Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages.

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.

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Sulfolipid-1 Biosynthesis Restricts Mycobacterium tuberculosis
Growth in Human Macrophages
Sarah A. Gilmore,‡Michael W. Schelle,†Cynthia M. Holsclaw,∥Clifton D. Leigh,†Madhulika Jain,#
Jeffery S. Cox,#Julie A. Leary,∥,⊥and Carolyn R. Bertozzi*,‡,†,§
†Department of Chemistry,‡Department of Molecular and Cell Biology, and§Howard Hughes Medical Institute, University of
California, Berkeley, California 94720, United States
∥Section of Molecular and Cell Biology and⊥Department of Chemistry, University of California, Davis, California 95616, United
States
#Department of Microbiology and Immunology, University of California, San Francisco, California 94143, United States
*
S Supporting Information
ABSTRACT: Mycobacterium tuberculosis (Mtb), the causative
agent of tuberculosis, is a highly evolved human pathogen
characterized by its formidable cell wall. Many unique lipids
and glycolipids from the Mtb cell wall are thought to be
virulence factors that mediate host−pathogen interactions. An
intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid
that has been implicated in Mtb pathogenesis, although no
direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0
that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine
macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore,
we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro,
despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific
phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.
M
enzymes, radical ion species, and antibiotics. This is especially
true of the human pathogen Mycobacterium tuberculosis (Mtb),
which possesses a formidable cell wall composed of many
natural products important to its lifecycle.1Distinctive features
of the mycobacterial cell wall include long chain mycolic acids
(C60−90) covalently linked to an arabinogalactan core that serve
to noncovalently anchor an outer membrane of lipids and
glycolipids.1In addition to functioning as a protective barrier,
many components of the Mtb cell wall are implicated in
modulating host−pathogen interactions. For example, glyco-
lipids such as phenolic glycolipids (PGL), lipoarabinomannan
(LAM), and trehalose dimycolate (TDM) interact with
immune cells to modulate the host response.2−4
The sequencing of the Mtb genome led to the identification
of genes involved in lipid biosynthesis, enabling their targeted
disruption and subsequent evaluation of roles in Mtb
pathogenesis.5Results from these experiments demonstrated
that cell wall metabolites can contribute to Mtb virulence.2,6,7
Despite this progress, the biological function of one class of cell
wall glycolipids, the sulfatides, remains elusive. Sulfolipid-1 (SL-
1), the most abundant sulfatide, is a tetra-acylated trehalose-
based glycolipid located on the mycobacterial outer membrane
of Mtb (see Figure 1).8Provocatively, SL-1 is uniquely
expressed in pathogenic mycobacteria, and its amounts have
been positively correlated with strain virulence.9,10The linkage
icrobial pathogens have engineered their cell surfaces to
serve as protective barriers against host degradative
between SL-1 and Mtb virulence inspired a search for functions
of SL-1, which over the span of 50 years has resulted in
attribution of numerous, sometimes conflicting, biological
activities to this sulfatide. For example, in cell culture models
purified SL-1 has been proposed to alter phagosome−lysosome
fusion, disrupt mitochondrial oxidative phosphorylation, and
activate as well as suppress the production of cytokines and
reactive oxygen species produced by human leukocytes.11−17
More recently, knowledge of the genes involved in SL-1
biosynthesis has allowed the generation of Mtb strains deficient
in SL-1 production.18−22However, strains of Mtb lacking fully
elaborated SL-1 do not appear to have consistent phenotypes
or phenotypes distinguishable from wild-type Mtb in animal
models of infection. This observation calls into question both
the physiological relevance of the in vitro activities ascribed to
SL-1 and the ability of animal infection to legitimately model
the role of SL-1 in human tuberculosis.19−21,23
To directly address the role of SL-1 in Mtb pathogenesis, we
utilized SL-1 purified as a natural product from Mtb whole cells
as well as a fully synthetic analogue of SL-1, termed SL-A (see
Figure 1),24to probe the previously reported ability of SL-1 to
modulate leukocyte function. Interestingly, we find that SL-1
Received:
Accepted:
Published: February 24, 2012
August 23, 2011
February 14, 2012
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and SL-A elicit a unique transcriptional signature distinct from
known pro-inflammatory agonists. To evaluate the role of SL-1
in the context of its native environment, the Mtb cell envelope,
we engineered a strain of Mtb in which SL-1 biosynthesis is
abolished at its first committed step,22sulfation of trehalose by
the sulfotransferase Stf0 (see entire pathway elaborated in
Supplementary Figure 1). Mtb strains deficient in SL-1
production demonstrated augmented survival in human
macrophages. Furthermore, the stf0-deletion mutant exhibited
increased resistance in vitro to a human cationic antimicrobial
peptide, LL-37, which suggests a mechanism for the improved
intracellular survival of Δstf0. Both murine macrophages and a
murine model of tuberculosis failed to recapitulate this
phenotype, indicating that SL-1 may contribute to pathogenesis
in a species-specific manner during infection.
Figure 1. Structure of SL-1 and a synthetic analogue. The synthetic analogue of SL-1 is composed of a T2S core esterified with fatty acyl groups at
the 2′, 3′, 6′, and 6 positions, similar to SL-1. However, due to the complex synthesis of the fatty acyl substituents found on the natural product, SL-A
lacks the hydroxyl groups and multimethyl branched units found on SL-1.
Figure 2. SL-1 induces a unique transcriptional signature in human leukocytes. The transcript profiles of hiDCs stimulated with P3K, SL-1, SL-A, or
vehicle only were assessed by microarray. The 50 most highly upregulated and statistically significant transcripts (p < 0.02) induced by SL-1, SL-A, or
P3K as compared to vehicle only treated cells were organized according to function. Due to limited overlap in genes upregulated by each stimulus,
different functional categories were chosen. The percent overlap of gene identities between P3K vs SL-1, P3K vs SL-A, and SL-1 vs SL-A is
highlighted. The identities of individual genes are listed in Supplementary Table 1.
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■RESULTS AND DISCUSSION
SL-1 Stimulates a Unique Transcriptional Signature in
Host Cells. SL-1 is situated on the outer membrane of Mtb
where it is poised to interact with host cells during the course of
Mtb infection. To test this possibility, we purified SL-1 directly
from Mtb whole cells (Supplementary Figure 2) and monitored
its effect on human leukocytes using whole-genome expression
profiling. Since any metabolite purified from cells may contain
trace levels of bioactive contaminants, we also tested the activity
of a fully synthetic analogue of SL-1,24termed SL-A, in these
assays (see Figure 1). Human primary immature dendritic cells
(hiDC) were derived from peripheral blood monocytes of four
independent donors and treated with either SL-1, SL-A, vehicle
only, or the well-characterized Toll-like receptor (TLR)
lipoprotein agonist Pam3Cys-Ser-(Lys)4(P3K) as a positive
control. First, we analyzed our microarray data to identify the
top 50 statistically significant (p < 0.02) transcripts upregulated
by SL-1, SL-A, or P3K as compared to vehicle treated hiDCs.
As expected, P3K induced a robust pro-inflammatory gene
expression profile in hiDCs, with the majority of induced
transcripts involved in mounting an immune response (Figure
2 and Supplementary Table 1). SL-1 and SL-A, conversely, did
not induce a similar pattern of pro-inflammatory gene
expression in hiDCs like P3K; instead, SL-1 and SL-A
stimulated the upregulation of a unique set of transcripts with
24% concordance in genes upregulated in both SL-1 and SL-A
treated hiDCs (Figure 2 and Supplementary Table 1). Thus,
our microarray data suggests that SL-1 is not acting as a pro-
inflammatory stimulus as previously suggested.13To further
Figure 3. Sulfation of trehalose by Stf0 is the first committed step in SL-1 biosynthesis. (A) The sulfotransferase Stf0 sulfates free trehalose to initiate
SL-1 biosynthesis. (B) SL-1 is absent in the Δstf0 strain. Surface exposed lipids were removed by hexanes extraction of WT, Δstf0, and
complemented (Δstf0 + pstf0) Mtb strains and analyzed by FT-ICR MS. SL-1 is observed as a collection of lipoforms that vary in the lengths of
their acyl groups (±14 mass units).
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validate the observation that purified SL-1 plays little role in
mediating a pro-inflammatory immune response, we measured
by ELISA amounts of the pro-inflammatory cytokine tumor
necrosis factor (TNF) induced by SL-1 and SL-A in a variety of
cell types, including hiDCs, human primary macrophages, the
human monocyte THP-1 cell line, and the murine macrophage
RAW 264.7 cell line. Neither SL-1 nor SL-A stimulated
production of TNF in any of the cell types we examined
(Supplementary Figure 3), which corroborated with our
microarray data to provide further evidence that purified SL-1
is not a classical pro-inflammatory stimulus. Reasons for these
discrepancies with previous data could include the presence of
small levels of impurities in previous SL-1 preparations,
differences in the concentrations of SL-1 used, or differences
in the culture conditions of mammalian cells.
To determine a broad subset of genes whose transcription is
influenced by SL-1, we further expanded the analysis of our
microarray data to include all of the statistically significant (p <
0.02) genes that are transcriptionally regulated (i.e., genes
induced and repressed) by SL-1 and SL-A. This analysis
revealed 789 genes reproducibly regulated by SL-1 and 432
genes regulated by SL-A with 172 genes commonly regulated
by both SL-1 and SL-A in hiDCs (Supplementary Table 2).
While we feel that the concordance of genes regulated by SL-1
and SL-A is strong, it is important to note that SL-1 and SL-A
are not structurally identical. Due to synthetic challenges, SL-A
is composed of straight chain lipid moieties at its 3′, 6, and 6′
positions instead of the fully elaborated phthioceranoyl and
hydroxyphthioceranoyl substituents of SL-1 (see Figure 1).24
Nonetheless, to be rigorous we defined a set of SL-1-regulated
genes as those whose expression is seen to be influenced by
both SL-1 and SL-A in these experiments (Supplementary
Table 2). Notably, in this set of SL-1-regulated genes we
observed SL-1-dependent upregulation of CD1D, a gene
involved in activation of NKT cells during Mtb infection,25as
well as SL-1-dependent downregulation of IL8, a chemokine
important for neutrophil chemotaxis.26Also, SL-1 and SL-A
caused upregulation of transcripts involved in phosphatase/
kinase activation, PTPRE, PTPRN, PPP1R11, and PPP1R14A,
which could influence unidentified signaling pathways in
mycobacterial infection (Supplementary Table 1). Overall, we
did not see modulation of classic pro- and anti-inflammatory
genes such as TNF, MIP2, IL10, NFKB, CD83, CD86, or
CD80, which have been previously reported to be regulated by
the mycobacterial lipids TDM and LAM.3,4Interestingly, many
of the SL-1-regulated genes are of unknown function, and thus
these genes will guide future studies to improve our knowledge
of key host−pathogen interactions involved in Mtb patho-
genesis.
Disruption of stf0 Creates a Strain of Mtb for
Evaluating the Contribution of SL-1 to Virulence. With
evidence of an SL-1 induced transcriptional response in
immune cells, we decided to evaluate the role of SL-1 during
pathogenesis in the context of its natural environment, the Mtb
cell envelope. Using bioinformatics methods, we previously
identified the sulfotransferase Stf0 (CAA17370; Rv0295c) in
the Mtb strain H37Rv22that installs a sulfate group onto
trehalose to form trehalose-2-sulfate (T2S) as the first
committed step in SL-1 biosynthesis (Figure 3, panel A). In
order to address the role of SL-1 biosynthesis in Mtb
pathogenesis, we constructed an stf0 knockout in the Erdman
strain of Mtb, because it has recently been reported that the
H37Rv strain can undergo spontaneous loss of the well-
established virulence factor lipid phthiocerol dimycocerosate
(PDIM) during in vitro culture.27Indeed, when we constructed
an Erdman strain of Mtb in which stf0 was disrupted, it no
longer biosynthesized T2S or SL-1, but PDIM biosynthesis
remained intact (Figure 3, panel B; Supplementary Figure 4,
panel a, b). SL-1 production was restored by complementing
the Δstf0 mutant with a plasmid expressing stf0 under the
control of its native promoter (Δstf0 + pstf0) (Figure 3, panel
B).
Loss of SL-1, which makes up approximately 1% of Mtb’s dry
weight,28could alter growth, cell envelope permeability and
integrity, or surface lipid composition. We observed that the
growth of Mtb in liquid culture was not affected by disruption
of stf0 or complementation of the Δstf0 strain (Supplementary
Figure 5). To assess cell envelope integrity and permeability, we
challenged the bacteria with detergents or antibiotics,
respectively. As shown in Table 1, the minimal concentration
of the detergents sodium dodecyl sulfate (SDS) and
deoxycholate (DOC) that inhibited 50% of bacterial growth
(MIC50) were similar for the Δstf0 mutant and WT Mtb.
Likewise, the MIC50 values of the antibiotics isoniazid,
rifampicin, amikacin, and kanamycin were similar between
Δstf0 and WT Mtb (Table 1). These data suggest that loss of
the major cell envelope component SL-1 does not compromise
the overall integrity or permeability of the Mtb cell envelope in
vitro. Additionally, production of other surface extractable lipids
including polyacyltrehalose (PAT)29and phthiocerol dimyco-
cerosate (PDIM) remained robust as determined by qualitative
FT-ICR MS analysis of the stf0 mutant (Supplementary Figure
4, panel b).
SL-1 Biosynthesis Enhances Mtb’s Susceptibility to
the Antimicrobial Peptide LL-37. Because SL-1 is an
anionic species found in the mycobacterial outer membrane, we
reasoned that it could facilitate interactions with cationic
antimicrobial peptides (AMP), as has been demonstrated for
other negatively charged bacterial lipid species including LPS in
the outer membrane of Gram-negative bacteria and phospha-
tidylglycerol (PG) in the plasma membrane of Mtb.30,31AMPs
are small, cationic peptides that are important components of
the innate immune defense against invading pathogens,
including Mtb,32and exert antimicrobial effects by inserting
into and disrupting bacterial cell membranes.33Therefore, we
examined the in vitro susceptibility of the Δstf0 and WT strains
as well as another SL-1-deficient strain (ΔpapA2, Supplemental
Figure 1) to LL-37/CAP-18, a human AMP expressed in
macrophages with known antimicrobial activity against
Table 1. Δstf0 Does Not Exhibit Global Defects in Cell Wall
Integritya
WT
Δstf0
0.02
0.007
0.0125
0.027
0.30
1.5
Δstf0 + pstf0
0.02
0.008
0.0125
0.030
0.40
>7.5b
detergents (% w/v)SDS
DOC
rifampicin
isoniazid
amikacin
kanamycin
0.02
0.010
0.025
0.025
0.30
1.1
antibiotics (μg/mL)
aMtb was grown in the presence of each detergent or antibiotic. The
minimal inhibitory concentration that inhibited 50% of Mtb growth
was noted after 7 days. These data are representative of at least two
independent experiments each performed in duplicate.bΔstf0 + pstf0
contains a kanamycin resistance gene.
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Mtb.32,34As previously demonstrated, LL-37 reduced the
viability of WT Mtb (Figure 4). Conversely, stf0 and papA2
mutants lacking SL-1 proved more resistant to LL-37-mediated
killing in vitro, a phenotype that could be complemented by
expressing stf0 or papA2 (Figure 4 and Supplemental Figure 6).
The resistance of the stf0 and papA2 mutants to LL-37 was not
concentration-dependent in the range of concentrations tested,
suggesting that the interaction of Mtb with LL-37 is dependent
on SL-1 as well as other negatively charged components that
may reside in the Mtb cell envelope. Thus, we conclude that
the susceptibility of Mtb to LL-37 is mediated in part by the
highly abundant, anionic Mtb cell envelope component SL-1.
Stf0 Negatively Regulates the Intracellular Growth of
Mtb in Human Macrophages. The intracellular growth of
Mtb in human macrophages is limited by antimicrobial peptides
such as LL-37, in contrast to murine macrophages that rely
mostly on the production of nitric oxide radicals for bactericidal
activity.32,34,35Thus, we hypothesized that the Δstf0 strain
lacking the negatively charged SL-1 may be less susceptible
than WT Mtb to antimicrobial agents produced by human
macrophages thereby enhancing its intracellular survival. To
test this idea, we infected human THP-1 and murine
RAW264.7 macrophage cell lines with WT, Δstf0, or Δstf0 +
pstf0 strains and evaluated their intracellular growth.
Interestingly, the stf0 mutant displayed increased intracellular
survival in THP-1 cells as demonstrated by a 2.5-fold increase
in cfu counts 6 days post infection (Figure 5, panel A). This
growth phenotype could be partly complemented by expression
of stf0 in the mutant strain (Δstf0 + pstf0) (Figure 5, panel A).
Partial complementation of the stf0 mutant phenotype could
result from use of an integrating plasmid containing the
putative native promoter for stf0 that may not recapitulate wild
type transcriptional regulation of stf0. The augmented survival
of Δstf0 was likely not due to enhanced infectivity of this
mutant strain, as no significant difference in cfu counts was seen
4 h post infection (day = 0) (Figure 5, panel A). In contrast to
their different growth phenotypes in human macrophages, the
Δstf0 and WT strains had indistinguishable growth phenotypes
in RAW264.7 murine macrophages up to 6 days post infection
(Figure 5, panel B). Using a murine model of tuberculosis, we
found that Δstf0 and WT cells proliferated at similar rates in
the lungs, livers, and spleens of BALB/c mice infected via
aerosolization (Figure 5, panel C and Supplementary Figure 7).
Figure 4. Δstf0 is more resistant to LL-37 compared to WT Mtb. The
indicated strains of Mtb were exposed to increasing concentrations (0,
6.5, and 65 μg/mL) of LL-37. After 3 days, Mtb viability was measured
by plating bacteria on solid agar to enumerate cfu counts. Data are
representative of one of three independent experiments each
performed in triplicate. * P = 0.74 (not significant) for the comparison
of Δstf0 versus WT; ** P = 0.012 for the comparison of Δstf0 versus
WT. Error bars correspond to the means (±SD); an error of less than
∼2% is not visible on this graph (SD of Δstf0 at 65 μg/mL = 1.0%; SD
of Δstf0 + pstf0 at 65 μg/mL = 1.27%).
Figure 5. Stf0 restricts the intracellular growth of Mtb in human macrophages. (A) Human THP-1 macrophages or (B) murine RAW macrophages
were infected with the indicated strain of Mtb. After 4 h (day 0) and 6 days post infection, adherent macrophages were lysed and plated on solid agar
to determine the number of viable intracellular bacteria by cfu counts. Data represent the average cfu count obtained from three independent
experiments performed in triplicate (±SD). * P < 4 × 10−5for the comparison of Δstf0 versus WT; ** P < 0.03 for the comparison of Δstf0 versus
Δstf0 + pstf0 (A) or one of three independent experiments performed in triplicate (B) (±SD). Δstf0 behaves similarly to WT Mtb in a murine
model of infection. (C) Lung cfu counts for BALB/c mice infected via aerosol with WT or Δstf0 Mtb. Each data point represents the average cfu
count from 4−5 mice, and error bars indicate the standard deviation from the mean. (D) Time-to-death for mice infected with WT or Δstf0 Mtb.
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