The Two-Domain LysX Protein of Mycobacterium
tuberculosis Is Required for Production of Lysinylated
Phosphatidylglycerol and Resistance to Cationic
Erin Maloney1., Dorota Stankowska1., Jian Zhang2, Marek Fol1¤, Qi-Jian Cheng3, Shichun Lun3,
William R. Bishai3, Malini Rajagopalan1, Delphi Chatterjee2, Murty V. Madiraju1*
1Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, United States of America, 2Department of Microbiology, Immunology and
Pathology, Colorado State University, Fort Collins, Colorado, United States of America, 3Department of Medicine; Center for Tuberculosis Research, Johns Hopkins
University School of Medicine, Baltimore, Maryland, United States of America
The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as
phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic
species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component
of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase
(lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows
increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane
potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF
alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both
proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and
showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our
results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and
for survival of the pathogen upon infection.
Citation: Maloney E, Stankowska D, Zhang J, Fol M, Cheng Q-J, et al. (2009) The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for
Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides. PLoS Pathog 5(7): e1000534. doi:10.1371/journal.ppat.1000534
Editor: Eric J. Rubin, Harvard School of Public Health, United States of America
Received February 4, 2009; Accepted July 8, 2009; Published July 31, 2009
Copyright: ? 2009 Maloney et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: We gratefully acknowledge the support of National Institutes of Health (www. nih.niaid.gov) grants and contracts [AI41406, AI73966 (MM); AI48417
(MR); AI37139 (DC); AI 30036, AI 36973, and AI 37856 (WB)]. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Department of Immunology and Infectious Biology, University of Lodz, Lodz, Poland
. These authors contributed equally to this work.
Mycobacterium tuberculosis (Mtb), the causative agent of tubercu-
losis, is a successful human pathogen that has infected more than
one-third of the world’s population. The success of Mtb as an
infectious agent relies, in part, on its ability to modulate the
expression of bacterial factors in response to infection so that it can
successfully multiply within the hostile host environment . The
characteristic lipid-rich cell envelope of Mtb is one of the factors
believed to contribute to its survival in vivo [2,3]. It is generally
believed that Mtb polar lipids (PoLs) consisting of acidic
phospholipids (PL) such as cardiolipin (CL), phosphatidylglycerol
(PG), phosphatidylinositol and its mannoside derivatives, in
addition to basic PL such as phosphatidylethanolamine, are
important constituents of the Mtb membrane . Mtb PLs are
known to function as important immune modulators  and have
been shown to be released within phagosomes and transferred into
lysosomes [5,6]. It is interesting to note that PG, which is an
abundant PL in other bacteria, is only a minor species in
Mycobacteria, whereas CL is a major species [2,3] with a high
turnover rate .
The relative ratio of acidic to basic PLs is one of the
determinants of net membrane charge. In some Gram-positive
pathogens such as Staphylococcus aureus and Listeria monocytogenes, a
fraction of the PG or CL molecules, or both, are lysinylated by the
esterification of a glycerol hydroxyl group to lysine. Lysinylation
imparts a net positive charge to these acidic PLs. This could, in
turn, influence the ratio of acidic to basic PLs, resulting in an
altered membrane charge. This could explain the bacterial
susceptibility to cationic antibiotics (CAMAs) and peptides
(CAMPs) [8,9]. Although Mtb PLs have been well characterized
for more than four decades, it is unknown if lysinylated PLs are a
subset of the Mtb PLs and, if so, what the consequences associated
with the absence of these lysinylated PLs might be. The present
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study demonstrates that the Mtb lysX gene, encoding a two-domain
protein, is required for the production of lysinylated PG (L-PG),
and the absence of L-PG is associated with changes in membrane
potential, increased sensitivity to CAMAs and CAMPs, and
growth defects in vivo.
Identification of Lysinylated PLs in Mtb
In order to detect lysinylated PLs in Mtb, actively growing
cultures were incubated with14C-lysine for 3 days; total lipids were
extracted, and PoLs were resolved by thin layer chromatography
(TLC). A distinct radiolabeled lipid was evident, indicating that
lysinylated PLs are members of the Mtb PoL pool (Fig. 1A, lane i).
In S. aureus, the mprF gene is responsible for L-PG production .
Homology searches of the Mtb genome identified Rv1640c as lysX,
which encodes an mprF-like gene as a fusion to a lysyl-tRNA
synthetase (lysU). The latter gene is distinct from the essential
housekeeping tRNA synthetase (Rv3598c). The mprF gene in S.
aureus encodes a protein with potential lysyl transferase activity
. In order to evaluate the function of lysX, we created a lysX
mutant strain, Rv-80lys, by replacing the majority of the coding
region comprising the mprF and lysU domains with a gentamycin
resistance cassette using homologous recombination (see Methods
section). A complementing derivative of this strain, Rv-81ami, was
created by integrating a plasmid expressing the intact lysX gene
under the control of the amidase promoter . The lysX mutant
strain was found to be defective in the production of L-PoLs
(Fig. 1A, lane iv compared with lane i). L-PoL production was
restored, however, in the lysX complemented strain Rv-81ami
(Fig. 1A, see lane vii), confirming that the lysX gene product is
responsible for the production of L-PoLs. Staining TLC plates
with iodine (lanes ii, v, viii and xi) or ninhydrin (lanes iii, vi, ix and
xii), on the other hand, did not detect L-PoLs, indicating that they
may not be an abundant lipid species. We cultured Mtb in the
presence of14C-acetic acid and extracted total lipids, followed by
TLC separation and subsequent quantification of L-PoL relative
to total input radioactivity, and found that L-PoL accounts for
approximately 0.3% of the total lipids (data not shown).
Structural analysis of the lysinylated polar lipid
In order to determine the nature of the L-PoL, preparative 2D-
TLC was carried out to collect L-PoLs. Structural analysis of the
L-PoL was carried out using a combination of MALDI-MS, amino
acid analysis and NMR (Fig. 2A–D). The MALDI-MS analysis in
negative-ion mode revealed m/z 681 ([M-H]2) to be the molecular
ion peak (Fig. 2A). The1H-NMR results confirmed the presence of
an acetyl group at dCH32.1 ppm and dCH2from the primary
amine in lysine at d 2.4 ppm (Fig. 2B), whereas the31P-NMR
spectrum showed a shift in the phosphorus resonance spectrum at
d 214.96 ppm (Fig. 2B inset). Fatty acid analysis demonstrated
that the molecule was C18 (data not shown), and amino acid
analysis following acid hydrolysis confirmed the presence of lysine
(Fig. 2C). Together, these data demonstrate that lysine is
covalently linked to PG with the predicted structure shown in
Fig. 2D. The L-PoL in the text is referred to hereafter as L-PG.
Similar structural analyses of the corresponding unlabeled PoL of
the slower migrating radioactive lipid species of lysX (Fig. 1A, lane
iv) could not be done, given that it was present in negligible
quantities (not shown). The thermal decomposition products of
lysine are well characterized . We speculate that the PoL
accumulation in the lysX strain is a consequence of lysine
degradation. Further studies are required to clarify the nature of
the lipid species accumulating in the lysX mutant.
An intact lysX gene containing lysU and mprF domains is
necessary for the production of L-PG
As previously noted, the Mtb lysX is a fusion gene encoding both
mprF and lysU activities, with mprF located at the 59 end of the lysX
gene (see Fig. 1B). The Gram-positive bacteria that have been
shown to produce L-PG, however, contain only mprF. The Mtb
MprF and S. aureus MprF share three domains of unknown
function, DUF470, DUF471 and DUF472. In order to evaluate
whether L-PG production in Mtb requires the activities of both the
LysU and MprF domains, we generated Rv-82med, a lysX
complemented derivative that produces only the MprF domain
(see Methods) and evaluated its ability to produce L-PG following
the incubation of actively growing cells with radiolabeled lysine.
The Rv-82med strain, much like Rv-80lys, was defective in L-PG
production (see Fig. 1A, lane x and compare with lane iv).
Quantitative real-time PCR analysis using primers and TaqMan
probes targeted to the mprF region of lysX (compared to the 16S
rRNA housekeeping gene) revealed that the expression of mprF in
Rv-82med was comparable to that in Rv-03 wild type and Rv-
81ami (data not shown). Together, these results indicate that Rv-
82med expresses mprF and that the MprF domain alone is not
sufficient for the production of L-PG in Mtb.
Phenotypes associated with the absence of L-PG
Gram-positive organisms such as S. aureus and B. subtilis are
sensitive to cationic antimicrobial antibiotics (CAMAs) such as
vancomycin (Van) and polymyxin-B (PMB) and to cationic
antimicrobial peptides (CAMPs) such as human neutrophil peptide
(HNP-1) and lysozyme. On the other hand, Mtb is generally
tolerant to these compounds. HNP-1 and lysozyme are produced
in neutrophils and macrophages, respectively. It is generally
believed that CAMPs induce cell death by interfering with the
integrity of the negatively charged membrane. Furthermore, the
ability of intracellular pathogens to resist the action of CAMPs
The human pathogen Mycobacterium tuberculosis (Mtb)
survives in the hostile intracellular environment, in part, by
withstanding the actions of host-induced cationic antimi-
crobial peptides (CAMPs). Membrane phospholipid com-
position and the resultant charge could play an important
role in Mtb survival within the host. Acidic phospholipids
such as cardiolipin, phosphatidylinositol and its mannoside
derivatives, phosphatidylglycerol, and a single basic
species, phosphatidylethanolamine, are constituents of
the Mtb membrane bilayer. We demonstrate that lysiny-
lated phosphatidylglycerol (L-PG) represents another basic
phospholipid and that the lysX gene, which encodes a
two-domain protein with lysyl transferase and lysyl-tRNA
synthase activities, is necessary for L-PG production. We
show that L-PG is required for maintenance of an optimal
membrane potential and resistance towards CAMPs.
Phagosomes containing the lysX mutant showed an
increased association with lysosomes, and the lysX mutant
showed growth defects in mouse and guinea pig lungs,
indicating that LysX activity is required for full virulence.
Collectively, our results suggest that LysX activity, which is
responsible for the production of L-PG, is necessary for
maintenance of an optimal membrane potential such that
the pathogen can grow optimally upon infection, presum-
ably by withstanding the actions of CAMPs.
Mtb lysX and Lysinylated Lipid
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Figure 1. Polar lipid and Southern blot analysis of the lysX mutant strain. A: Mtb strains were grown in the presence and absence of14C-
lysine. Total lipids were extracted in chloroform:methanol (2:1 v/v) and resolved by TLC on Silcia Gel 60 (EMD Chemicals, New Jersey) in a solvent
system of chloroform:methanol:water (65:25:4 v/v/v). TLC plates were either visualized by autoradiography (lanes i, iv, vii and x), exposed to iodine
vapors (lanes ii, v, viii and xi), or stained with ninhydrin (lanes iii, vi, ix and xii). B: Southern blot analysis of Mtb lysX mutant strains. B-i: The ClaI
fragment bearing the wild type lysX gene (3.5 kb) with the locations of the mprF and lysU regions marked. The dark box designated as ‘‘probe 1’’ is an
approximately 750 bp fragment that hybridizes with the 59-end of lysX and 160 bp of the lysX coding region. The ClaI fragment bearing the mutant
lysX gene disrupted with the gentamycin cassette (0.9 kb) is also shown. The dark band designated as ‘‘probe 2’’ is the 900 bp gentamycin gene that
hybridizes with the mutant lysX gene. B-ii: Southern blot analysis of ClaI-digested Mtb genomic DNA hybridized with probe 1. The 7 kb and 4 kb
band positions represent Rv-03 and Rv-80lys, respectively. Note that the complemented copy contains a band corresponding to the integrated copy
of lysX gene plus the flanking plasmid sequence. B-iii: Southern blot analysis of ClaI-digested Mtb genomic DNA (see Fig. 1B-iii) hybridized with
probe 2. pMMR85 is a positive control plasmid containing the mutant lysX gene plus flanking regions.
Mtb lysX and Lysinylated Lipid
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produced by the host is, in part, responsible for pathogen
proliferation upon infection . The presence of lysine groups
on the acidic PG would impart a net positive charge and,
therefore, could affect the net ratio of positively charged to
negatively charged PL species. Thus, the absence of L-PG could
make the Mtb membrane relatively acidic, thereby sensitizing the
bacterium to the action of CAMAs. To test this possibility, actively
growing Rv-03, Rv-80lys, Rv-80ami and Rv-82med were exposed
to Van and PMB, and growth and viability were measured
(Fig. 3A). Van and PMB interfered with the growth and viability of
Rv-80lys and Rv-82med (see Fig. 3A-ii and 3A-iii), inset showing
viability after 3 days of exposure; and Figure S1 showing viability
after 6 days of exposure). Comparisons of growth, measured as the
change in optical density (OD), and viability, measured as the
change in CFU, revealed that while the lysX mutant was relatively
more sensitive to Van and PMB than Rv-03, it was able to recover
when grown in the absence of antibiotics, indicating that Van and
PMB do not exert potent bactericidal activity. All of the strains
grew well in the absence of antibiotics, although the lysX mutant
showed a small reduction in growth rate in the absence of
antibiotics (Fig. 3A-i), inset shows an approximately 0.3 log
reduction in viability). Visualization of Rv-80lys cells following
nucleoid staining and bright field or fluorescence microscopy did
not reveal any significant differences in cell morphology or
nucleoid organization (data not shown).
The increased sensitivity of the lysX strain to antibiotics suggests
an enhanced association between the two. To test this possibility,
actively growing lysX and Rv-03 cells were stained with
fluorescent-vancomycin (Fl-Van), and the staining patterns were
visualized by fluorescence microscopy. Earlier studies revealed that
in stained Mtb cells, Fl-Van associates with the nascent growth
zones, primarily at the poles and mid-cell septa . These studies
also indicated that not all Mtb cells could be stained with Fl-Van
. We found that a higher percentage of lysX cells were stained
with Fl-Van compared to Rv-03 (see Figure S2). Approximately
52% of lysX cells showed staining patterns not only at the mid-cell
Figure 2. Structural analysis of L-PoL. A: MALDI mass spectrometry analysis of L-PG in negative-ion mode. The m/z 681 [M-H]2represented the
molecular ion peak. B: Structural analysis of L-PG.1H-NMR spectrum of L-PG. The acetyl group can be found at dCH32.1 ppm, and dCH2from the
primary amine in lysine was detectable at 2.4 ppm. The inset shows the
214.96 ppm. C: The amino acid profile of L-PG. Pure lipid was hydrolyzed with 6 N HCl for 24 h, and the soluble hydrolytic product was analyzed in
an amino acid analyzer. Note that the elution of lysine at 54.33 min coincides with the standard (not shown). The small peak at position 41 min
corresponds to a buffer change during the run, and the ammonia peak at 56.11 min is from the buffer used to run the analyzer. D: The proposed L-
PG structure. The proposed structure of L-PG with a C18fatty acid identified from fatty acid analysis (data not shown).
31P-NMR spectrum, in which the phosphorus resonance shifted at
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and polar septa but also over the entire length; meanwhile, only
32% of wild type cells showed such a staining pattern (see Figure
S2 legend for details). These results are consistent with the idea
that Van is able to gain better access to Rv-80lys cells compared to
Next, we examined whether the lysX mutant was also sensitive
to lysozyme and HNP-1. Similar to the results seen with the
CAMAs, HNP-1 and lysozyme significantly reduced the viability
of Rv-80lys compared to Rv-03 and Rv-81ami (Fig. 3B-i and 3B-
ii), see legends for P values). The phenotype of Rv-82med was
found to be similar to that of Rv80lys (Fig. 3B). Together, these
results indicate that the absence of L-PG production is associated
with increased sensitivity of the bacterium to the actions of
CAMPs and CAMAs. Importantly, these experiments also showed
that complementation of the lysX mutant restored the wild type
growth phenotype under these conditions (Fig. 3A and 3B).
Altered membrane potential of Mtb lysX mutants
We wished to test whether the absence of L-PG production in
Rv-80lys cells was associated with changes in the properties of the
PL bilayer (e.g., membrane potential). The membrane potential of
the Rv-80lys cells was determined using a slow-response
membrane potential-sensitive dye, DiOC2(3), and comparing with
Rv-03 cells. This cationic cyanine dye exhibits green fluorescence
(Ex=488 nm and Em=520 nm) in the monomeric state and red
fluorescence (Ex=488 nm and Em=620 nm) in the aggregated or
oligomeric state. As a negative control, the membrane potential
was measured following exposure of the cells to the proton
ionophore m-chlorophenylhydrazone (CCCP), which is known to
eliminate the proton gradient across the membrane. As seen in
Figure 4, the membrane potentials (measured as the ratio of red to
green fluorescence) of the lysX mutant Rv-80lys and Rv-82med
were 21% and 17%, respectively, higher than that of the Rv-03
and complemented Rv-81ami (P,0.002). The increased ratio of
red to green fluorescence observed in lysX mutants suggests
accumulation of the positively charged lipophilic dye on the
negatively charged membrane. The red to green fluorescence ratio
in all strains was decreased to similar levels (,41%) in the presence
of CCCP (P=0.001). Presumably, this reduction reflects the
completely depolarized state of the membrane. Together, these
results indicate that the membranes of Rv-80lys and Rv-82med
are hyperpolarized relative to Rv-03 and Rv-81ami.
Figure 3. Phenotype of lysX strains. Panel A: The growth and viability of the Mtb lysX strains in 7H9 broth in the absence of antibiotics -i. A-ii:
The growth and viability of cultures grown in the presence of 1.0 mg/ml Van. At the indicated times, growth was measured. After 3 days of growth in
broth, viability was determined by plating cells on Middlebrook 7H11 agar and determining the CFU. Symbols: Filled diamonds – Rv-03; grey squares
– Rv81-ami; white triangles – Rv-80lys; crosses – Rv-82med. The inset shows the viable cell count. Black bars - Rv-03, grey bars - Rv-81ami, white bars -
Rv-80lys and dashed bars - Rv82-med. A-iii: The growth and viability of cultures grown in the presence of 100 units/ml PMB. * Represents a P
value,0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method); bars represent means6standard error. Panel B: The growth and viability
of Mtb strains in the presence of 1 mg/ml lysozyme -i- or HNP-1 -ii. There was no significant reduction in viability compared to cultures grown
without TFA (data not shown). The stars represent P,0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method). The bars represent the
Mtb lysX and Lysinylated Lipid
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lysX mutant phenotype in macrophages
We next examined whether the Mtb lysX mutant Rv-80lys
showed proficient growth in macrophages upon infection of the
THP-1 macrophage cell line. The Rv-80lys showed a modest
growth defect in macrophages compared to Rv-03 and comple-
mented Rv-81ami (see Fig. 5, P=0.01 for day 3, P=0.006 for day
6). Similar results were also noted for Rv-82med (Fig. 5, P,0.014
for day 3 and P,0.03 for day 6 compared to Rv-03).
Intracellular replication of Mtb is, in part, due to its ability to
resist the delivery of its phagosomes to lysosomes . This
process can be visualized by examining the co-localization of Mtb
with the lysosome-associated-membrane protein (LAMP-1). In
order to address whether the lysX mutant had a phagosome-
lysosome fusion defect, we infected macrophages with Mtb strains
expressing green-fluorescent protein and visualized co-localization
with LAMP-1. Increased association of phagosomes containing
Rv-80lys with lysosomes was evident compared to Rv-03 and
complemented Rv-81ami (Fig. 6, p,0.001). Rv-82med behaved
like Rv-80lys, indicating that the full-length lysX gene is required
for functional activity. These results are consistent with the
hypothesis that the lysX mutant is not as proficient as the Rv-03
strain in preventing fusion of phagosomes with lysosomes, which
could contribute to defects in intramacrophage replication.
The production of inflammatory cytokines tumor necrosis
factor-alpha (TNF-a), IL-6 and IL-10 is necessary to mount a
protective immune response against Mtb infection [16,17,18].
TNF-alpha restricts the growth of Mtb in alveolar macrophages
, and the multiplication of virulent Mtb in monocyte-derived
macrophages (MDMO) is associated with suppression of TNF-a
production during the early periods after infection. To test selected
pro-inflammatory cytokine responses of macrophages, MDMO
were infected with Rv-80lys and Rv-03 strains, and the production
of TNF-a and IL-6 was measured (Fig. 7). As can be seen, the
secretion of TNF-a was elevated after infection with the lysX
mutant compared to the wild type and complemented strains,
similar to MDMO cells exposed to PMA (Fig. 7A). Similarly, the
secretion of IL-6 was also increased following infection with the
lysX mutant compared to the wild type and complemented strains
lysX has growth defects in vivo
To evaluate the phenotype of lysX in vivo, C57BL/6 mice and
Hartley strains of guinea pigs were aerosol infected with lysX, and
the viability of the pathogen was measured (Fig. 8). The lysX
mutant showed only a modest growth defect in mice (Fig. 8A) but
was clearly attenuated in guinea pigs (Fig. 8B) and showed reduced
dissemination to the spleen (Figure S4). Gross pathology and
histopathology of the lungs of infected mice at 28 days (Fig. 8C
and E) and guinea pigs at 42 days (Fig. 8D and F) showed distinct
differences between the wild type and the lysX mutant.
Hematoxylin-eosin staining confirmed that the lungs infected with
Rv-03, but not those infected with Rv-80lys, had extensive
inflammation in both species and showed caseating granulomas in
guinea pigs. The vast differences in the growth kinetics and
pathology between the wild type and lysX mutant pathogens
indicate that LysX activity is required for full virulence.
Interestingly, the lysX complemented strain Rv-81ami behaved
like the lysX mutant with respect to in vivo growth and pathology
(data not shown), indicating that the complemented strain is not
able to restore lysX function in vivo.
The ability of Mtb strains to produce complex cell wall-
associated lipids called phthiocerol dimycocerosates (PDIM) and to
bind and reduce neutral red dye is associated with virulence.
Avirulent and attenuated strains are defective in these processes.
Furthermore, virulent Mtb strains propagated in the laboratory
often lose these properties [2,19,20,21]. The neutral red reduction
and PDIM profiles of the lysX mutant were comparable to those of
wild type cells (Figure S5), indicating that the observed in vivo
growth defects of the lysX mutant are not due to a loss of PDIM
and defect in neutral red reduction.
The primary conclusion of our data is that L-PG is one of the
basic PLs in Mtb and that the lysX gene, encoding the two-domain
LysX protein, is responsible for its production. Although L-PG is a
Figure 5. The viability of lysX in macrophages. THP-1-derived
macrophages were infected with Mtb strains; at the indicated times
following infection, the macrophages were lysed, and the Mtb viability
was determined. The white bars represent Rv-80lys; the dashed bars
represent Rv-82med; the grey bars represent Rv-81ami; and the black
bars represent Rv-03. Data are mean6standard error from three
independent experiments, and the Mann-Whitney Rank Sum Test was
used for data analyses.
Figure 4. Determination of the membrane potential of Mtb
strains. The relative membrane potential was calculated using the
average mean fluorescence intensity (the ratio between the average red
fluorescence and the average green fluorescence). The graph shows the
average of four experiments. Wild type and lysX mutant strains were
incubated with 3 mM DiOC2(3) for 5 h in either the presence (+) or
absence (2) of 100 mM CCCP. The bars represent the mean6standard
error. * Represents P,0.002 versus Rv-03 and Rv-81ami (Student-
Newman-Keuls Method), whereas # refers to P=0.001 compared to
untreated with CCCP.
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Figure 6. Co-localization of Mtb with LAMP-1-expressing phagosomes. Panel A: THP-1-derived macrophages were infected with GFP-
expressing Mtb strains (Rv-03, Rv-80lys, Rv-81ami and Rv-82med). Bacteria (green spots) inside LAMP-1-positive phagosomes (red) produce a yellow
signal indicating co-localization (merged). Panel B: The percent co-localization was determined by visual scoring of yellow spots after 72 h of
infection. We analyzed 126 macrophages for Rv-80lys, 164 for wild type, 168 for complemented, and 127 for Rv-82med and scored 534 bacterial cells
for Rv-80lys, 1,307 for wild type, 848 for Rv-81ami, and 402 for Rv-82med. Mtb Rv-03 (black bar; 21.261.6%), Rv-80lys (white bar; 52.063.0%), Rv-
81ami (grey bar; 26.462.2%) and Rv-82med (dashed bar; 52.662.8%). * P,0.001 versus Rv-03 and Rv-81ami using the Student-Newman-Keuls
Method; bars represent mean6standard error.
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minor PL species of Mtb, its absence has several consequences, one
of which is an alteration of the membrane potential. This
underscores the role of LysX activity in maintaining optimal
membrane function. Presumably, the absence of L-PG in the lysX
mutant shifts the ratio of acidic to basic PLs, thereby hyperpo-
larizing the membrane. A consequence of the absence of L-PG is
the increased sensitivity of the pathogen to lipophilic antibiotics
such as PMB and Van. It is likely that hyperpolarization of the
membrane in the lysX mutants due to its net negative charge
promotes interactions with cationic peptides and antibiotics
produced by the host immune system, which in turn could lead
to the killing of the invading pathogens [13,22]. It is known that
host-induced CAMPs are one of the frontline defenses against
invading pathogens. Therefore, sensitization of lysX mutant Mtb
cells to the action of CAMPs suggests that maintenance of the
optimal membrane potential is necessary for Mtb growth in vivo.
In partial support of this claim, we found that the lysX mutant
showed defects in intracellular replication (Fig. 5) and that
infection of macrophages with lysX led to increased production
of pro-inflammatory cytokines (Fig. 7). We also found that the lysX
mutant showed increased co-localization with LAMP-1 vesicles
(Fig. 6). Finally, we showed that the lysX mutant was attenuated in
guinea pig lungs and had a modest growth defect in mouse lungs
(Fig. 9). Together, these results are consistent with the hypothesis
that LysX activity is required to maintain an optimal membrane
potential and possibly to promote pathogen survival upon
infection. Notably, the gross pathological differences between the
lysX mutant and wild type were striking compared to the modest
differences in growth in vitro and ex vivo (see Figs. 3, 5 and 8). The
reduced bacterial burden and the reduced pathology and size of
granulomas in the lungs of guinea pigs clearly suggest that LysX
activity is required for bacterial multiplication and virulence.
Evaluation of the host-induced cytokine response following
different stages of infection with wild type and lysX mutant
pathogens could provide valuable insights into lysX function. Our
studies also showed that the lysX mutant, like wild type, retained
the ability to produce PDIMs and reduce neutral red (Fig. S5). It
remains to be evaluated, however, if other membrane and cell
wall-associated lipids are modulated in the lysX background.
The production of L-PG is believed to involve two biochemical
steps: the generation of lysyl-tRNA by the LysU protein and the
transfer of a lysine group from the lysyl-tRNA to PG by MprF, a
membrane-bound lysyl-transferase protein . The Gram-
positive bacteria shown to produce L-PG carry a single
housekeeping lysU gene that encodes a cytosolic LysU protein
[8,9,24]. E. coli does not contain L-PG, but ectopic expression of
the S. aureus mprF gene allows E. coli to accumulate L-PG in their
membranes, suggesting that cytosolic LysU and membrane-bound
MprF cooperate to produce L-PG [10,25]. Mtb contains two lysU
genes, one encoded by Rv3598c, which is an essential gene, and
the other encoded by the lysU domain of lysX . Since
expression of the mprF fragment of lysX does not lead to the
production of L-PG (Fig. 2), it appears that in Mtb, unlike in other
bacteria, the cytosolic LysU and the membrane-bound MprF do
not cooperate to produce L-PG.
This raises the question as to why a dedicated lysU gene
product, distinct from the housekeeping gene, is required for L-PG
production in Mtb. One possibility is that the lysinylation reaction
occurs on the membrane, and the local presence of LysU and
MprF activities are required to transfer lysine from the lysyl-tRNA
to the membrane-bound PG. If the cytosolic lysyl-tRNA could not
diffuse through the Mtb plasma membrane, a separate activity
would be needed to replace it. Nonetheless, such dedicated
activities imply that PG lysinylation in Mtb is a tightly regulated
reaction. The temporal expression profile of Mtb genes upon
infection in mice shows that lysX is upregulated during acute and
chronic infection . Presumably, increased expression levels of
lysX would ensure that sufficient levels of L-PG were produced to
maintain the optimal ratio of acidic to basic PLs. This would, in
turn, ensure that the optimal membrane potential required for Mtb
proliferation upon infection is maintained. Another possibility,
although unlikely, is that the demand for lysyl-tRNA required for
lysinylation and protein synthesis cannot be met by a single
housekeeping enzyme. Clearly, however, further studies are
required to address this issue.
While this manuscript was in preparation, Vandal et al.
reported the characterization of several transposon mutants of
Mtb that were hypersensitive to acidic pH, one of which was lysX
[28,29]. Their transposon mutants were hypersensitive to
antibiotics and other stressors such as heat, SDS and DETA-
NO. Although the lysX mutant was moderately sensitive to DETA-
NO, its growth was not attenuated in murine lungs. It is unknown
whether L-PG is produced in the lysX transposon mutant and
whether the lysX mutant shows any residual activity. As shown in
Fig. 1A, our lysX mutant was generated by removing most of the
coding sequence responsible for producing the mprF and lysU
activities. We demonstrated that L-PG was not produced in the
lysX mutant and that maintenance of the membrane potential and
resistance to CAMPs were dependent on LysX activity. Impor-
tantly, we showed that LysX activity was required for full virulence
Figure 7. Select cytokine responses of macrophages. Panel A:
MDMO were infected with Mtb strains, and TNF-a release was measured
by ELISA. PMA was used as a positive control. TNF-a levels in panel A
were measured after 24 h. * Represents P,0.05 compared to Rv-03, Rv-
81ami, control (untreated cells) and PMA. Panel B: Secretion of IL-6 by
MDMO after 48 h of infection. * P,0.05 compared to Rv-03, Rv-81ami
and control. The experiments were done in duplicate, and represen-
tative results are shown. Data are mean6standard error. Student-
Newman-Keuls Method was used.
Mtb lysX and Lysinylated Lipid
PLoS Pathogens | www.plospathogens.org8 July 2009 | Volume 5 | Issue 7 | e1000534
in mice and guinea pigs. These results underscore the importance
of lysX function in Mtb survival upon infection. One limitation of
our results, however, is that the complemented Rv-81ami was not
able to restore the lysX defect in vivo, although it did restore
defects in other assays reported in this study. One possibility is that
the expression of lysX in-trans at an attB locus was not sufficient to
restore the LysX activity to optimal levels, and small changes in
activity could have consequences for the complementation
phenotype in vivo. Further studies are required to address this
L-PG appears to be a minor lipid species, yet the loss of L-PG
production affected membrane potential and Mtb growth in vitro
and in vivo. It is interesting to note that PG, the purported
substrate of L-PG, is also a minor lipid species in Mtb and other
mycobacterial species [2,3,30,31]. This raises the question of how
the lysinylation of a minor PL species contributes to the observed
phenotype. It is known that PG is a biosynthetic intermediate of
CL, one of the major PL species of mycobacteria. Indeed, the
enzymatic activities responsible for CL production from PG pools
have been detected in mycobacteria . PG also accumulates as
a result of CL catabolism and, if unregulated, could be further
processed to produce a diacylglycerol intermediate via the action
of phospholipases [33,34]. Accordingly, we speculate that the
lysinylation step helps to prevent PG degradation such that the
optimal membrane potential required for Mtb survival upon
infection is maintained. Our results also suggest that changes in
membrane potential are a potential mechanism for regulating
CAMP sensitivity in Mtb and possibly in the mprF mutants of other
bacteria; therefore, this could be exploited to develop novel
antimicrobial compounds. It is tempting to speculate that by
Figure 8. Growth of lysX and WT strains in vivo. Panel A: C57BL/6 female mouse lungs were infected with lysX (triangles) and WT (dark
diamonds) strains via an aerosol route. The mean CFU counts were obtained from lung homogenates of at least three mice per group and plated on
Middlebrook 7H10 agar plates. All plates were incubated at 37uC for at least three weeks before colonies were counted. Panel B: Guinea pigs (Hartley
strain) were infected with lysX (open symbols) or WT (dark symbols) strains via an aerosol route. Five guinea pigs were sacrificed at days 1, 21 and 42
days after infection, and the survival of Mtb strains in the entire lung homogenate was determined by plating on agar medium as described above.
Panels C, D: Gross pathology of the lungs infected with the Rv-80lys and Rv-03 Mtb strains. Lungs from mouse -C- and guinea pig -D- were excised,
stored in 10% formalin, embedded, and stained with hematoxylin and eosin for histopathological analysis. Note the presence of tubercles on the
surface of the lungs for Rv-03 compared to Rv-80lys. Panels E, F: Histopathology of mouse -E- and guinea pig -F- lungs infected with the Rv-80lys
and Rv-03 Mtb strains. Hematoxylin-eosin staining confirmed that the lungs infected with Rv-03, but not those infected with Rv-80lys, had extensive
inflammation in both species and showed caseating granulomas in guinea pigs.
Mtb lysX and Lysinylated Lipid
PLoS Pathogens | www.plospathogens.org9 July 2009 | Volume 5 | Issue 7 | e1000534
manipulating LysX activity, we could promote the action of other
conventional antibiotics against Mtb.
Mouse and guinea pig infection protocols were approved by the
Animal Care and Use Committee at Johns Hopkins School of
Medicine, Baltimore, MD for mice and at Texas A & M
University, College Station, TX for guinea pigs, under the NIH
contract (Tuberculosis Animal Research and Gene Evaluation
Strains, media and culture conditions
The Mtb strains were cultured in Middlebrook 7H9 broth
supplemented with 10% OADC (oleic acid, albumin, dextrose,
catalase) and 0.05% Tween-80. As needed, 50 mg/ml hygromycin
(hyg), 10 mg/ml kanamycin (kan) or 50 mg/ml gentamycin (gm)
was added. For the determination of viable colonies and the
scoring of recombinants, bacterial cells were plated on Mid-
dlebrook 7H11 agar plates containing the appropriate antibiotics.
In some experiments, cultures were grown in the presence of L-
[U-14C]-lysine (300 mCi/mmol, Amersham Pharmacia Biotech)
or [1,2-14C] acetic acid (46 mCi/mmol, PerkinElmer), and total
lipids were extracted and resolved by TLC. The radioactivity
present in the L-PG spot was determined and normalized relative
to total in put radioactivity.
Construction of the lysX deletion and complemented
The lysX coding region was cloned downstream of the amidase
promoter in an integration-proficient, hygromycin-resistant plas-
mid and electrotransformed into Mtb in order to generate the lysX
merodiploid strain. The chromosomal copy of the lysX gene was
disrupted in the lysX merodiploid background by homologous
recombination as described previously . Using this approach,
90% of the lysX coding region was replaced with a 900-bp
gentamycin resistance cassette. This strain, designated as Rv-
81ami, was the lysX complemented strain. Next, the resident
integrated plasmid encoding the functional lysX gene was replaced
with an empty kanamycin-resistant plasmid to generate the lysX
mutant strain, designated as Rv-80lys, as described [14,35,36,37].
A cartoon depicting the lysX mutant and complemented strain
construction is shown (Fig. 9). All strains were confirmed by PCR
and Southern blot analysis.
For the generation of the lysX complemented strain expressing
the mprF domain, a 1,950 bp lysX gene encoding the mprF domain
was amplified by PCR using the primers MVM530lysF (59-
lys650MM606R (59-AGC AGCAAGCTTCTAGAATCACGC-
CAACCGCTCGGGACTGC-39) and cloned into the pJFR19
vector under the control of the amidase promoter . The
integrity of cloned insert was verified by DNA sequencing. This
recombinant plasmid was used to replace the resident empty
plasmid in the Rv-80lys mutant to generate Rv82-med. This strain
was confirmed by PCR and Southern blot analysis.
Intracellular growth measurements
The human monocyte cell line THP-1 (American Type Culture
Collection, Rockville, Maryland) was used. Cells were grown in
RPMI 1640 (Invitrogen, CA) supplemented with 2 mM L-
glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum
(Invitrogen) and 100 U/ml penicillin G (Sigma, MO). The
viability of the macrophages was determined using trypan blue
Figure 9. Cartoon showing construction of the lysX mutant and
complemented strains. A- refers to the wild type strain carrying the
lysX gene at its native locus. B- refers to the Rv-80 merodiploid (hygr)
strain produced by the integration of a plasmid expressing the lysX
gene from the amidase promoter. C- refers to a single crossover (SCO,
hygr, kanr, gmr) recombinant produced by integration of the suicide
recombination plasmid. D- refers to a mutant double crossover (DCO,
hygr, kans, gmr) produced following a reciprocal recombination event.
E- refers to Rv-80lys (kanr, gmr) produced following switching of the
resident integrated plasmid (hygr) with the incoming pMV306 plasmid
(kanr). F- refers to Rv-82med (hygr, gmr) produced following switching
of the resident pMV306 plasmid (kanr) with the incoming plasmid
expressing the mprF fragment from the amidase promoter (hygr).
Mtb lysX and Lysinylated Lipid
PLoS Pathogens | www.plospathogens.org 10July 2009 | Volume 5 | Issue 7 | e1000534
staining. Monocytes were differentiated into macrophages by
exposure to 50 nM PMA (phorbol 12-myristate 13-acetate; Sigma)
and 7.5 ng/ml IFN-c (human interferon-gamma, Peprotech) for
24 h, followed by a 24 h incubation with 50 nM PMA alone. The
macrophages were washed three times with RPMI 1640 medium
and incubated in medium that was not supplemented with PMA
or IFN- c for the next 24 h. Single cell suspensions of Mtb strains
in RPMI 1640 media were used to infect 4.56105macrophages in
triplicate in a 24-well plate at a multiplicity of infection of 1:2–4.
After 3 hours of infection, macrophages were lysed in 0.09% SDS,
and viability was determined to get a t0 count. No statistical
differences in viability among these strains were noted at t0.
Subsequently, macrophages at 3 and 6 days post-infection were
also processed in order to determine Mtb viability. The lysX strains
were no more sensitive than the other strains in terms of the
concentrations of SDS used to lyse the macrophages and process
them for viability determination (see Figure S3).
Co-localization of M. tuberculosis with phagosomes
THP-1 macrophages (56105) attached to glass coverslips were
infected with GFP-producing Mtb (Rv-03, Mtb Rv-80lys, Rv-
81ami and Rv-82med). The macrophages were fixed, blocked and
incubated with H4A3 monoclonal antibodies to LAMP-1,
followed by a rhodamine-conjugated goat anti-mouse IgG, as
described . Bacterial co-localization with LAMP-1-positive
vesicles appeared as yellow spots. The experiments were done in
duplicate, and representative images are shown.
Cytokine measurement in MDMO
Peripheral blood mononuclear cells (PBMC) were isolated from
healthy volunteers by differential gradient centrifugation on Ficoll-
Paque Plus (Amersham Biosciences). Adherent monocytes were
isolated by seeding 56106cells in 24-well plates in MDMO-media
(RPMI 1640 supplemented with 10% heat-inactivated human
serum) and incubating for 90 min at 37uC in 5% CO2. Following
the removal of non-adherent cells, MDMO-media was added, and
cells were incubated at 37uC for 4 days to mature into
macrophages and then used for infection with Mtb strains.
56105macrophages were infected in triplicate in 24-well plates
at a multiplicity of infection of 1:5 as described previously . At
indicated periods of infection, supernatants were removed, and the
TNF-a and IL-6 levels were measured using ELISA assays
(eBioscience, Inc., CA) according to the manufacturer’s instruc-
tions. In some experiments, MDMO were stimulated with 150 nM
PMA, and the secretion of TNF-a was measured.
Isolation of mycobacterial lipids
The extraction of total lipids from whole cells using chlor-
oform:methanol (2:1 v/v) and the separation of polar lipids in a
solvent system containing chloroform:methanol:water (65:25:4) in
the first dimension and chloroform:methanol:acetic acid:water
(80:12:16:4) in the second dimension were performed as described
previously [7,32,39]. Polar lipids were visualized by exposing the
plates to iodine vapors or staining them with ninhydrin in order to
detect amino acid-containing lipids. In some experiments,
autoradiography was used to detect radiolabeled lipids. For
PDIMs analysis hexane:diethylether:acetic acid (80:20:1, vol/
vol/vol) solvent system was used.
MALDI-MS, ESI-MS and ESI-MS/MS
MALDI-MS was performed using an UltraFlex TOF/TOF
(Bruker Daltonics, Billurica, CA) as described previously . The
L-PG sample in acetonitrile was mixed 1:1 with 2,5-dihydrox-
ylbenzoic acid matrix for spotting onto the target plate.
Fatty acid analysis
The L-PG was hydrolyzed with 3 N HCl in methanol for 4 h at
80uC. The sample was dried and treated with silylation reagent
(TRI-SIL, Pierce Biotechnology, Rockford, IL) for 30 min at room
temperature. The trimethylsilylated derivatives were analyzed by
GC/MS. Specifically, the sample was applied to a DB-5 column at
an initial temperature of 60uC for 1 min, then increased to 130uC
at a rate of 30uC/min, and finally increased to 280uC at a rate of
1H and31PNMR were performed at a concentration of 2 mg L-
PG sample per 0.6 mL of CDCl3on a Varian Inova 400 MHz
Amino acid analysis
The purified L-PG was incubated overnight at 100uC in 6 N
HCl in a heat-block. Samples were cooled, evaporated to dryness,
resuspended in water and subjected to amino acid analysis.
Neutral red assay
Neutral red chemical staining of Mtb wild type and lysX mutants
was carried out following the protocol described by Soto et al.
Experiments evaluating the lysX phenotype
In order to evaluate the growth inhibitory effects of cationic
compounds, Van (1 ug/mL), PMB (100 units/uL), human
neutrophil peptide-1 (25 ug/mL) or lysozyme (0.5 mg/mL) was
added to the growth media. The human neutrophil peptide stock
was dissolved in 0.1% tri-fluoro acetic acid (TFA), and the cultures
contained 0.025% TFA. No growth inhibition was noted at this
concentration of TFA. The cultures were initially diluted to an
OD600of 0.05, dispensed into a 96-well microplate (100 uL per
well) and incubated at 37uC with rotation at 60 rpm. At the
indicated time periods, the change in the optical density (A600) was
measured, and viability was determined. Low dose aerosol
infection experiments of mice (C57BL/6 female mice) and guinea
pigs (Hartely strains) for evaluating the growth and viability of lysX
strain were essentially as described previously .
Membrane potential analysis
Cytoplasmic membrane potential changes were determined
using the slow response, membrane potential-sensitive cyanine dye
DiOC2(3) (Sigma). Briefly, actively growing cultures of Mtb strains
(OD600=0.8) were incubated with 3 mM DiOC2(3) for 5 h.
Spectrofluorometry was used to detect the red fluorescence
(488 nm/620 nm) associated with aggregates of DiOC2(3), which
exhibits green fluorescence (488 nm/520 nm) in the monomeric
state. The assay was performed using white 96-well microtiter
plates (Perkin Elmer; Waltham, MA) and a Cary Eclipse
spectrofluorometer (Varian; Palo Alto, CA). A negative (depolar-
ized) control of 100 mM m-chlorophenylhydrazone CCCP (Sig-
ma), a proton ionophore that destroys the proton gradient and
eliminates the bacterial membrane potential, was included. The
membrane potential was measured as the ratio of red fluorescence
(associated with membrane potential changes) to green fluores-
cence (a cell size-dependent, membrane potential-independent
signal). Preliminary optimization studies revealed that 5 h
incubation was optimal for the measurements.
Mtb lysX and Lysinylated Lipid
PLoS Pathogens | www.plospathogens.org 11July 2009 | Volume 5 | Issue 7 | e1000534
Differences between groups were analyzed by multiple com-
parison procedures (Student-Newman-Keuls Method) with a
simple one-way ANOVA or Mann Whitney Rank Sum Test,
using SIGMASTAT (SPSS Science, Inc., Chicago, IL). A P value
of less than 0.05 was considered significant.
presence -B- of Van 1.0 mg/ml. At day six of growth, viability was
determined by plating cells on Middlebrook 7H11 agar and
counting. The bars represent mean6standard error.
Found at: doi:10.1371/journal.ppat.1000534.s001 (1.57 MB TIF)
Viability of Mtb strains in the absence -A- and
cultures (optical density ,0.01 to 0.04) were grown with
fluorescent vancomycin BODIPY (Invitrogen) at a final concen-
tration of 1 mg/mL for 20 hours. The cells were harvested by low
speed centrifugation, fixed in 4% paraformaldehyde for 24 h, and
imaged on a Nikon Eclipse microscope with a CCD camera;
bright field (BF) and fluorescent images (Van) were acquired.
Magnification was 1006, and data were analyzed using Meta-
morph software. At least 100 cells were imaged for each strain; the
images were scored for staining patterns and the presence or
absence of fluorescent staining. Approximately 35% of wild type
and 25% of lysX cells were not stained under these conditions. For
clarity, only select staining patterns are shown: defined dye
accumulation at the cell poles -A, D- and mid-cell (as in D) or
diffuse accumulation throughout the cell - B, E. In some cells, both
types of accumulation were observed -C, F. For the lysX mutant,
about 52% of cells showed diffuse staining, but this was only seen
in about 32% of wild type cells.
Found at: doi:10.1371/journal.ppat.1000534.s002 (2.97 MB TIF)
Visualization of Fl-Van stained cells. Actively growing
viability of Mtb strains was examined under the same conditions
used for macrophage infection and lysis in the presence of 0.09%
SDS. Actively growing cultures of Mtb strains were harvested,
exposed to 0.09% SDS for 3 min, diluted and spread on
Middlebrook 7H11 agar plates. Cells untreated with SDS were
processed similarly. All plates were incubated at 37uC, and
Effect of SDS on the viability of the lysX mutant: The
colonies were counted. No statistically significant differences
between the SDS treated and untreated groups were noted. Data
shown are mean 6 standard error.
Found at: doi:10.1371/journal.ppat.1000534.s003 (1.05 MB TIF)
guinea pigs. Growth of the lysX mutant and Rv-03 strain in the
spleens of mice -A- and guinea pig -B. Following aerosol infection
of mice and guinea pigs, spleens were harvested at the indicated
time points. Homogenates were prepared, and viability was
determined on agar plates. The lysX mutant showed reduced and/
or delayed dissemination compared to wild type in both animal
Found at: doi:10.1371/journal.ppat.1000534.s004 (1.64 MB TIF)
Growth of the lysX mutant in the spleen of mice and
Wild type and lysX mutant cells were stained with neutral red and
photographed. As a control, the attenuated strain Mtb H37ra was
used. Panel B: The lipids were separated by silica thin-layer
chromatography (TLC) with
(80:20:1, vol/vol/vol) as solvent system. The lipids were visualized
by spraying with 10% phosphormolybdate in ethanol followed by
heating to about 110uC for 15 min.
Found at: doi:10.1371/journal.ppat.1000534.s005 (5.65 MB TIF)
Neutral red staining and PDIM analysis: Panel A:
We thank Dr. Barry Starcher for performing the amino acid analysis;
Renata Dziedzic for help with the microscopy experiments; Dr. Paul
Converse for his interest and helpful discussions at various stages of this
work; Dr. Buka Samten for advice and help with the monocyte isolation
protocols; and Dr. Chris Fancklyn for discussions during earlier stages of
this work. The H4A3 hybridomas used in the present study were obtained
from the Developmental Studies Hybridoma Bank under the auspices of
the NICHD and maintained by the University of Iowa, Department of
Biological Sciences, Iowa City, IA 52242.
Conceived and designed the experiments: WB MR DC MVVM.
Performed the experiments: EM DS JZ MF QJC SL. Analyzed the data:
EM DS JZ MF QJC SL WB MR DC MVVM. Wrote the paper: EM DS
WB MR DC MVVM.
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PLoS Pathogens | www.plospathogens.org13 July 2009 | Volume 5 | Issue 7 | e1000534