., 2001), differential regulation by STPKs
has been reported (Molle et al., 2006). For instance,
phosphorylation decreases the activity of KasA, while the
enzymic activity of KasB is enhanced (Veyron-Churlet
et al., 2009). This differential effect of phosphorylation
allows M. tuberculosis to produce immature mycolates by
inhibiting KasA activity but enhancing KasB activity, which
ensures the full-length mycolic acids required for bacilli
virulence and intracellular survival (Veyron-Churlet et al.,
2009). A similar scenario may occur during PDIM
biosynthesis in which different enzymes are phosphory-
lated by STPKs. Two studies have investigated the
involvement of two STPKs in PDIM biosynthesis regu-
lation. In vitro assays have shown that PknB is able to
phosphorylate PapA5 on threonine residues and undergo
reversible phosphorylation by Mstp (Gupta et al., 2009),
indicating that the transfer of mycocerosic acids onto the
phthiocerol moiety is regulated by the PknB–Mstp protein
complex. However, the in vivo effect of phosphorylation by
PknB cannot be assessed since it is an essential gene
product like PknA. Likewise, MmpL7, which is involved in
PDIM transport, was found to be an endogenous substrate
of PknD (Pe
´rez et al., 2006), suggesting that phosphoryla-
tion of MmpL7 can regulate deposition of PDIMs onto the
cell wall.
The iTRAQ experiment has given insights into the nature
of the signalling cascade mediated by PknH. Proteomic
analysis revealed that seven proteins involved in PDIM
synthesis are differentially regulated. Noteworthy is the
level of PpsE expression which was maintained in
untreated and treated cultures among the other proteins
detected in the iTRAQ experiment. PpsE, a PKS, is the final
enzyme involved in synthesis of the b-diol backbone
PDIMs (Trivedi et al., 2005). The upregulation of this
enzyme suggests its importance in PDIM biosynthesis. In
fact, the interaction of PpsE with TesA and MmpL7 has
been shown (Jain & Cox, 2005; Rao & Ranganathan, 2004).
The authors suggested that MmpL7 acts not only as a
transporter but also as a scaffold to couple PDIM synthesis
and transport (Jain & Cox, 2005). PpsE also interacts with
the type II thioesterase TesA; the latter enzyme might be
involved not only in releasing the growing product from
PpsE, but also in housekeeping functions that remove
inappropriate acyl units and/or aberrant acyl intermediates
(Rao & Ranganathan, 2004). These data indicate that PpsE
may act as an activator or inhibitor during final PDIM
synthesis. On the other hand, the upregulation of PpsE in
DpknH strains treated with nitric oxide might be due to the
protein sensing the incorrect synthesis of PDIMs, because
of the lack of signalling by PknH. Synthesis of PDIMs
represents a high energy cost due to replication, transcrip-
tion and translation of a gene cluster ~50 kbp. Thus, it is
tempting to suggest that phosphorylation, mediated by
STPKs and protein–protein complexes, may efficiently
coordinate PDIM synthesis.
Further experiments are needed to address PDIM regu-
lation via PknH phosphorylation; however, our study
provides insights into the multiple in vivo signalling
cascades that it employs. On the other hand, M. tuberculosis
synthesizes and secretes diverse and complex lipids that
interact with the host (Kremer & Besra, 2005). The
outermost layer of the cell wall is composed of free lipids
that include the trehalose ester family (sulfolipids,
diacyltrehaloses, triacyltrehaloses and polyacyltrehaloses),
the recently characterized mannosyl-b-1-phosphomycoke-
tides, the phenolthiocerol and phthiocerol dimycocero-
sates, and the closely related phenolic glycolipids
(Minnikin et al., 2002). Therefore, based on this complex-
ity, the study of particular lipids hinders in vivo lipid-
specific studies, since there is not a unique and highly
specific method to detect all M. tuberculosis cell-wall
components. The development of scFv antibodies against
PDIMs allowed us to monitor biosynthesis and localization
at the bacterial cell surface. This new sensitive technique
can specifically track a single-family lipid class; thus, it has
potential for use in biochemical studies and studies of lipid
biosynthesis during infection.
ACKNOWLEDGMENTS
This study was funded by the Canadian Institute of Health Research
(CIHR) via operating grant no. MOP-106622 (awarded to Y. A.-G.).
We would like to acknowledge the British Columbia Centre for
Disease Control for providing access to a containment level 3
laboratory, the Genome BC Proteomics Centre (University of
Victoria) for performing lipidomic analysis and Jeffrey Helm for
helpful discussions. G. S. B. acknowledges support from the Medical
Research Council (UK) and the Wellcome Trust. G. S. B. also
acknowledges support in the form of a Personal Research Chair from
Mr James Bardrick and a Royal Society Wolfson Research Merit
Award.
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