Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule.
ABSTRACT Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.
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ABSTRACT: Mycolic acids (MAs) are α-alkyl, β-hydroxy long-chain fatty acids found in abundance in the cell envelope of the Mycobacterium tuberculosis complex (MTBC). MAs form an efficient permeability barrier, modulate host innate immune responses, and are the targets of several anti-tuberculosis drugs. Using mass spectrometry, we measured the relative abundance of 80 MA species across 36 clinical isolates of MTBC covering four major phylogenetic lineages. We found significant variations in the MA patterns between different MTBC strains and lineages. MA patterns of "ancient" lineages contrasted those from "modern" lineages, with a lower representation of alpha-mycolates among Lineage 6 strains and an inversion of the methoxy: keto-mycolates ratio in Lineage 1 strains. By interrogating the whole genome sequences of these MTBC strains, we identified relevant single-nucleotide polymorphisms that may sustain the lineage-specific MA patterns. Our results show that the strain genetic background influences MA metabolism and suggests that strain diversity should be considered in the development of new anti-tuberculosis drugs that target MA synthesis.MicrobiologyOpen. 09/2014;
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ABSTRACT: Tuberculosis is a disease of the lung, and efficient transmission is dependent on the generation of a lesion in the lung, which results in a bacterium-laden cough. Mycobacterium tuberculosis (Mtb) is able to manipulate both the innate and acquired immune response of the host. This manipulation results in an effective CD4(+) T cell response that limits disease throughout the body but can also promote the development of progressively destructive lesions in the lung. In this way Mtb infection can result in an ambulatory individual who has a lesion in the lung capable of transmitting Mtb. The inflammatory environment within the lung lesion is manipulated by Mtb throughout infection and can limit the expression of acquired immunity by a variety of pathways.Nature Immunology 12/2014; 16(1):57-63. · 24.97 Impact Factor
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ABSTRACT: Infection by Mycobacterium tuberculosis causes tuberculosis, a disease characterized by alteration of host innate and adaptive immunity. These processes are mediated by a series of bacterial biomolecules, among which phenolic glycolipids (PGLs) and the related p-hydroxybenzoic acid derivatives have been suggested to play important roles. To probe the importance of structural features of these glycans on cytokine modulation, we synthesized three M. tuberculosis PGL analogues (1–3), which differ from the native glycoconjugates by possessing a simplified lipid algycone. The ability of 1–3 to modulate the release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and nitric oxide (NO) was evaluated. None of the compounds stimulated the secretion of these signalling molecules. However, all showed a Toll-like Receptor 2-mediated, concentration-dependent inhibition profile that was related to the methylation pattern on the glycan.ChemBioChem 05/2014; · 3.06 Impact Factor
The Journal of Experimental Medicine
JEM © The Rockefeller University Press
Vol. 201, No. 4, February 21, 2005 535–543
immune activation through cyclopropane
modification of a glycolipid effector molecule
controls host innate
and Michael S. Glickman
Steven A. Porcelli,
Division of Infectious Diseases and
New York, NY 10021
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center,
evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear
whether these cell envelope compounds affect pathogenesis through a structural role in the
cell wall or as pathogenesis effectors that interact directly with host cells. Here we show
that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate
(TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM
modification by the cyclopropane synthase
proinflammatory activation of macrophages during early infection. Purified TDM isolated
from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and
induced less severe granulomatous inflammation in mice, demonstrating that the fine
structure of this glycolipid was critical to its proinflammatory activity. These results
established the fine structure of lipids contained in the Mtb cell envelope as direct effectors
of pathogenesis and identified temporal control of host immune activation through
cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.
(Mtb) infection remains a global health crisis. Recent genetic
was both necessary and sufficient for
a major global health emergency, which has
not been controlled by present therapeutic mo-
dalities. More effective antimicrobials or vaccines
to combat Mtb infection will only be possible
through greater understanding of the molecular
strategies used by Mtb to facilitate long-term
persistence in vivo. An abundance of recent
studies have established the
envelope as a critical determinant of Mtb–host
interactions (1–3). Specific mutations in Mtb
that lead to alterations of cell envelope lipids
and glycolipids have revealed that some of
these may lead to marked reductions in viru-
lence. This has been observed for mutations
that lead to a deficiency or failure to secrete
phthiocerol dimycocerosate (4, 5), changes in
mycolic acid carbon chain length (6) or oxy-
genation (7), and lack of mycolate modification
by cyclopropyl rings (8). However, the defects
in growth and pathogenesis observed in mutant
(Mtb) infection remains
strains lacking these diverse cell envelope prod-
ucts are distinct, suggesting that each com-
pound of the complex Mtb cell envelope has a
specialized role in pathogenesis. For example,
deficiency of oxygenated mycolic acids or
phthiocerol dimycocerosate confers replication
defect in mice (4, 5, 7), whereas deficiency of
mycolate cyclopropanation confers a persis-
tence defect (8). A central unresolved question
is whether individual cell envelope compounds
mediate pathogenesis indirectly through struc-
tural effects on properties of the cell envelope
(9) or alternatively act directly as effector mole-
cules that modify host immune responses or in-
terfere with antimicrobial activity (10–15).
gene (Rv0470c) is one recently
defined genetic determinant of Mtb virulence
and persistence that encodes an
methionine–dependent methyltransferase that
catalyzes proximal cyclopropanation of
colate, the major mycolic acid subclass of the
Mtb cell envelope (8). Mycolic acids are
hydroxy–branched fatty acids that are
found in mycobacteria and related taxa and can
exceed 80 carbons in length. These lipids serve
V. Rao and N. Fujiwara contributed equally to this work.
N. Fujiwara’s present address is Department of Host Defense,
Osaka City University Graduate School of Medicine, 1-4-3
Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
Michael S. Glickman:
Abbreviations used: Mtb,
CYCLOPROPANATED LIPID PATHOGENESIS EFFECTOR | Rao et al.
a major structural role in the cell wall of the bacterium, and
have also been identified as targets for the adaptive immune
response via presentation to T cells by the human CD1b pro-
tein (16). In the murine model of infection,
) fails to persist, is attenuated for viru-
lence, and invokes less severe immunopathology than wild-type
Mtb. These results suggested that the site specific cyclopro-
pane modification of mycolic acids is an important determinant
of Mtb-host interactions. As the cyclopropyl modification of
mycolic acids is absent in nonpathogenic mycobacteria, the
phenotypes of the
mutant suggest that this lipid modi-
fication system evolved to mediate important pathogenic
functions such as interaction with host innate immune recep-
tors. To investigate this hypothesis we focused on trehalose
dimycolate (TDM), an inflammatory glycolipid that contains
mycolic acids. Here, we show that the cyclopropyl modifica-
tion of mycolates on TDM modified innate immune recogni-
tion of Mtb and had a major effect on the role of these lipids
as direct effectors of virulence and pathogenesis.
Modulation of the early innate response by
M. tuberculosis infection in vivo
Whereas our prior results specifically implicated cyclopro-
pane modification of mycolic acids as a contributor to Mtb-
induced immunopathology, the mechanism by which
affected pathogenesis was not identified (8). To explore the
role of innate host immune recognition in the
type, we examined in greater detail the behavior of the Mtb
mutant during the early stages of infection in the
lungs. C57BL/6 mice were infected by aerosol inoculation
100 of either wild-type Mtb or the
and bacterial titers were determined at weekly intervals. Both
sets of mice received identical inocula (Fig. 1 A, day 1 time
point). Although our previous studies did not demonstrate
any growth defect in vivo at 3 wk of infection, a more de-
tailed examination at earlier time points revealed a dramatic
initial delay in the growth of
After 1 wk of infection, titers of the
were 50-fold lower than wild-type titers, whereas at 2 and 4
wk after infection, wild-type and mutant titers equalized.
The early growth defect of the mutant was reversed in the
complemented strain (Fig. 1 A, right, comp), demonstrating
that the transient early growth defect was due to loss of
function. These results indicated that the
transiently defective for early lung growth, but not intrinsi-
cally defective for replication in vivo, defining
porally restricted determinant of bacterial growth after air-
borne lung infection. In addition, they suggested a possible
interaction between the
-dependent structural features
of mycolic acids and the innate immune mechanisms acti-
vated in the earliest stages of infection in a naive host.
mutant bacilli (Fig. 1 A).
as a tem-
Dependence on TNF of the attenuation of the
TNF is an important regulator of immune responses and is
critically important for host defense against Mtb infection in
mice and humans (17, 18). In addition to contributing to
immune-mediated control of infection, TNF is an important
determinant of Mtb-induced granuloma structure, and is
likely to be important in preventing reactivation of latent in-
fection (19, 20). Paradoxically, TNF can also facilitate early
growth of Mtb in macrophages (21, 22), suggesting that
TNF has pleiotropic effects on Mtb that may depend on the
stage of infection or cell type examined. Given the effects of
mutation on initial growth of Mtb in vivo, we
tested whether the early growth defect of the
depended on TNF. Infections in TNF-deficient mice re-
vealed that the early growth defect of the
TNF dependence. (A) Wild-type C57BL/6 mice were infected by aerosol
with wild-type Mtb (black bars) or the ?pcaA mutant (open bars), and
bacterial titers were determined at the indicated time points by serial dilu-
tion and plating of lung homogenates. Each bar is the mean of bacterial
loads from four mice per group and error bars are SEM. When error bars
are not visible they are within the plotted bar. P values (Student’s t test)
for the comparison of WT vs ?pcaA mutant Mtb are indicated above each
bar set. The 1- and 7-d time points at the far right are a repeat experiment
including a complemented pcaA mutant (comp, hatched bar). (B) TNF?/?
mice were infected with either wild-type or ?pcaA mutant Mtb as in A,
and CFUs of lung tissue were determined at the indicated times. Each bar
is the mean of four mice per group. (C) Survival kinetics for TNF?/? mice
infected with wild-type or ?pcaA Mtb. 10 mice each were infected with
either wild-type (black line, square symbol) or ?pcaA Mtb (gray line, triangle
symbol) via the aerosol route and the time to death was recorded.
Effect of pcaA on early colonization of the lung and its
JEM VOL. 201, February 21, 2005
only evident in the presence of TNF, as wild-type and mu-
tant bacterial titers in infected lung were the same at all time
points (Fig. 1 B). To test whether the attenuated host mor-
tality of the
mutant infection was also TNF depen-
dent, we infected TNF-deficient mice with the wild-type or
mutant strains of Mtb and recorded host morbidity.
Wild-type mice displayed the predicted accelerated mortality
to Mtb infection that has been reported previously (17). In
contrast to the attenuation of virulence reported previously
mutant in wild-type mice (8),
tant–infected TNF-deficient mice succumbed to the infec-
tion with the same kinetics as mice infected with wild-type
Mtb (Fig. 1 C). Thus, the phenotypic difference between
mutant Mtb disappeared in mice lack-
ing TNF, suggesting that the mechanism by which cyclo-
propane modification of mycolic acids contributed to viru-
lence was linked to the induction of TNF during the early
phase of infection.
Reduction of macrophage cytokine responses to
Because our results suggested that the
ification of the cell envelope altered innate immune recogni-
tion of Mtb in a temporally restricted period during the first
week of murine infection, we infected murine bone mar-
row–derived macrophages in vitro with wild-type and the
mutant bacteria and measured both cytokine produc-
tion and bacterial survival during the first few days of infec-
tion. Wild-type Mtb induced high levels of interleukin-6
and TNF beginning at 24 h after infection (Fig. 2 A, black
bars). In contrast, the
mutant induced 5.3-fold lower
levels of TNF and 1.5-fold lower levels of IL-6 in culture
supernatant at 24 h (Fig. 2 A, open bars). Remarkably, the
hypostimulatory activity of the
rally restricted such that mutant and wild-type strains in-
duced identical levels of TNF by 96 h after infection. Ge-
netic complementation of the
reversed the mutant phenotype (Fig. 2, striped bars).
To more precisely characterize the temporal restriction of
the mutant phenotype, we measured TNF production in
culture supernatants of infected macrophages by removing
supernatants every 24 h such that only the newly synthesized
TNF was measured. These experiments confirmed that the
pcaA mutant was hypoinflammatory during the first 48 h
of infection but not thereafter (Fig. 2 B). These results indi-
cated that the
mutant was differentially recognized by
the innate immune system at the earliest times after infec-
tion, but that
was dispensable for innate immune rec-
ognition later in infection.
To directly ask whether mycolic acids deficient in cyclo-
propanation were hypoinflammatory during infection, we
delipidated the outer surface of live bacteria using extraction
with petroleum ether and infected macrophages with these
delipidated organisms. Delipidation of Mtb by this method
does not affect bacterial viability and removes a subfraction
of cell wall lipids that is
90% TDM when examined by
mutant was tempo-
mutant with wild-type
thin layer chromatography (unpublished data; 23, 24). De-
lipidation of the live bacilli markedly reduced TNF produc-
tion by macrophages infected with wild-type bacteria, but
had no effect on the relatively weak TNF production stimu-
mutant bacteria during early infection. (Fig. 2
A), demonstrating that the difference in innate immune rec-
ognition between wild-type Mtb and the
mediated by an extractable lipid.
To directly test whether the innate immune recognition
mutant bacteria resulted in more effective bacterial
killing, we measured bacterial growth within macrophages.
ates temporally restricted macrophage activation. (A) Requirement of
pcaA for early proinflammatory activation of macrophages by Mtb infec-
tion. Murine bone marrow–derived macrophages were left uninfected
(stippled bar), or infected with wild-type Mtb (black bars), ?pcaA mutant
Mtb (open bars), or the complemented ?pcaA mutant (striped bars). At the
indicated time points after infection, TNF (top), and IL-6 (bottom) levels
were determined by ELISA of culture supernatants. The same experiments
were performed with delipidated bacteria, pictured on the right side of A.
*, P ? 0.01. (B) Temporal sequence of pcaA-dependent macrophage acti-
vation. Murine bone marrow–derived macrophages were infected as
described in A and supernatants were removed and replaced with fresh
media every 24 h. Supernatants from the indicated time periods were
assayed for TNF by ELISA. Stippled bar is uninfected, black bar is wild-type
Mtb, and white bar is ?pcaA mutant infected. **, P ? 0.001. (C) Effect of
pcaA on intracellular replication of Mtb in macrophages. Bone marrow–
derived macrophages from wild-type C57BL/6 mice (WT) or TNF-deficient
mice (TNF?/?) were infected with wild-type Mtb (black bars) or the ?pcaA
mutant (open bars) and intracellular bacterial titers were determined at
the indicated time points. #, P ? 0.02.
pcaA-dependent modification of extractable lipids medi-
CYCLOPROPANATED LIPID PATHOGENESIS EFFECTOR | Rao et al.
Wild-type bacteria replicated rapidly in murine macrophages
(Fig. 2 C). Intracellular replication of wild-type bacteria was
markedly reduced by delipidation (unpublished data) or in
macrophages derived from TNF-deficient mice (Fig. 2 C),
consistent with previous reports documenting that impor-
tance of extractable lipids and host TNF in Mtb intracellular
replication (22, 24). In contrast,
defective for intracellular growth compared with wild-type
bacteria, but delipidation did not further reduce intracellular
replication of mutant bacteria (unpublished data). These re-
sults demonstrated that
-dependent modification of the
extractable lipids of the Mtb cell envelope mediated proin-
flammatory innate immune recognition and facilitated early
growth within macrophages.
To test whether
-modified or wild-type–extractable
lipids were sufficient to determine the innate immune recog-
nition of Mtb during infection, we performed a lipid transfer
experiment. Wild-type or
?pcaA mutant bacteria were delip-
idated and the resulting lipid extract was transferred either
back onto the parent strain or onto the opposite strain. Re-
markably, the hypoinflammatory phenotype of the ?pcaA
mutant was transferred to delipidated wild-type bacteria re-
constituted by ?pcaA lipids, demonstrating that ?pcaA mu-
tant–extractable mycolates directly mediated the early hy-
poinflammatory phenotype (Fig. 3, ?lipid ? opposite, black
bar). Conversely, delipidated ?pcaA mutant bacteria were
recognized as wild type when reconstituted with wild-type
lipids (Fig. 3, ?lipid ? opposite, white bar). These data were
consistent with the conclusion that the altered inflammatory
activity of ?pcaA-extractable lipids directly mediated the al-
tered innate immune recognition of this strain by host mac-
rophages that was apparent within the first 24 h of infection.
mutant bacteria were
Reduced macrophage stimulation by purified ?pcaA TDM
Trehalose dimycolate from Mtb has long been suspected to
be a virulence determinant, in part due to its ability to pro-
duce granulomatous pathology similar to the pathology of
Mtb infection. TDM contains a hydrophilic trehalose head
group and two mycolic acids esterified at the six positions of
each glucose (structure shown in Fig. 4 A). In the absence of
pcaA, TDM lacks a single cyclopropyl ring in its ? mycolates
and has an overabundance of ketomycolates (Fig. 4 C). Be-
cause the innate immune recognition of wild-type and mu-
tant Mtb can be transferred by petroleum ether–extractable
lipids, which are composed predominantly of TDM (Fig. 3),
we hypothesized that the phenotype of the ?pcaA mutant
could be completely or partially attributable to changes in
the inflammatory activity of TDM. To test this directly, we
purified TDM to homogeneity from wild-type Mtb and the
?pcaA mutant (Fig. 4 B) and tested the potency of these pu-
rified glycolipids in stimulating cultured macrophages.
Initial studies demonstrated that TDMs added directly to
the medium of macrophage cultures did not induce any
detectable responses. However, when purified TDM was
coated onto the surface of tissue culture plates, it became
highly stimulatory to macrophages. Wild-type TDM in-
duced high levels of TNF, as measured both by ELISA of se-
creted TNF in cell culture supernatants (Fig. 5 A, black
bars), and by flow cytometry with intracellular cytokine
staining for TNF (Fig. 5 B). In contrast, ?pcaA TDM in-
duced significantly lower levels of TNF from both bone
marrow–derived macrophages and the macrophage cell line
RAW264.7 (Fig. 5 A, open bars, and Fig. 5 B, lower left). A
dose response curve revealed that the hypostimulatory activ-
ity of ?pcaA mutant TDM was present across a wide dose
range and was reversed by restoration of the pcaA gene
flammatory phenotype of the pcaA mutant. Murine bone marrow–
derived macrophages were infected with wild-type Mtb (black bar) or
?pcaA mutant Mtb (open bars) as in Fig. 2 A. Bacteria were either un-
treated (native), delipidated with petroleum ether (?lipid), delipidated and
reconstituted with lipids from the same strain (?lipid ? self), or delipidated
and reconstituted with lipids from the opposite strain (?lipid ? opposite).
TNF levels after 24 h of infection are shown. *, P ? 0.001.
Extractable lipids are sufficient to transfer the hypoin-
mutant TDM. (A) Chemical structure of TDM. (B) Thin layer chromatography
of purified TDM from wild-type and ?pcaA mutant M. tuberculosis devel-
oped with chloroform/methanol/water (90:10:1, vol/vol/vol). (C) Mycolate
subclass analysis of TDM from wild-type and pcaA mutant strains. 14C-labeled
TDM-derived mycolates were analyzed by high performance thin layer
chromatography developed by running three times with hexane/ethyl
acetate (95:5, vol/vol). Mycolate subclasses (?, methoxy, and ketomyco-
lates) are labeled with arrowheads.
Purification and characterization of wild-type and ?pcaA
JEM VOL. 201, February 21, 2005
through direct genetic complementation (Fig. 5 C). This
difference was not due to generalized failure to stimulate the
cells, as levels of IL-10 from TDM-stimulated macrophages
were the same for both wild-type and ?pcaA TDMs (unpub-
lished data). These results demonstrated that the cyclopro-
pane content of TDM was an important determinant of the
inflammatory activity of this glycolipid in macrophages, and
identified the pcaA-dependent cyclopropanation of the my-
colates of TDM as a proinflammatory lipid modification and
a target for recognition by innate immunity.
Proinflammatory effect of pcaA-modified TDM in vivo
To test whether pcaA modification of TDM regulates in vivo
inflammatory responses, we tested the potency of these puri-
fied glycolipids for inducing granuloma formation in mice.
Consistent with the known properties of TDM (25, 26),
wild-type Mtb TDM invoked granulomatous pathology in
the lungs and liver when injected intravenously in mice as a
water–oil–water emulsion (Fig. 6, left). This lung pathology
peaked at day 7 and was characterized by mixed inflamma-
tory infiltrates that obliterated the normal air spaces. Strik-
ingly, ?pcaA mutant TDM was at least twofold less potent
than wild-type TDM at inducing pulmonary granuloma
(Fig. 6). These data demonstrated that the fine chemical
structure of TDM could dramatically alter its inflammatory
potency in vivo, and that pcaA-dependent modification of
TDM was directly proinflammatory. These results also sup-
ported the hypothesis that the hypoinflammatory pathologic
phenotype of the pcaA mutant strain in mice and macro-
phages was directly attributable to the altered inflammatory
properties of cyclopropane-deficient ?pcaA mutant TDM.
The results presented here establish a causal relationship be-
tween the fine chemical structure of mycolic acids and in-
nate immune recognition of M. tuberculosis at the earliest pe-
riod after aerosol infection. Whereas previous studies on the
role of pcaA in virulence emphasized the importance of this
gene in the persistence of Mtb and the pathology of the later
stages of infection, the current study focused on the effects of
deletion of pcaA on the earliest events after infection. This
has led to the identification of the pcaA-dependent modifica-
tion of mycolic acids, and in particular of the mycolic acids
incorporated into TDM, as a critical proinflammatory lipid
modification that regulated host-innate immune recognition
during the first week of the murine infection in vivo and the
first 24 h of macrophage infection in vitro.
Trehalose dimycolate, also named “cord factor,” has
been an intensely studied cell envelope compound of M. tu-
berculosis for over 50 yr. TDM was the first virulence deter-
minant proposed for M. tuberculosis when it was identified in
a petroleum ether extract of M. tuberculosis and found to in-
hibit the migration of neutrophils (27–32). The biologic ac-
tivity designated cord factor was later identified as TDM and
was thought to be responsible both for the cording morphol-
ogy and mycobacterial virulence. This postulated important
role for TDM became less plausible when TDM was isolated
from all mycobacteria that produce mycolic acids, most of
which are nonpathogenic and do not form serpentine cords
(33). However, interest in TDM has remained intense due
to its powerful adjuvant properties, chemical properties
when interacting with membranes (23, 34, 35), and ability to
induce granulomatous inflammation in experimental animals
that mimics whole Mtb infection (25, 26, 36).
By analyzing the activities of purified TDMs in vitro and
in vivo, the results from the current study also strongly sup-
ported the view that the cyclopropane modification of TDM
recognition by macrophages. (A) RAW 264.7 murine macrophage cell
line or murine bone marrow–derived macrophages were stimulated with
vehicle (stippled bar), or a monolayer of wild-type Mtb TDM (black bar),
pcaA mutant TDM (open bar), or complemented mutant TDM (striped bar).
TNF (ng/ml) in culture supernatants was measured 48 h after stimulation.
*, P ? 0.001. #, P ? 0.03. (B) RAW 264.7 cells were stimulated with indi-
cated TDMs for 3 h, incubated for 3 h with Brefeldin A, and stained with
monoclonal antibody to TNF. In the upper left plot, the shaded histogram
is vehicle stimulated cells, whereas the open histogram is LPS-stimulated
cells. Numbers above each plot are the percentage of TNF-positive and
-negative cells using the gates established on unstimulated cells in the
upper left panel. P ? 0.01 for the comparison of WT versus ?pcaA TDM by
t test. (C) Dose response curve for TNF induction by wild-type TDM (triangle),
?pcaA mutant TDM (square), or complemented TDM (circle) from RAW
cells measured after 24 h of stimulation.
Effects of pcaA modification of TDM on innate immune
M. TUBERCULOSIS CYCLOPROPANATED LIPID PATHOGENESIS EFFECTOR | Rao et al.
in the Mtb cell envelope acts directly as an effector of patho-
genesis, rather than by inducing indirect effects due to struc-
tural modifications of the cell envelope. As such, this study
provides proof of principle that the chemical diversity of the
Mtb cell envelope has evolved to interact specifically with
host cells and not solely as a structural scaffold, as has been
noted with other cell envelope mutants with impaired viru-
lence (11). Cyclopropane modification of membrane lipids
has been defined in E. coli and other bacteria and affects re-
sistance to cold shock and acid (37, 38). However, the im-
munomodulatory function for cyclopropane modification of
bacterial lipids identified in the current study is a novel func-
tion for this chemical entity. M. tuberculosis expresses a large
family of mycolic acid methyl transferases/cyclopropane syn-
thases that modify mycolic acids (39–41), two of which are
known to be important for pathogenesis (7, 8). However,
the pathogenetic mechanism of cyclopropanation for bacte-
rial virulence or pathogenesis has been unclear. Mycolates
are recognized by T cells when presented on CD1, but evi-
dence to date indicates that this recognition is independent
of cyclopropane modification (42). Instead, our findings
demonstrated that cyclopropane modification of mycolic ac-
ids acted directly to promote the virulent behavior of myco-
bacteria by modulating innate immune activation of macro-
phages and potentially other cell types during infection. The
particular macrophage receptor molecules responsible for
these responses to TDM have not yet been identified, and
this important point will require further study.
Our results strongly point to TNF as a key mediator of
the effects of the normally cyclopropanated TDM molecules
of wild-type Mtb on the host immune response. Thus, the
reduction in growth rate of the ?pcaA mutant seen in the
first week of infection was reversed in TNF-deficient mice,
and the difference observed previously in survival of mice
infected with wild-type versus ?pcaA bacilli was also absent
in TNF-deficient mice. Because the ?pcaA mutant elicited a
markedly reduced TNF response compared with wild-type
Mtb, these findings were consistent with the recent proposal
that one effect of TNF may actually be to facilitate the
growth of the bacilli early in the course of infection (22).
Thus, the reduced stimulation of TNF production by ?pcaA
TDM leads to less abundance of TNF during initial infec-
tion, and reduced bacterial growth in the first week. The
critical importance of TNF in antimycobacterial defense is
well established in mice (17) and humans (18). However,
the apparent protective effect of TNF is partially due to the
defective immune regulation that results from its absence,
leading to massive TH1 type immune activation, tissue ne-
crosis, and death (19, 20). A direct role for TNF in antimy-
cobacterial activity of macrophages has been controversial,
and recent data suggest that TNF facilitates growth of viru-
lent, but not attenuated strains of Mtb (21, 22) in cultured
human macrophages, suggesting that induction of TNF may
be an important virulence strategy of Mtb. Previous studies
of Mtb infection in TNF-deficient mice have shown that
bacterial burdens are unaffected during the first 2 wk of in-
fection, suggesting either that TNF has no role in early
growth of Mtb, or that TNF has equal and opposing effects
on bacterial growth during early infection in vivo. In this
latter model, loss of a growth promoting effect of TNF in
in mice. C57BL/6 mice were injected intravenously with a water–oil–water
emulsion of TDM purified from wild-type (two left panels) or ?pcaA mu-
tant (two right panels) Mtb at a dose of 300 ?g per mouse. The histology
Reduced pulmonary granuloma formation by ?pcaA TDM
of the lungs was examined at 7, 14, and 21 d after injection. The area of
granulomatous inflammation was quantitated as described in Materials
and methods and plotted against time. Diamonds represent wild-type
TDM whereas squares represent ?pcaA mutant TDM. *, P ? 0.05.
JEM VOL. 201, February 21, 2005
macrophages would be counterbalanced in vivo by loss of a
growth restricting effect of TNF produced by other cell
types (43). This model is consistent with the data presented
here in which the growth of the pcaA mutant is restricted in
wild-type mice but recovers to wild-type Mtb growth levels
in TNF-deficient mice. The data presented here indicate
that Mtb has evolved cyclopropane lipid modification to
manipulate the host TNF axis. In the case of the ?pcaA mu-
tant, defective growth in the first week after aerosol infec-
tion and altered innate immune recognition during this pe-
riod attenuated the later pathology of the infection. As
shown in our previous study (8), this dramatically alters the
course of chronic Mtb infection, and thus emphasizes the
powerful interrelationship between innate and adaptive im-
munity in this infection.
Our results expand earlier studies that examined the role
of cell envelope lipids in immunopathogenesis of Mtb infec-
tion. A clinical strain of Mtb that was hypervirulent for mice
induced lower levels of TNF in mouse lung at 28 d (44) and
was hypoinflammatory in cultured macrophages in vitro over
the course of a 96-h infection (45). Recent work indicates
that these phenotypes are due to production of phenolic gly-
colipid by this clinical strain (46). Thus, the accumulated data
indicate the prolonged suppression of host TNF by PGL
promotes bacterial virulence, whereas temporally restricted
suppression of host TNF during the first weeks of infection
through loss of the pcaA modification of TDM is advanta-
geous to the host. These data are consistent with a model in
which structurally distinct lipid components of the cell enve-
lope promote or inhibit host inflammatory responses at dis-
tinct time periods during the course of infection for the ulti-
mate purpose of achieving microbial symbiosis.
Our past and present results provide new insight into the
relationship between TDM and mycobacterial pathogenesis.
Our previous work demonstrated that inactivation of the cy-
clopropane synthase pcaA abolished cording and attenuated
Mtb in mice (8). In light of prior work with cord factor
these results suggested that the cyclopropane modification of
TDM was necessary for the cording morphology and ex-
plains the lack of cording of saprophytic mycobacteria that
contain TDM because these mycobacteria lack cyclopropane
modification of mycolic acids. Our present results indicate
that pcaA modification of TDM with cyclopropyl groups is a
proinflammatory modification both in the context of puri-
fied glycolipid and whole bacilli. Strikingly, this pathoge-
netic function of this lipid modification is temporally re-
stricted to early infection. This demonstrates not only that
cell envelope glycolipids of Mtb are direct effectors of patho-
genesis, but that each cell envelope effector may have dis-
tinct functions at restricted time points during infection.
Although cyclopropane synthases of Mtb are clearly not es-
sential for in vitro growth and viability, the findings of the
current study suggest that pharmacologic inhibition of mem-
bers of this enzyme family could reverse pathogen-induced
immunomodulation, thereby enhancing host immunity and
control or eradication of infection.
MATERIALS AND METHODS
Media, strains, and culture conditions. All M. tuberculosis strains were
grown in Middlebrook 7H9 liquid media (Becton Dickinson) supple-
mented with 10% oleic acid/albumin/dextrose/catalase (OADC) (Becton
Dickinson), 0.5% glycerol (Fisher Scientific) and 0.05% Tween-80 (Sigma-
Aldrich). Where appropriate, hygromycin (Roche) was added at a final
concentration of 50 ?g/ml. The wild-type M. tuberculosis strain used in this
study was M. tuberculosis Erdman, which has been passaged in mice and
minimally passaged in vitro. The M. tuberculosis ?pcaA mutant and the ?pcaA
mutant complemented with a single copy of pcaA under its native promoter
have been described previously (8). Solid media for the growth of Mtb was
Middlebrook 7H10 (Becton Dickinson) with 10% OADC and 0.5% glyc-
erol, and cultures were incubated at 37?C with 5% CO2.
RAW 264.7 cells and L929 cells were obtained from American Type
Culture Collection and were cultured in DMEM and RPMI-1640, respec-
tively, supplemented with 10% FBS, L-glutamine, Pen-Strep, 50 ?g/ml
Gentamicin, Hepes, and 2-mercaptoethanol (Gibco-BRL). All culture me-
dia and cells were tested for LPS by the Limulus amebocyte assay: QCL-
1000 (Cambrex Biosciences) and were below the limit of detection of the
assay (0.1 EU/ml). All cell lines and tissue culture reagents were tested rou-
tinely for mycoplasma contamination using a PCR-based assay, as described
Purification of TDM from Mtb. TDM was purified from M. tuberculo-
sis grown in liquid media. Cells were harvested by centrifugation and auto-
claved to kill viable bacteria. Autoclaved pellets were weighed and soni-
cated in chloroform/methanol (4:1, vol/vol) for 15 min on ice. Water was
added (1/20 total volume) and the organic phase was collected. The aque-
ous phase was sequentially reextracted with chloroform/methanol (3:1 and
2:1, vol/vol) and the organic phases combined and evaporated completely.
The dried pellet was extracted with acetone and the insoluble phase con-
taining TDM was collected by centrifugation. The TDM fraction was pre-
cipitated from chloroform by dropwise addition of methanol at 4?C to a
final ratio of 1:2 chloroform/methanol (vol/vol). This precipitate was dis-
solved in tetrahydrofuran and reprecipitated by dropwise addition of metha-
nol at 4?C to a final ratio of 1:2 tetrahydrofuran/methanol (vol/vol). The
precipitated TDM fraction was then dissolved in chloroform/acetone (8:2,
vol/vol), loaded onto a column of silica gel, and eluted with chloroform/
methanol (9:1, vol/vol). The final product was weighed and the purity and
quantity were examined by TLC using 10 ? 10 cm HPTLC plates (Alltech
Associates, Inc.) developing with chloroform/methanol/water (90:10:1,
vol/vol/vol). Products were visualized by spraying with 20% sulfuric acid in
ethanol and charring for 15 min at 110?C.
For analysis of mycolates derived from TDM, logarithmically replicat-
ing bacilli were incubated with 50 ?Ci 2-[14C] acetic acid (PerkinElmer)
for 18 h. Labeled bacilli were harvested by centrifugation and extracted
with 2:1 chloroform-methanol (vol/vol) for 12 h The chloroform-metha-
nol extract was dried under nitrogen and mycolic acids were prepared as de-
scribed previously (39). Mycolates were analyzed by high performance thin
layer chromatography using three developments of hexanes/ethyl acetate
(95:5) and plates were visualized by autoradiography using a Bio Max Tran-
Screen LE (Eastman Kodak Co.).
Response of macrophages to TDM. Purified TDM was used to stimu-
late either RAW 264.7 cells or bone marrow–derived macrophages by a
modification of the protocol described previously (48). In brief, TDM was
suspended at a concentration of 1 mg/ml in isopropanol and sonicated in a
bath sonicator (model 3510; Branson Ultrasonic Corporation) for 5 min.
This suspension was then incubated at 60?C for 10 min and sonication re-
peated. The resulting solution was layered onto 24-well tissue culture plates
at the indicated concentrations and incubated at 37?C in order to ensure
complete evaporation of the solvent. Control wells were layered with sol-
vent without TDM and incubated at 37?C. To this layer of TDM, either
RAW 264.7 cells or bone marrow–derived macrophages were added at a
concentration of 106 cells in 100 ?l of medium and incubated at 37?C. At
M. TUBERCULOSIS CYCLOPROPANATED LIPID PATHOGENESIS EFFECTOR | Rao et al.
various time intervals after stimulation, supernatants were collected for anal-
ysis of cytokine production by using the commercial ELISA duo-set kit
(BD Biosciences) according to the manufacturer’s recommendations. Intra-
cellular cytokine staining was performed with the Cytofix/Cytoperm system
(BD Biosciences) according to the manufacturer’s instructions. Antibodies
used were FITC-labeled anti-CD11b (Mac-1) antibody and APC-labeled
anti–TNF-? antibody (BD Biosciences) and CD11b? cells were analyzed
for expression of TNF in an LSR flow cytometer (BD Biosciences).
Isolation of bone marrow macrophages. Marrow cells were isolated
from both hind limbs of mice and cultured at a concentration of 2–5 ? 106
cells/ml in RPMI-1640 containing 20% FBS and 30% L929 cell supernatant
at 37?C in Petri-dishes (OPTILUXTM; BD Discovery Labware; BD Bio-
sciences). After 2 d, the plates were washed with sterile HBSS in order to
remove the nonadherent cells. The adherent population was further incu-
bated in macrophage growth media (RPMI 1640 ? 20% FBS ? 30% L929
cell supernatant) for 3–4 d after which the cells were harvested.
Preparation of inoculum and infection of macrophages. Before in-
fection with mycobacteria, bone marrow–derived macrophages were
seeded at a concentration of 2 ? 105 cells/well in a 24-well tissue culture
dish in RPMI 1640 medium containing 10% FBS without antibiotics and
incubated at 37?C in an atmosphere of 5% CO2 for 16–18 h. The mycobac-
terial strains for infection of macrophages were cultured in Middlebrook
7H9 broth at 37?C to mid-log phase of growth (A600 of 0.5–0.8). The cells
were harvested by centrifugation at 3,000 g and washed twice with PBS
containing 0.05% Tween-80 in order to remove excess media components.
The cells were resuspended in PBS–Tween-80, sonicated for 5 s to disperse
clumps, and adjusted to a concentration of 107/ml based on the A600. Mac-
rophages were infected at an multiplicity of infection of 5 for 6 h at 37?C.
The cells were washed twice with sterile HBSS in order to remove extra-
cellular bacteria. The levels of secreted cytokines in the culture supernatants
were estimated by ELISA. For estimation of the bacterial load, macrophages
were lysed by addition of a solution of PBS containing 0.05% SDS and se-
rial dilutions of the lysates were plated onto Middlebrook 7H10 and incu-
bated at 37?C for 3 wk.
Infection of mice by mycobacteria. 6-8-wk-old C57BL/6 mice ob-
tained from Jackson ImmunoResearch Laboratories and maintained on
standard feed and specific pathogen-free conditions were infected aerogeni-
cally with mycobacterial strains in a Middlebrook inhalation exposure sys-
tem (Glas-Col). All animal procedures were approved by the Memorial
Sloan Kettering Institutional Animal Care and Use Committee. The myco-
bacterial inoculums for murine infection were prepared as described earlier
for infection of macrophages. Mtb cells were suspended at a concentration
of 4 ? 108 CFU in 10 ml of sterile distilled water. Mice were infected with
a volume of suspension and exposure time calibrated to deliver ?100 CFU
per animal. The extent of infection was estimated by plating the lung ho-
mogenates of animals killed at 24 h after aerosol infection. At various time
intervals after infection the lungs and spleen were homogenized in PBS ?
0.05% Tween-80 and serial dilutions were plated onto Middlebrook 7H10
agar. After incubation at 37?C in a 5% CO2 atmosphere, mycobacterial col-
onies were counted and the bacterial burdens in the organs were calculated.
Delipidation and lipid reconstitution of mycobacteria. Delipida-
tion of bacilli was performed by using petroleum ether using a modification
of published methods (24). After two 5-min extractions with petroleum
ether, delipidated cells were centrifuged and suspended in PBS/0.05%
Tween-80 for infection of macrophage cultures. Reconstitution of lipids
was done by incubating delipidated cells with the petroleum ether extracts
of mycobacterial cultures of identical cell number for 30 min at 25?C, fol-
lowing which cells were harvested by centrifugation and suspended in PBS/
0.05% Tween-80. Macrophage cultures were infected with the native, de-
lipidated, and reconstituted bacilli at a multiplicity of infection of 5 as de-
Induction of pulmonary granulomatous inflammation in mice.
Granulomatous inflammation was induced by systemic injection of mice
with a suspension of purified TDM. To prepare 1 ml of suspension, 1.5 mg
of purified TDM was dried completely and then redissolved in 32 ?l of In-
complete Freund’s Adjuvant (Difco Laboratories), to which 32 ?l of PBS
was added. Normal saline with 0.2% Tween-80 was then added to a total
volume of 1 ml, and the suspension was extensively mixed using a rotary
homogenizer to form a water–oil–water emulsion. C57BL/6 mice were in-
jected intravenously through the tail vein with 200 ?l of water–oil–water
emulsion containing 300 ?g of TDM. Mice were killed at day 7, 14, and 21
after injection. The lungs were removed and fixed with 10% formalin. The
sections were paraffin embedded and stained with hematoxylin–eosin. The
areas of granulomatous inflammation were calculated by digital image pro-
cessing using the Scion Image program (Scion Corp.). The level of granulo-
matous inflammation was quantitated by determining the area within each
section that showed a pixel density greater than a threshold value that was
two standard deviations above the average for the entire section. This area
was divided by the total lung area in the section and multiplied by 100 to
obtain a percentage value for the area of diseased lung.
The authors thank Feng Gao and Paola Bongiorno for outstanding technical assistance.
This work was supported by National Institutes of Health grants AI53417 (M.S.
Glickman), AI 45889 (S.A. Porcelli), and AI48933 (S.A. Porcelli). M.S. Glickman is the
recipient of the Ellison Medical Foundation New Scholars Award in Global Infectious
Diseases, and a grant from the Speakers’ fund for Biomedical Research awarded by
the City of New York. N. Fujiwara was supported by grants from the Ministry of
Health, Labour and Welfare in Japan (Research on Emerging and Re-emerging
Infectious Diseases, Health Sciences Research Grants) and the Mitsubishi Pharma
The authors have no conflicting financial interests.
Submitted: 18 August 2004
Accepted: 16 November 2004
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