The trans-Golgi SNARE syntaxin 6 is recruited to
the chlamydial inclusion membrane
Elizabeth R. Moore,3 David J. Mead, Cheryl A. Dooley, Janet Sager
and Ted Hackstadt
Received 29 September 2010
Revised29 October 2010
Accepted 22 November 2010
Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, National Institute of Allergy
and Infectious Diseases, Rocky Mountain Laboratories, 903 South 4th Street, Hamilton, MT 59840,
Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a
parasitophorous vacuole termed an inclusion. The chlamydial inclusion is isolated from the
endocytic pathway but fusogenic with Golgi-derived exocytic vesicles containing sphingomyelin
and cholesterol. Sphingolipids are incorporated into the chlamydial cell wall and are considered
essential for chlamydial development and viability. The mechanisms by which chlamydiae obtain
eukaryotic lipids are poorly understood but require chlamydial protein synthesis and presumably
modification of the inclusion membrane to initiate this interaction. A polarized cell model of
chlamydial infection has demonstrated that chlamydiae preferentially intercept basolaterally
directed, sphingomyelin-containing exocytic vesicles. Here we examine the localization and
potential function of trans-Golgi and/or basolaterally associated soluble N-ethylmaleimide-
sensitive factor attachment protein receptor (SNARE) proteins in chlamydia-infected cells. The
trans-Golgi SNARE protein syntaxin 6 is recruited to the chlamydial inclusion in a manner that
requires chlamydial protein synthesis and is conserved among all chlamydial species examined.
The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma
membrane retrieval signal (YGRL). Thus in addition to expression of at least two inclusion
membrane proteins that contain SNARE-like motifs, chlamydiae also actively recruit eukaryotic
Chlamydiae are significant human pathogens responsible
for a number of distinct diseases. Chlamydia trachomatis
comprises 15 serologically defined variants or serovars
associated with diverse disease states including endemic
blinding trachoma, sexually transmitted diseases, and a more
invasive granulomatous disease, lymphogranuloma vene-
reum (Schachter, 1999). Chlamydia psittaci causes zoonotic
diseases that occasionally are transmitted to humans.
Chlamydia pneumoniae contributes to the two to five million
cases of respiratory pneumonia per year, although the actual
incidence of C. pneumoniae-induced disease is unknown
Chlamydiae have evolved a unique biphasic developmental
cycle. The infectious, metabolically dormant form, termed
the elementary body (EB), is endocytosed by the host cell
and remains within a vesicle termed the inclusion, where it
differentiates into a metabolically active but non-infectious
reticulate body. The inclusion membrane grows to accom-
modate the increasing number of organisms, while allow-
ing the organisms to acquire essential amino acids,
nucleotides and lipids from the host cell (Hackstadt
et al., 1995; Hatch, 1975a, b; McClarty, 1994; Moulder,
1991; Wylie et al., 1997). A fundamental question of
chlamydial biology relates to the mechanisms that allow the
inclusion to create a unique intracellular organelle
permitting survival and replication of the parasite.
Upon infection, the nascent inclusion membrane sur-
rounding the infectious EB is plasma membrane derived,
but within a few hours, chlamydial type III secreted
proteins modify the inclusion membrane (Fields et al.,
2003; Rockey et al., 1995, 2002; Shaw et al., 2000). These
modifications are evidenced by the initiation of a number
of interactions with the host cell, including dynein-
dependent trafficking to the microtubule-organizing centre
(Clausen et al., 1997; Grieshaber et al., 2003), and
separation of the inclusion from the classical endosomal
pathway, including restricted fusion with lysosomes (Al-
Younes et al., 1999; Fields & Hackstadt, 2002; Hackstadt,
Abbreviations: EB, elementary body; FBS, fetal bovine serum; PMRS,
plasma membrane retrieval signal; SNARE, soluble N-ethylmaleimide-
sensitive factor attachment protein receptor.
3Present address: Division of Basic Biomedical Sciences in the School
of Medicine, The University of South Dakota, 414 East Clark Street,
Vermillion, SD 57069-2390, USA.
Microbiology (2011), 157, 830–838
830045856Printed in Great Britain
1999; Taraska et al., 1996; van Ooij et al., 1997; Wyrick,
2000), and fusion with Golgi-derived vesicles delivering
sphingomyelin and cholesterol to the developing chlamy-
diae (Carabeo et al., 2003; Hackstadt et al., 1996; Scidmore
et al., 1996b).
The properties of lipid acquisition suggest that this
trafficking is vesicular in nature (Hackstadt, 1999,
Carabeo et al., 2003; Hackstadt et al., 1996; Scidmore
et al., 1996b). The specificity of this trafficking only to the
chlamydial inclusion (Heinzen et al., 1996), a requirement
for chlamydial modification of the inclusion membrane
(Scidmore et al., 1996b), and the lack of disruption of
normal Golgi processing and export of protein (Scidmore
et al., 1996a) suggest a unique trafficking pathway. The
acquisition of sphingomyelin, but not glucosylceramide, by
chlamydiae further implies specificity of this lipid-traffick-
ing pathway (Moore et al., 2008). Development of a
polarized epithelial cell model of chlamydial infection
demonstrated that in chlamydia-infected polarized cells,
the sphingomyelin retained by the chlamydiae is derived
predominantly from the basolateral trafficking pathway,
indicating that the chlamydial inclusion preferentially
intercepts Golgi-derived, basolaterally targeted exocytic
vesicles (Moore et al., 2008). This finding has led us to
focus on proteins that govern fusion along basolateral
trafficking pathways. Soluble N-ethylmaleimide-sensitive
factor attachment protein receptor (SNARE) proteins
constitute the predominant mechanism of membrane
fusion (Parlati et al., 2002). In this study we examine
syntaxins, a family of SNARE proteins, which are
associated with trans-Golgi and basolaterally directed
membrane fusion events. We demonstrate a specific
interaction between syntaxin 6, a trans-Golgi SNARE,
and the chlamydial inclusion membrane.
Organisms and cell culture. HeLa 229 cells [American Type Culture
Collection (ATCC; Manassas, VA; CCL-2.1)], cultivated in RPMI
1640 (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS)
(Hyclone) and 10 mg gentamicin ml21(Gibco-BRL), were used to
propagate Chlamydia trachomatis serovar L2 (LGV 434), C.
muridarum (MoPn/Weiss strain), C. pneumoniae (AR-39) and C.
psittaci (caviae) GPIC (HC/BW). Infectious EBs were purified from
HeLa cells using a Renografin (Braco Diagnostics) gradient, as
described by Caldwell et al. (1981). Chlamydial titres were determined
as described by Furness et al. (1960), by utilizing indirect
immunofluorescence with a polyclonal rabbit anti-C. trachomatis L2
EB, followed by an anti-rabbit Alexa Fluor-conjugated secondary
antibody (Molecular Probes). Multiplicities of infection (m.o.i.) for
all experiments are based on inclusion-forming units (i.f.u.)
determined in HeLa cells. Coxiella burnetii Nine Mile phase II was
propagated and purified from Vero cells (ATCC; CCL-81) as
previously described (Hackstadt et al., 1992).
HeLa and C2BBe1 cell lines were cultured at 37 uC in 5% CO2.
C2BBe1 (ATCC CRL-2102) cells were cultivated in DMEM+2 mM
GlutaMax (Invitrogen) supplemented with 10% FBS, 4 mM
L-glutamine, 0.01 mg human transferrin ml21(Invitrogen) and
10 mg gentamicin ml21. All eukaryotic cells were passaged based on
ATCC-suggested protocols using a 0.25% trypsin, 0.53 mM EDTA
Examination of localization of syntaxin 6 to the chlamydial
Endogenous syntaxins. To examine localization of endogenous
syntaxins to the chlamydial inclusion, C2BBe1 cells were seeded onto
glass coverslips in 24-well plates, 48 h prior to infection with C.
trachomatis L2. At 18 h post-infection, cells were fixed in absolute
ethanol at 220 uC for 30 min. Samples were then processed for
indirect immunofluorescence using rabbit anti-IncG (inclusion
membrane protein), mouse anti-syntaxin 4 (BD Biosciences),
mouse anti-syntaxin 16 (Synaptic Systems) or mouse anti-syntaxin
6 (BD Biosciences). All secondary antibodies were conjugated to
Dylight Fluors and obtained from Jackson ImmunoResearch
Laboratories. Coverslips were mounted to slides using ProLong
Gold antifade reagent (Invitrogen). Samples were visualized with an
LSM 510 Laser Module Zeiss Axiovert 200M confocal microscope
(Carl Zeiss MicroImaging).
eGFP-syntaxin 6. To examine the localization of eGFP-syntaxin 6
(kindly proved by Jeffrey Pessin, Albert Einstein College of Medicine,
Bronx, NY, USA) (Watson & Pessin, 2000) to chlamydial inclusions,
C2BBe1 cells were diluted and plated onto glass coverslips in 24-well
plates the day before the transfection. DNA was diluted to 250 ng per
100 ml of Optimem (Invitrogen), and the PLUS and Lipofectimine-
LTX reagents (Invitrogen) were used according to the manufacturer’s
protocol. Cells were incubated with the DNA–lipid complexes for a
minimum of 4 h prior to recovery in culture medium. Cells were then
infected with either C. trachomatis L2, C. muridarum or C. caviae for
an additional 18 h prior to fixation in 3% paraformaldehyde,
permeabilized with 0.1% Triton X-100 and 0.5% SDS in PBS, and
processed for indirect immunofluorescence to detect intracellular
bacteria. To examine localization of eGFP-syntaxin 6 to C.
pneumoniae or Coxiella burnetii Nine Mile Phase II, cells were
infected for 36–72 h prior to transfection with the eGFP-syntaxin 6
construct. An antibodymade
paraformaldehyde-fixed C. trachomatis serovar L2 or C. caviae EBs
was used to detect C. trachomatis serovar L2 and C. muridarum, or C.
caviae, respectively. Detection of C. burnetii was achieved using an
antibody against whole paraformaldehyde-fixed organisms raised in
rabbits. A mouse monoclonal antibody raised against C. pneumoniae
was kindly provided by Harlan Caldwell (NIAID, Rocky Mountain
Laboratories, Hamilton, MT, USA).
3XFLAG-syntaxin 6 wild-type and mutants. To examine which
domain of syntaxin 6 is involved in localizing the protein to the
chlamydial inclusion, eGFP-syntaxin 6 (Watson & Pessin, 2000) was
used as a template to make the following syntaxin 6 deletion
constructs: DH1 (helical domain, encoding amino acids 47–71),
DH2 (helical/SNARE domain, encoding amino acids 166–225) and
DYGRL (tyrosine motif encoding amino acids 140–143). The
GeneTailor Site-Directed Mutagenesis System (Invitrogen) was
used in the production of all constructs, with the primers listed
in Table 1. To complete the construction of the DH1 and DH2
syntaxin 6 mutants, PCR products were digested with HindIII (New
England Biolabs), followed by ligation with T4 DNA ligase (New
England Biolabs) and transformed into One-Shot MAX Efficiency
DH5a-T1R (Invitrogen). All deletion constructs and wild-type
syntaxin 6 were subcloned into p3XFLAG-CMV 7.1 expression
vector (Sigma Aldrich) using Phusion High Fidelity Polymerase
(New England Biolabs) and primers 7 and 8 (Table 1). All
mutations were confirmed by sequencing (SeqWright). 3XFLAG-
syntaxin 6 constructs were transformed into C2BBe1 cells as
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Edited by: P. C. F. Oyston
E. R. Moore and others
838 Microbiology 157