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Chlamydia trachomatis Utilizes the Mammalian CLA1 Lipid Transporter to
Acquire Host Phosphatidylcholine Essential for Growth

John V. Cox1, Yasser M. Abdelrahman1,2, Jan Peters1,3, Nirun Naher1, and Robert J. Belland1

Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health
Science Center, Memphis, TN 38163 1; Department of Microbiology and Immunology,
Faculty of Pharmacy, Cairo University, Cairo, Egypt 2; Regional Biocontainment Laboratory,
University of Tennessee Health Science Center, Memphis, TN 381633

Running title: Host Phosphatidylcholine Acquisition by Chlamydia
Key words: Chlamydia, CLA1, ABCA1, lipid transporter, phosphatidylcholine

Corresponding author: John V. Cox, Department of Microbiology, Immunology, and
Biochemistry, University of Tennessee Health Science Center, 858 Madison Avenue,
Memphis, TN, USA, Tel.:(901)-448-7080; Fax:(901)-448-7360; E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article
as doi: 10.1111/cmi.12523

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SUMMARY
Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be
acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1)
receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the
inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was
inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function.
Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug
on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for
CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine
analog to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function indicating a
critical role for these transporters in phosphatidylcholine acquisition by this organism. Our
analyses using a dual-labeled fluorescent phosphatidylcholine analog and mass
spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was
unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts
host phospholipid transporters normally used to assemble lipoproteins to acquire host
phosphatidylcholine essential for growth.
INTRODUCTION
The obligate intracellular pathogen, Chlamydia trachomatis, undergoes a unique bi-phasic
developmental cycle. The infectious form of the organism, which is termed an elementary
body (EB), is endocytosed by cells and retained within a vesicle termed the inclusion where
the organism differentiates into the non-infectious reticulate body (RB) (Gutter et al., 1973,
Moulder, 1991, Shaw et al., 2000, Nicholson et al., 2003, Abdelrahman et al., 2005). As the
developmental cycle proceeds, the replicating RBs differentiate back into EBs, which are
released from the cell (Hybiske et al., 2007), initiating another round of infection. The growth
of Chlamydia within the inclusion is dependent upon its ability to acquire nutrients, including
lipids (Hackstadt et al., 1995, Wylie et al., 1997, Carabeo et al., 2003, Beatty, 2006, Derre et
al., 2011, Elwell et al., 2011) from the host.

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Phosphatidylcholine accounts for ~40% of the total phospholipids in the membrane of C.
trachomatis EBs (Wylie et al., 1997). Yet, Chlamydia lack the genes necessary for
phosphatidylcholine biosynthesis (Aktas et al., 2010). The phosphatidylcholine present in
the chlamydial membrane is derived from the host and the import of phosphatidylcholine into
the inclusion of infected cells was shown to be associated with its deacylation at the sn-2
position by the host calcium-dependent phospholipase A2 (cPLA2). This deacylation
reaction releases lysophospholipid, which is reacylated by a bacterial branched chain fatty
acid prior to its incorporation into the bacterial cell membrane (Wylie et al., 1997). Studies
using inhibitors of host cPLA2 (Su et al., 2004), cPLA2 knock down cells (Vignola et al.,
2010), mouse embryonic fibroblasts from cPLA2 knockout mice (Vignola et al., 2010) and
mass spectrometry analysis of chlamydial phospholipids (Yao et al., 2015a) have yielded
conflicting data regarding the importance of this PC processing step for the growth of
Chlamydia trachomatis serovar L2 within infected cells.
Our studies indicated that C. trachomatis serovar D hijacks the host high-density
lipoprotein (HDL) biogenesis machinery to acquire PC from the host cell (Cox et al., 2012).
Plasma HDL is assembled and disassembled by the movement of lipids between the plasma
membrane and extracellular apoA-I by the phospholipid flippase ABCA1 (Oram et al., 2000,
Wang et al., 2000, Zannis et al., 2006) and the bidirectional lipid transporter CLA1, the
human SR-B1 scavenger receptor (Calvo et al., 1993, Urban et al., 2000, Rhainds et al.,
2004, Van Eck et al., 2005). Localization studies indicated that ABCA1 and CLA1 and their
lipid acceptor apoA-I are recruited to the inclusion of C. trachomatis-infected cells (Cox et al.,
2012). Treatment of C. trachomatis-infected cells with glyburide, a drug that inhibits the
ability of both ABCA1 (Nieland et al., 2004, Smith et al., 2004) and CLA1 (Smith et al., 2004)
to efflux lipids to extracellular apoA-I, inhibits the accumulation of PC within the inclusion of
infected cells and prevents chlamydial growth (Cox et al., 2012). The knockdown of ABCA1
expression also inhibits the growth of C. trachomatis in infected HeLa cells (Cox et al.,
2012). These data suggest that ABCA1, and perhaps CLA1, in conjunction with their lipid

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acceptor apoA-I are required to transport PC to the inclusion of infected cells where it is
utilized for chlamydial growth.
The goal of this study was to determine if CLA1 transport activity is necessary for the
acquisition of host PC by Chlamydia trachomatis serovar D and to determine the structure of
the PC associated with the chlamydial membrane. We used the CLA1-specific inhibitor,
BLT-1, and CLA1 shRNA knockdown analyses to show that CLA1 activity is important for PC
acquisition and the growth of Chlamydia within infected cells. Fluorescent PC analogues
and mass spectrometry further show that PC acquired from the host is not modified prior to
its incorporation into the bacterial membrane. Thus, C. trachomatis co-opts host lipid
transporters involved in HDL biogenesis to import intact PC into the inclusion to support
chlamydial growth and development.

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RESULTS
Chlamydial Growth is Dependent upon CLA1
B1 type scavenger receptors, such as CLA1, are multi-functional proteins that can direct
cholesterol efflux to extracellular HDL, and mediate the uptake of both cholesterol esters and
PC from HDL particles (Rhainds et al., 2004, Van Eck et al., 2005). The CLA1-dependent
uptake of lipids from extracellular HDL relies upon the ability of CLA1 to transfer lipids from
HDL to cellular membranes in the absence of HDL endocytosis (Rhainds et al., 2004). Our
previous studies indicated that CLA1 is recruited to the inclusion of C. trachomatis serovar
D-infected cells (Cox et al., 2012). While additional localization analyses confirmed this
result, the percentage of endogenous CLA1 that is present in the plasma membrane (arrows
in Fig. 1A), the inclusion membrane (arrowheads in Fig. 1A), and the lumen of the inclusion
(asterisk in Fig. 1A) was variable in fixed cells. A similar result was observed in live cells
expressing a CLA1-DsRed fusion (data not shown). To address whether the lipid trafficking
activities of any of these populations of CLA1 were involved in chlamydial growth regulation
within infected cells, we determined whether chlamydial growth was inhibited by BLT-1, a
drug that blocks the lipid efflux and uptake activities of B1 type scavenger receptors while
having no effect on ABCA1-dependent lipid efflux (Nieland et al., 2002, Nieland et al., 2004).
Others have shown that a concentration of BLT-1 in excess of 10M was necessary to
inhibit SR-B1-dependent cholesterol efflux by ~90% in HEK293 cells (Nieland et al., 2004).
In our analyses, HeLa cells were infected with C. trachomatis serovar D in media containing
BLT-1 concentrations ranging from 10nM to 25M. The drug was added to the infected cells
at 8 hours post-infection to avoid possible effects of the drug on the entry of C. trachomatis
into cells. The maximal inhibition was observed in cells treated with 25M BLT-1, which
yielded approximately a 4-log reduction in IFUs compared to the untreated control (Fig. 1B).
Higher concentrations of BLT-1 affected HeLa cell doubling times and were not included in
the analysis.

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The substitution of a serine for cysteine 384 in the extracellular domain of murine SR-B1
renders the protein insensitive to the inhibitory effects of BLT-1 (Yu et al., 2011). To
determine whether the effect of BLT-1 on chlamydial growth was primarily due to its ability to
inhibit CLA1 function, we mutated cysteine 384 in CLA1-DsRed (Cox et al., 2012) to serine.
Confocal analyses indicated that the CLA1(C384S) mutant was efficiently recruited to the
inclusion membrane where it co-localized with the inclusion membrane protein IncA, and the
lumen of the inclusion of cells infected with C. trachomatis serovar D (Fig. 2A). To
determine whether this mutant could suppress the inhibitory effects of BLT-1, mocktransfected HeLa cells or HeLa cells transfected with wild type CLA1-DsRed or with
CLA1(C384S)-DsRed were infected with C. trachomatis in the presence of 25M BLT-1 and
fixed at 48 hours post-infection. The cells were then stained with chlamydial Hsp60-specific
antibodies to assess the effect of the fusions on chlamydial growth. BLT-1-treated cells that
were either mock-transfected or transfected with wild type CLA1-DsRed had small inclusions
at 48 hours post-infection (Fig. 2B). However, in cells expressing CLA1(C384S)-DsRed,
chlamydial growth occurred in the presence of 25M BLT-1 as the transfected cells
contained much larger inclusions (Fig. 2B). Quantification of the inclusion diameter in drugtreated cells revealed that the CLA1-C384S mutant significantly suppressed the inhibitory
effect BLT-1 on chlamydial replication (Fig. 2C). IFU measurements were used to more
directly assess the effect of the CLA1 mutant on chlamydial growth. Mock transfected HeLa
cells or cells transiently transfected with the C384S mutant construct were infected with C.
trachomatis serovar D in the absence or presence of 25M BLT-1 and Chlamydia were
harvested at 42 hours post-infection. Approximately 15% of the HeLa cells expressed
detectable levels of the CLA1(C384S)-DsRed fusion in this transient transfection assay, and
immunoblotting analyses indicated that endogenous CLA1 (Fig. 2D - marked by a dash) was
more abundant than CLA1(C384S)- DsRed or CLA1- DsRed (Fig. 2D, lanes 2 and 3 fusions are marked by an asterisk) in transfected cell lysates. Bacteria harvested from mock
transfected HeLa cells yielded a slightly reduced number of IFUs compared to control cells

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(Fig. 2E). A similar slight reduction in IFUs was observed in cells transfected with
CLA1(C384S) (Fig. 2E). The addition of 25M BLT-1 to mock transfected cells resulted in
~4-log reduction in IFUs compared to the control (Fig. 2E), similar to the results observed in
Fig. 1B. However, expression of the CLA1(C384S) mutant resulted in ~2-log increase in the
number of infectious organisms recovered from BLT-1-treated cells (Fig. 2E). This result
coupled with the fact that only ~15% of the infected cells expressed detectable levels of the
CLA1(C384S) mutant indicate that the ability of BLT-1 to block chlamydial growth is in large
part the result of its inhibitory effect on CLA1.
As an alternative approach for verifying that CLA1 is required for chlamydial growth in
HeLa cells, we knocked down the expression of this transporter using shRNAs. For these
studies, we used a transient transfection and fluorescence-based assay (Cox et al., 2012) to
measure the effect of CLA1 shRNAs on CLA1 protein levels and chlamydial growth in
infected cells. These experiments revealed that the expression of high levels of the control
shRNA (arrows in Fig. 3A) had no apparent effect on CLA1 or chlamydial Hsp60 levels in
infected cells. However, CLA1 and Hsp60 levels were both lower in cells expressing the
CLA1-specific shRNAs (arrows in Fig. 3B). Quantification of these knockdown studies
revealed that the control shRNA had no effect on CLA1 or Hsp60 levels in infected cells (Fig.
3C), while CLA1 and Hsp60 levels in cells expressing high levels of the CLA1-specific
shRNAs were reduced to ~2% and ~5%, respectively, of controls (Fig. 3C). There was also
a dramatic reduction in inclusion size in infected cells expressing CLA1 shRNAs (Fig. 3D).
In contrast to its effect on chlamydial growth, the CLA1 shRNAs had no apparent effect on
actin levels (Supp. Fig. S1A) or actin organization (Supp. Fig. S1B). Together the
knockdown and BLT-1 studies indicate the growth of C. trachomatis serovar D within
infected HeLa cells is dependent upon the host CLA1 transporter.

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CLA1 and ABCA1 are Required for Chlamydial Phosphatidylcholine Acquisition
Our data indicated that the lipid transport and/or lipid transfer activities of CLA1 may be
necessary for the delivery of essential lipids to the inclusion of infected cells. To test this
hypothesis, we used a previously described assay (Cox et al., 2012) to determine whether
BLT-1 inhibited the trafficking of a fluorescent analogue of PC, β-BODIPY FL C5-HPC, which
has a modified fluorescent fatty acid at the sn-2 position (Supp. Fig. S2A). This analysis
revealed that BLT-1 treatment inhibited the accumulation of β-BODIPY FL C5-HPC in the
inclusion of infected cells (arrows in Fig. 4A). Quantification of this experiment (Cox et al.,
2012) revealed approximately a 50% reduction in the total cellular level of β-BODIPY FL C5HPC and approximately a 65% reduction in inclusion-associated β-BODIPY FL C5-HPC in
BLT-1 treated cells (Fig. 4B). These results indicated that in addition to ABCA1 (Cox et al.,
2012) the lipid trafficking activities associated with plasma membrane and/or inclusionassociated CLA1 were required for the trafficking of PC to the inclusion of C. trachomatisinfected cells.
Although glyburide (Cox et al., 2012) and BLT-1 (Figs. 1 and 4) inhibited the recruitment of
β-BODIPY FL C5-HPC to the inclusion of C. trachomatis-infected HeLa cells and blocked
chlamydial growth, the fluorescent PC analogue still trafficked to the inclusion of infected
cells grown in the presence of these drugs. To address whether the inclusion-associated
population of BODIPY FL C5-HPC in drug treated cells was incorporated into Chlamydia, the
incorporation of fluorescence into bacteria harvested from cells incubated in the presence or
absence of the drugs was monitored by confocal microscopy. In addition to fluorescent
bacteria, the lysates from infected cells also contained a very limited number of fluorescent
membrane vesicles. To distinguish the fluorescent C. trachomatis from the fluorescent
vesicles, the preparations were stained with Sytox Orange to visualize chlamydial DNA.
Control experiments demonstrated that all of the Sytox Orange-positive structures
corresponded to C. trachomatis that could be stained with antibodies directed against
MOMP (Caldwell et al., 1982), the chlamydial major outer membrane protein (Supp. Fig.
S2B). Confocal analysis revealed that both BLT-1 and glyburide inhibited the incorporation

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of fluorescence into chlamydial cell membranes (Fig. 5A). Using the Sytox Orange staining
to define the boundaries of the chlamydial cells, we quantified the fluorescence associated
with C. trachomatis under the different experimental conditions. This analysis revealed that
BLT-1 treatment of infected cells reduced the incorporation of fluorescence into bacterial cell
membranes by ~80%, while the glyburide treatment almost completely blocked the
incorporation of fluorescence into bacterial cell membranes (Fig. 5B). A virtually identical
result was obtained with control and glyburide-treated cells that were density gradient
purified prior to confocal analysis (Fig. 5C). These data strongly suggested that the lipid
transport activities associated with CLA1 and ABCA1 were necessary for the incorporation of
host PC into chlamydial cell membranes.
Unmodified Host Phosphatidylcholine is Incorporated into Chlamydial Membranes
We next determined the structure of the chlamydial PC acquired by the ABCA1/CLA1dependent transport pathway. Although previous metabolic labeling studies indicated that
host PC was deacylated at the sn-2 position and reacylated with a branched-chain fatty acid
containing an odd number of carbons by C. trachomatis serovar L2 prior to its incorporation
into the bacterial cell membrane (Wylie et al., 1997), recent mass spectrometric data
indicated that C. trachomatis serovar L2 incorporates unmodified host PC into its
membranes (Yao et al., 2015a). The deacylation of β-BODIPY FL C5-HPC would remove
the fluorescent fatty acid moiety from this PC analogue and render it undetectable in our
assays, so it was not a useful probe for monitoring this putative deacylation/reacylation
reaction. Therefore we carried out a series of lipid trafficking assays using Red/Green
BODIPY PC-A2, which contained a modified fatty acid at the sn-2 position that fluoresces
green and a modified fatty acid at the sn-1 position that fluoresces red (Supp. Fig. S2A).
Control experiments indicated that BLT-1 and glyburide had very similar effects on the
incorporation of β-BODIPY FL C5-HPC (Fig. 5B) and Red/Green BODIPY PC-A2 (Fig. 5D)
into chlamydial membranes. C. trachomatis serovar D were labeled with Red/Green
BODIPY PC-A2 to assess the extent of modification at the sn-2 position during acquisition
from the host. Labeled C. trachomatis were harvested and pelleted through a 40% sucrose

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cushion to remove the majority of host cell membranes as assessed by Sytox Orange
staining prior to fractionating their lipids by HPLC. The profile in Fig. 6A illustrates that the
lipids isolated from C. trachomatis contained a single fluorescent peak that co-migrated with
the original starting substrate. This fluorescent peak was absent in the control sample
prepared from mock-infected HeLa cells (Fig. 6A). In addition, the ratio of green to red
fluorescence in the fluorescent lipid recovered from the chlamydial membranes was very
similar to the input Red/Green BODIPY PC-A2 (Fig. 6C). These results indicated that the
fluorescent PC analogue was trafficked to Chlamydia without undergoing deacylation.
Control experiments indicated that Red/Green BODIPY PC-A2 can be cleaved by purified
cPLA2 in vitro generating a fluorescent species that does not co-migrate with the starting
substrate in HPLC analyses (Fig. 6B).
The analysis of the fluorescent probes suggested that unmodified PC was being trafficked
to Chlamydia. Additional experiments employed mass spectrometry to determine molecular
species of PC in infected and uninfected HeLa cells, and in density gradient purified C.
trachomatis serovar D EBs isolated from HeLa cells at 42 hours post-infection (Fig. 7). A
comparison of the PC species present in the infected cell system (Fig. 7B, (HeLa + C.
trachomatis)) to the uninfected cell system (Fig. 7A, HeLa) showed no difference in the
distribution of PC molecular species. The PC molecular species fingerprint in purified EBs
(Fig. 7C) was also virtually identical to uninfected HeLa cells. PC species that underwent
selective deacylation at the sn-2 position and reacylation with a branched chain fatty acid
would have been detected in this analysis. Since these species were not observed, we
conclude that like C. trachomatis serovar L2 (Yao et al., 2015a) host PC is trafficked to C.
trachomatis serovar D without modification. Furthermore, the transport of PC to Chlamydia
is dependent upon ABCA1 and CLA1.

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Chlamydial Acquistion of Host Cholesterol and Sphingomyelin is not Dependent upon
ABCA1 and CLA1
In addition to phospholipids, C. trachomatis acquires cholesterol from the host (Carabeo et
al., 2003). Since ABCA1 and CLA1 can both efflux cytosolic cholesterol to extracellular
apolipoprotein particles (Zannis et al., 2006) and CLA1 mediates the uptake of cholesteryl
esters from HDL (Nieland et al., 2002, Nieland et al., 2004), we investigated whether these
transporters are involved in cholesterol acquisition by C. trachomatis. For these assays, we
monitored BODIPY-cholesterol trafficking in HeLa cells infected with C. trachomatis serovar
D that had been incubated in the absence and presence of glyburide or BLT-1. As
expected, inhibiting the cholesterol efflux activities of ABCA1 and CLA1 with these drugs
stimulated an increase in the total cellular level of cholesterol in infected cells (Fig. 8A).
Quantification of these studies revealed a small but significant increase in the level of
inclusion-associated cholesterol in glyburide-treated cells (Fig. 8B). Additional analyses also
monitored the incorporation of BODIPY-cholesterol into chlamydial cell membranes in
infected cells incubated in the presence and absence of drug. Although BLT-1 treatment did
not significantly alter the incorporation of cholesterol into chlamydial cell membranes (Fig.
8B), glyburide treatment resulted in a ~6-fold increase in the amount of cholesterol
incorporated into bacterial membranes (Fig. 8B). Similar assays using BODIPY-ceramide
were carried out to monitor sphingomyelin trafficking in infected cells. This fluorescent
ceramide analogue is converted to sphingomyelin and delivered to the chlamydial inclusion
where it is incorporated into chlamydial membranes (Hackstadt et al., 1995, Derre et al.,
2011, Elwell et al., 2011). Our analyses revealed that total cellular levels and inclusionassociated fluorescence derived from BODIPY-sphingomyelin were increased in glyburidetreated cells relative to controls (Supp. Fig. S3A). However, neither drug had a significant
effect on the incorporation of BODIPY-sphingomyelin into bacterial cell membranes (Supp.
Fig. S3B). The mechanism responsible for the enhanced accumulation of cholesterol in
bacterial cell membranes of glyburide-treated cells is not understood at this time. However,
these data clearly indicate that among the classes of lipids derived from the host, which

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includes cholesterol, sphingolipids, and phosphatidylcholine, ABCA1 and CLA1 are only
required for the incorporation of host phosphatidylcholine into chlamydial cell membranes.

DISCUSSION
This work illustrates the importance of the host HDL biogenesis machinery in delivering PC
to Chlamydia. The growth of C. trachomatis is dependent upon its ability to acquire lipids
from the host (Hackstadt et al., 1995, Wylie et al., 1997, Carabeo et al., 2003, Beatty, 2006,
Derre et al., 2011, Elwell et al., 2011), and multiple redundant pathways contribute to the
acquisition of glycosphingolipids (Derre et al., 2011, Elwell et al., 2011) and cholesterol
(Carabeo et al., 2003). We have focused on PC because it cannot be made by C.
trachomatis and it is one of the most abundant phospholipids associated with isolated
bacteria (Wylie et al., 1997, Stephens et al., 1998). Previous studies indicated that PC
trafficking to the inclusion and chlamydial growth was dependent upon the host ABCA1
transporter, which is recruited to the inclusion membrane of C. trachomatis-infected cells
(Cox et al., 2012). The inhibitor and knock down data presented here illustrate that CLA1,
another transporter in the HDL biogenesis pathway, is also critical for PC acquisition and the
growth of C. trachomatis. Thus, C. trachomatis employs an ABCA1//CLA1 PC transport
pathway normally used for HDL biogenesis to acquire PC essential for growth. Consistent
with ABCA1 and CLA1 transporting unmodified PC to Chlamydia, lipidomics demonstrate
that PC in isolated C. trachomatis serovar D mimics the PC profile in the host cell.

PC

species that underwent selective deacylation at the sn-2 position by cPLA2 and reacylation
with a branched chain fatty were not detected in our analysis (Fig. 7). A recent study
indicated that unmodified host PC is also incorporated into C. trachomatis serovar L2
membranes (Yao et al., 2015a). These findings are consistent with results showing that
cPLA2 is not necessary for the growth of C. trachomatis serovar L2 within infected cells
(Vignola et al., 2010). However, our results are at odds with the conclusions reached using
radioactive isoleucine that indicated the presence of bacterially derived branched-chain fatty
acids on chlamydial PC (Wylie et al., 1997). While the basis for this discrepancy is unclear,

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it is possible that PC species with a branched-chain fatty acid at the sn-2 position are
associated with the bacterial membrane but are too rare to be detected by mass
spectrometry. Alternatively, the radioactive isoleucine that was incorporated into PC may
have arisen from host metabolism of isoleucine to acetate which is then used to produce
fatty acids. Unlike phosphatidylcholine, C. trachomatis possesses the genes necessary for
the synthesis of phosphatidylethanolamine and phosphatidylglycerol (Nicholson et al.,
2003), and analytical studies have indicated that C. trachomatis serovar L2 synthesizes the
phosphatidylethanolamine and phosphatidylglycerol present in its membranes (Yao et al.,
2015a). However, the fatty acids necessary for chlamydial phosphatidylethanolamine
biosynthesis are at least in part derived from the host (Yao et al., 2015b).
Our data demonstrate a role for ABCA1, apoA-I and CLA1 in the transfer of host PC to C.
trachomatis and lead to the model for host PC acquisition by this organism illustrated in Fig.
9. Our model posits that the roles of ABCA1, CLA1 and apoA-I in delivering host PC to C.
trachomatis are similar to the roles of these proteins in transporting PC in mammalian cells
(Fig. 9). The PC transport activity of ABCA1 transfers PC from the membrane to its lipid
acceptor, apoA-I (Fitzgerald et al., 2002). Localization studies show ABCA1 in the inclusion
membrane of cells infected with C. trachomatis, while apoA-I accumulates in the inclusion
membrane and within the lumen of the inclusion (Cox et al., 2012). The model illustrates the
ABCA1-dependent transport of PC from the inclusion membrane to apoA-I within the lumen
of the inclusion.

CLA1 catalyzes the transfer of PC from apoA-I to biological membranes

(Rhainds et al., 2004), and the model assigns inclusion membrane-associated CLA1 the
same role in facilitating the movement of PC bound to apoA-I to the bacterial membrane.
While the nature of the membrane association and the topology of lumenal CLA1 is not
known, it is possible that this population of CLA1 also catalyzes the transport of PC from
apoA-I to Chlamydia. The inhibitory effects of BLT-1 on chlamydial growth could also be in
part due to the reduced levels of total cellular and inclusion-associated PC observed in cells
grown in the presence of the drug. Future experiments will more precisely define the role of

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CLA1 in mediating the movement of PC from its origin in the host to the inclusion to support
the expansion of this unique organelle and its utilization by the bacteria.
EXPERIMENTAL PROCEDURES
Antibodies, Plasmids, and Reagents
Mouse monoclonal antibodies directed against MOMP were obtained from Argene, and
mouse monoclonal antibodies directed against Chlamydia Hsp60 protein and IncA were
provided by Dr. R. P. Morrison and Dr. D. D. Rockey, respectively. A rabbit polyclonal
antibody that recognizes human CLA1 (Calvo et al., 1993, Cox et al., 2012) and a mouse
monoclonal antibody directed against -actin were obtained from Novus. Various AlexaFluor
conjugated secondary antibodies, Hoechst, β-BODIPY FL C5-HPC, Red/Green BODIPY PCA2 complexed to BSA, and phalloidin conjugated to Alexa 594 were obtained from
Invitrogen. BODIPY cholesterol was obtained from Avanti. Glyburide and fatty acid-free
BSA were purchased from Sigma Chemicals. BLT-1 was obtained from Chembridge Corp.,
and cPLA2 was obtained from Worthington Biochemical Corp.
A scrambled control shRNA and 2 CLA1-specific shRNAs in the pGFP-V-RS vector were
purchased from Origene. The pGFP-V-RS vector also encodes GFP under the control of a
separate promoter. The CLA1-specific shRNAs target the sequences:
GGCTGAGCAAGGTTGACTTCTGGCATTCC (nucleotides 969-997) and
GAGGCACACTCCTTGTTCCTGGACATCCA (nucleotides 1349-1377) in the two variant
human CLA1 mRNAs. Cysteine 384 in the CLA 1-DsRed monomer fusion (Cox et al., 2012)
was mutated to a serine by PCR based mutagenesis. The resulting mutant was sequenced
prior to its use in transfection studies.
Cell Culture and Chlamydial Infections
HeLa cells grown in DMEM containing 10% fetal calf serum were infected with C.
trachomatis serovar D (strain UW-3/Cx) or serovar L2 (strain 434/Bu) and EBs were
harvested at 48 hours post-infection and purified on renograffin gradients as described
previously (Caldwell et al., 1981). Alternatively, HeLa cells were infected with C.

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trachomatis serovar D and at various times post-infection, the cells were fixed and
processed for immunostaining, or the cells were harvested and the number of inclusion
forming units (IFUs) was determined by a limiting dilution assay. In some instances, BLT-1
was added to infected cells at 8 hours post-infection and maintained in the media for the
duration of the infection.
HeLa cells were transfected with wild type CLA 1-DsRed or CLA1(C384S)-DsRed
encoding plasmids or the pGFP-V-RS plasmids encoding the CLA1-specific or control
shRNAs using the Effectene transfection reagent (Qiagen). Transfected cells were then
infected with C. trachomatis in the absence or presence of BLT-1 and fixed at various times
post-infection in 4% paraformaldehyde in phosphate buffered-saline (PBS) for 10 minutes.
Fixed cells were permeabilized by incubation in ice-cold methanol for 30 seconds and
permeabilized cells were then rinsed with PBS and incubated with primary antibodies as
described in the text.
Localization Analyses
HeLa cells infected with C. trachomatis serovar D were fixed at 24 hours post-infection
by incubation in 4% paraformaldehyde in phosphate buffered-saline (PBS) for 10 minutes.
Following rinsing in PBS, the cells were permeabilized in PBS containing 0.1% saponin prior
to incubation with monoclonal antibodies directed against IncA (Bannantine et al., 1998) and
rabbit polyclonal antibodies that recognize CLA1. The cells were then rinsed with PBS and
incubated with the appropriate AlexaFluor conjugated secondary antibodies prior to analysis
by confocal microscopy.
Immunoblotting Assays
Lysates were prepared from control HeLa cells or HeLa cells transfected with CLA1DsRed or CLA1(C384S)-DsRed by direct lysis in SDS sample buffer. The lysates were then
subjected to immunoblotting analysis with rabbit polyclonal antibodies that recognize
endogenous CLA1 from HeLa cells (Cox et al., 2012). Similar immunoblotting analyses
assessed actin levels in HeLa cells that were transfected with control scrambled shRNA or
CLA1-specific shRNAs. Immunoreactive species in blotting analyses were detected using

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HRP-conjugated secondary antibodies and chemiluminescence detection systems.
shRNA Knockdown Analyses
HeLa cells were transfected with pGFP-V-RS plasmids encoding the CLA1-specific and
control shRNAs. Twenty-four hours post-transfection the cells were infected with C.
trachomatis serovar D (MOI=2) and then fixed and processed for staining with CLA1 and
Hsp60 antibodies at 48 hours post-infection. Z-stacks were acquired for each transfected
cell and non-transfected control cells using a Zeiss LSM 510 confocal microscope. The
fluorescence intensity of the GFP reporter encoded by the pGFP-V-RS plasmid was used as
a measure of shRNA levels within an individual transfected cell. Cells expressing high levels
of the shRNAs were defined using the setting of the detector gain on the confocal
microscope required to reach a saturating level of fluorescence for the GFP reporter. The
setting for high shRNA expressers was <800. In each transfected and non-transfected cell,
the fluorescence intensity through the entire Z-stack that resulted from staining with the
CLA1 and Hsp60 antibodies was quantified using the imaging software for the Zeiss LSM
510 confocal microscope. The values presented in the text represent the average value
obtained from Z-stacks of at least 25 different cells. Additional analysis of transfected cells
revealed that there was no obvious difference in the morphology of cells expressing the
CLA1-specific and control shRNAs.
Fluorescent Lipid Labeling of C. trachomatis-Infected HeLa Cells
HeLa cells infected with C. trachomatis serovar D were incubated at 37oC for 28 hours.
At this time, the cells were incubated in the absence or presence of 25M BLT-1 or 300M
glyburide for 3 hours at 37oC. β-BODIPY FL C5-HPC, BODIPY cholesterol, or BODIPYceramide in fatty acid-free BSA was then added to the media of control and drug-treated
cells at a final concentration of 1.5M and the cells were incubated an additional hour at
37oC (Cox et al., 2012). The cells were then fixed in 4% paraformaldehyde in PBS and
imaged by confocal microscopy. In some experiments, HeLa cells were back-extracted in
media containing fatty acid-free BSA for 1 hour prior to microscopic analysis and identical

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results were observed. Z-stacks were acquired for control and drug-treated cells using
identical acquisition parameters and the levels of total and inclusion-associated fluorescence
in control and drug-treated cells were quantified using the imaging software for the Zeiss
LSM 510 confocal microscope. 2 micron slices were acquired to minimize photobleaching
and fluorescence was quantified from the 2 brightest slices in the z-stack. In the analysis,
the lines demarcating the boundaries of inclusions were drawn inside the inclusion
membrane to insure that cytoplasmic fluorescence was excluded from the inclusionassociated fluorescence value. The values shown in the text represent the average values
obtained from the analysis of at least 100 cells from two independent experiments. Under
the conditions used for image acquisition, there was no fluorescence detected in control cells
incubated in the absence of fluorescent lipids.
HeLa cells infected with C. trachomatis serovar D were incubated at 37oC for 28 hours. At
this time, the cells were incubated in the absence or presence of 25M BLT-1 or 300M
glyburide for 3 hours at 37oC. β-BODIPY FL C5-HPC, Red/Green BODIPY PC-A2, BODIPY
cholesterol, or BODIPY-ceramide in fatty acid-free BSA was then added to the media of
control and drug-treated cells at a final concentration of 1.5M and the cells were incubated
an additional 3 hours at 37oC. At this time, cells were harvested and C. trachomatis were
isolated and the incorporation of fluorescent lipid into the chlamydial cell membrane was
monitored by confocal microscopy. To discriminate fluorescent bacteria from the fluorescent
host cell membranes present in the lysate, the preparations were stained with Sytox Orange
to visualize chlamydial DNA. Control experiments revealed that all Sytox Orange positive
structures also stained with anti-MOMP antibodies (Supp. Fig. S2B). Cells from the different
experimental conditions were imaged by confocal microscopy using identical image
acquisition parameters. The Sytox Orange or MOMP staining were used to define the
boundaries of the chlamydial cells, and we quantified the fluorescence associated with C.
trachomatis using the imaging software for the Zeiss LSM 510 microscope. In some
instances, Chlamydia were density gradient purified (Caldwell et al., 1981) prior to analysis

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by confocal microscopy. For each experimental condition, at least 200 cells from 2
independent experiments were analyzed.
HeLa cells were mock-infected or infected with C. trachomatis serovar D. At 25 hours
post-infection, Red/Green BODIPY PC-A2 in fatty acid-free BSA was added to the cells and
they were incubated an additional 3 hours at 37oC. This fluorescent phosphatidylcholine
analogue contains a modified fatty acid at the sn-2 position that fluoresces green, and a
modified fatty acid at the sn-1 position that fluoresces red. C. trachomatis were harvested
from infected cells and pelleted through a 40% sucrose cushion by centrifuging at 12,000xg
for 30 mins. to remove a substantial fraction of contaminating host cell membranes as
assessed by staining with MOMP antibodies. An identical sample was also prepared from
mock-infected cells. The lipids were extracted from these preparations in 100% methanol
and the extracts were subjected to reverse phase HPLC using a Waters Breeze 2 HPLC
apparatus (Waters, Milford, MA). Fractions were collected every 1 min with a Waters
Fraction Collector III (Waters, Milford, MA) and the red (590nm) and green fluorescence
(528nm) associated with each fraction was monitored. Additional control studies
characterized the fractionation properties of the Red/Green Bodipy PC-A2 that was
incubated in the absence or presence of 1 U cPLA2 prior to HPLC analyses using identical
run conditions.
PC Molecular Species Profiling
Lipids were extracted from purified C. trachomatis EBs using the Bligh and Dyer method
(Bligh et al., 1959). PC molecular species fingerprints were determined using direct infusion
electrospray ionization-mass spectrometry technology (Ivanova et al., 2007, Krank et al.,
2007). Mass spectrometry analysis was performed using a Finnigan TSQ Quantum
(Thermo Electron, San Jose, CA) triple quadrupole mass spectrometer equipped with the
nanospray ion source. The lipid extracts were resuspended in 50:50 (v/v)
chloroform:methanol with 1% formic acid. The instrument was operated in positive ion
mode. Ion source parameters were: spray voltage 1000V, capillary temperature 270°C,
capillary offset 35 V, and the tube lens offset was set by infusion of the polytyrosine tuning
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and calibration solution (Thermo Electron, San Jose, CA) in electrospray mode. Parameters
for the analysis of phosphatidylcholine are scan range, 600-900 m/z; scan time, 0.3 s;
product loss, 184.1 m/z; collision energy; 40 V; peak width, Q1 and Q3 0.7 full width at halfmaximum (FWHM); and Q2 CID gas, 0.5 mT. Instrument control and data acquisition was
performed using the Finnigan Xcalibur software (Thermo Electron, San Jose, CA). Acyl
chain lengths were assigned from the mass based on predications from LipidMaps (Ivanova
et al., 2007).
ACKNOWLEDGEMENTS
This work was supported in part by a University of Tennessee Health Science Center
Pathogenesis Center of Excellence Grant (J.V.C.), National Institutes of Health Grant
AI070693 (R.J.B.). We thank Dr. Charles O. Rock and Dr. Jiangwei Yao at St. Jude
Children’s Research Hospital for performing the mass spectrometry analysis. Antibodies
against chlamydial Hsp60 and chlamydial IncA were kindly provided by Dr. R. P. Morrison
and Dr. D. D. Rockey, respectively.

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Fig. 1. CLA1 transport activity is required for chlamydial growth. HeLa cells were
infected with C. trachomatis serovar D (MOI=2). At 48 hours post-infection (A), the cells
were fixed with 4% paraformaldehyde in PBS. The cells were then permeabilized in PBS
containing 0.1% saponin and incubated with a mouse monoclonal antibody directed against
IncA and a rabbit polyclonal antibody that recognizes CLA1. Arrows in A indicate CLA1
associated with the plasma membrane of infected cells. Arrowheads in A indicate CLA1
associated with the inclusion membrane of infected cells, and the asterisk in A indicates
CLA1 in the lumen of the inclusion of an infected cell. The white bar in A is 10m. (B)
Alternatively, HeLa cells were infected with C. trachomatis serovar D. At 8 hours postinfection, the indicated amounts of BLT-1 in DMSO or DMSO alone (Cont) were added to the
cells and the infection was allowed to proceed for 48 hours at which time the number of IFUs
for each experimental condition was determined. Values in B are the average of three
independent experiments.

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Fig. 2. The effect of BLT-1 on chlamydial growth is primarily due to its ability to block
the transport activity of CLA1. HeLa cells were transfected with CLA1(C384S)-DsRed (A).
The cells were then infected with C. trachomatis serovar D and fixed in 4%
paraformaldehyde in PBS at 24 hours post-infection. The cells were then permeabilized and
stained with antibodies directed against IncA. Following washing and incubation with goat
anti-mouse IgG conjugated to Alexa 488, the cells were analyzed by confocal microscopy.
(B) HeLa cells were mock transfected or transfected with CLA1-DsRed or CLA1(C384S)DsRed. The cells were then infected with C. trachomatis serovar D and either untreated or
BLT-1 was added to the media at 8 hours post-infection. The cells were fixed at 48 hours
post-infection and incubated with antibodies specific for Chlamydia Hsp60 followed by goat
anti-mouse IgG conjugated to Alexa 488 (green) prior to analysis on a Zeiss Axioplan II
microscope. DNA was visualized with Hoechst staining (blue). Arrows indicate inclusions in
transfected cells. (C) The average diameter of inclusions in control (Cont) cells that were not
drug treated. The effect of BLT-1 on the average diameter of inclusions in mock-transfected,
CLA1-DsRed transfected, and CLA1(C384S)-DsRed transfected cells is also shown. (D)
Lysates prepared from mock transfected (D, lane 1), CLA1(C384S)-DsRed transfected (D,
lane 2), or CLA1-DsRed transfected HeLa cells (D, lane 3) were subjected to immunoblotting
analysis with CLA1-specific antibodies. The dash in D denotes endogenous CLA1, and the
asterisk in D denotes CLA1(C384S)-DsRed and CLA1-DsRed fusions. (E) HeLa cells were
untreated (Cont), mock-transfected, or transfected with CLA1(C384S)-DsRed. The cells
were then infected with C. trachomatis serovar D and incubated in the absence or presence
of BLT-1, and the effect of BLT-1 treatment on IFUs was determined at 48 hours postinfection. Values in E represent the average of three independent experiments. Staining
with Hsp60 antibodies revealed that equal numbers of cells were infected with C.
trachomatis under the various experimental conditions described in E. White bars in A and B
are 10M.
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Fig. 3. CLA1 knockdown inhibits chlamydial growth. HeLa cells were transfected with
GFP-V-RS plasmids encoding a scrambled control shRNA (A) or two CLA 1-specific
shRNAs (B). The shRNA-encoding plasmids encoded a GFP reporter. The cells were then
infected with C. trachomatis serovar D and fixed at 48 hours post-infection. The cells were
then permeabilized and incubated with rabbit antibodies specific for CLA1 and mouse
monoclonal antibodies specific for Chlamydia Hsp60 and imaged by confocal microscopy to
monitor the extent of chlamydial growth. Arrows indicate cells expressing the control
scrambled shRNA (A) or CLA1-specific shRNAs (B). Z-stacks were acquired for each
transfected cell and non-transfected control cells and the fluorescence intensity of the GFP
reporter encoded by the pGFP-V-RS plasmid was used as a measure of shRNA levels within
an individual transfected cell. Cells expressing high levels of the shRNA were chosen
(described in Experimental Procedures) for the quantification in panel C. In each transfected
and non-transfected cell, the fluorescence intensity through the entire Z-stack that resulted
from staining with the CLA1 and Hsp60 antibodies was quantified using the imaging software
for the Zeiss LSM 510 confocal microscope. The values shown in the histogram in C
represent the average value obtained from Z-stacks of at least 25 different cells. (D) The
average diameter of inclusions in non-transfected and in cells transfected with the control
scrambled shRNA or the CLA1-specific shRNAs. White bars in A and B are 5m.

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Fig. 4. CLA1 is required for the delivery of PC to the inclusion of infected cells. (A)
HeLa cells were infected with C. trachomatis serovar D. At 25 hours post-infection, the cells
were incubated in the absence or presence of 25M BLT-1 for 3 hours. β-BODIPY FL C5HPC was then added to control and drug-treated cells for 1 hour. The cells were then fixed
and imaged by confocal microscopy using identical image acquisition parameters. Total and
inclusion-associated β-BODIPY FL C5-HPC fluorescence in control and drug-treated cells
were quantified. The values in B represent the average from the analysis of at least 100 cells
from two independent experiments. *** in C indicates p values < 0.001 (two-tailed t-test).
Arrows in A point to inclusions. Bars in B are 10m.

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Fig. 5. CLA1 and ABCA1 are necessary for the chlamydial acquisition of host PC.
HeLa cells were infected with C. trachomatis serovar D. At 25 hours post-infection, the cells
were incubated in the absence or presence of 25M BLT-1 or 300M glyburide for 3 hours.
β-BODIPY FL C5-HPC (A, B and C) or Red/Green BODIPY PC-A2 (D) in fatty acid-free BSA
was then added to control and drug-treated cells for 1 hour. Control and drug treated cells
were then harvested and β-BODIPY FL C5-HPC labeled bacteria in the cell lysates were
stained with Sytox Orange prior to confocal analysis (A). Histograms reflect the average
level of β-BODIPY FL C5-HPC (B) or Red/Green BODIPY PC-A2 (D) from at least 200
different cells under the different experimental conditions from two independent experiments.
MOMP staining was used to identify bacterial cells labeled with Red/Green BODIPY PC-A2.
The histograms in panel D reflect the green fluorescence emitted by Red/Green BODIPY
PC-A2 associated with bacteria. In some instances labeled cells were density gradient
purified prior to microscopic analysis (C). *** in B - D indicates p values < 0.001 (two-tailed
t-test). Arrows in A indicate β-BODIPY FL C5-HPC-positive, Sytox Orange-negative
membranes that were excluded from the quantification. Bars in A are 1.5m.

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Fig. 6. Fluorescent PC is not modified during its incorporation into the chlamydial
membrane. HeLa cells were mock-infected or infected with C. trachomatis serovar D. At 25
hours post-infection, Red/Green BODIPY PC-A2 in fatty acid-free BSA was added to the
cells and the cells were incubated an additional 3 hours at 37oC. C. trachomatis were then
harvested from infected cells and pelleted through a 40% sucrose cushion to remove
contaminating host cell membranes. An identical sample was prepared from mock-infected
cells. The lipids were extracted from these preparations in 100% methanol and the extracts
were subjected to reverse phase HPLC. Fractions were collected every 1 min and the green
fluorescence (528nm) associated with each fraction was monitored (A). A similar profile was
obtained when red fluorescence (590nm) was monitored. The only fluorescent peak
recovered from chlamydial membranes, which was absent in the sample prepared from
mock-infected cells, co-migrated with the starting substrate in this HPLC analysis (A).
Red/Green BODIPY PC-A2 was incubated in the absence and the presence of purified
cPLA2 in vitro prior to HPLC analysis (B). The results shown in A and B are representative
of multiple analyses. The ratio of green (528nm)/red (590nm) fluorescence in the peak
obtained from chlamydial membranes and in the peak obtained from the starting substrate in
A is illustrated in C. FLU (fluorescence light units)

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Fig. 7. Mass spectrometric analysis of phosphatidylcholine species in chlamydialinfected cells and purified EBs. (A) PC molecular species profile in uninfected HeLa cells.
(B) Molecular species fingerprint of PC isolated from C. trachomatis serovar D-infected HeLa
cells at 48 hours post-infection. (C) PC molecular species profile of gradient purified C.
trachomatis serovar D EBs. Species are listed with the most probable composition of fatty
acids in the 1-position over the 2-position. Ether lipids are designated with the “O-“ prefix.

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Fig. 8. CLA1 and ABCA1 are not necessary for the chlamydial acquisition of
cholesterol from the host. (A) HeLa cells were infected with C. trachomatis serovar D. At
25 hours post-infection, the cells were incubated in the absence or presence of 25M BLT-1
or 300M glyburide for 3 hours. BODIPY-cholesterol was then added to control and drugtreated cells for 1 hour. The cells were then fixed and imaged by confocal microscopy using
identical image acquisition parameters. (B) The total cellular fluorescence and the inclusionassociated fluorescence derived from BODIPY-cholesterol in control and drug-treated cells
were quantified. (C) Alternatively, C. trachomatis were harvested from control and drugtreated cells as described above and the fluorescence derived from BODIPY-cholesterol was
quantified. The values in B represent the average from the analysis of at least 100 cells
from two independent experiments, while the values in C reflect the average from at least
200 cells from two independent experiments . *** indicates a p value < 0.001, while **
indicates a p value < 0.002 (two-tailed t-test).

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Fig. 9.

Model illustrating the role of ABCA1, apoA-I and CLA1 in facilitating the

acquisition of host PC by C. trachomatis serovar D. ABCA1 resides in the inclusion
membrane of infected cells and is responsible for transferring PC (phospholipids with yellow
head groups) from the inclusion membrane to a lipid-free apoA-I acceptor (light gray disk) in
the lumen of the inclusion.

CLA1 is also present in the inclusion membrane with its

extracellular domain oriented toward the inclusion lumen. The model illustrates that CLA1 is
responsible for transferring PC from lipidated apoA-I (dark gray disk) to the chlamydial
membrane. Analytical analyses of membranes prepared from C. trachomatis serovar L2
indicate that the phosphatidylethanolamine and phosphatidylglycerol in chlamydial
membranes (phospholipids with green head groups) are of bacterial origin.

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