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
Syntaxin 6 localizes to the chlamydial inclusion
mCherry-syntaxin 6. Syntaxin 6 was subsequently cloned into
Co-localization of syntaxin 6 with the chlamydial
C. trachomatis serovar L2 obtains sphingomyelin from
a Golgi-derived basolaterally targeted pathway via an
unknown mechanism (Moore et al., 2008). Given the
integral role of SNARE proteins in mediating vesicular
trafficking, we examined whether syntaxin family members
co-localized with the chlamydial inclusion in C. trachoma-
tis-infected C2BBe1 cells by indirect immunofluorescence.
The trans-Golgi-associated syntaxin 6 was found to asso-
ciate with the chlamydial inclusion (Fig. 1). Syntaxin 6
displays a distinct ring-like staining pattern around the
chlamydial inclusion similar to the inclusion membrane
protein IncG. Syntaxin 4, known to control fusion to the
basolateral plasma membrane (Low et al., 1996; Teng
et al., 2001), and syntaxin 16, a ubiquitious trans-Golgi-
associated syntaxin which mediates retrograde endosomal-
Golgi transport (Mallard et al., 2002), did not colocalize
with the chlamydial inclusion but can be observed on the
plasma membrane (Fig. 1a).
Table 1. Primers used in cloning syntaxin 6 (stx6)
Delete YGRL sequence from pEGFP stx6
Delete H1 domain from pEGFP stx6
Delete H2 domain from pEGFP stx6
Clone stx6 constructs into p3XFLAG-
CMV 7.1 expression vector
Fig. 1. Localization of endogenous syntaxin
C2BBe1 cells were seeded onto glass cover-
slips for 48 h prior to infection with C.
trachomatis serovar L2 (m.o.i. 6:1) for an
additional 18 h. Cells were fixed in absolute
ethanol for 30 min at ”20 6C and processed
essentially as described in Methods. Samples
were visualized with an LSM 510 Laser
Module Zeiss Axiovert 200M confocal micro-
scope. Arrows indicate chlamydial inclusions.
(b) C2BBe1 cells were transfected with
eGFP-syntaxin 6 and infected with C. tracho-
matis L2 for 18 h. Cells were fixed with
methanol and counterstained with an anti-EB
antiserum. Bars, 10 mm.
E. R. Moore and others
832 Microbiology 157
To confirm this interaction, HeLa cells were transiently
transfected with eGFP-syntaxin 6 and infected with
C. trachomatis. Syntaxin 6 was found to localize to the
chlamydial inclusion membrane (Fig. 1b). These findings
suggest that syntaxin 6 is specifically recruited to the
chlamydial inclusion membrane.
Syntaxin 6 recruitment is conserved among
Because sphingomyelin trafficking to the inclusion is a
conserved feature among chlamydial species (Hackstadt
et al., 1995; Rockey et al., 1996; Wolf & Hackstadt, 2001),
we next examined whether mCherry-syntaxin 6 would
colocalize to the inclusion membrane of other Chlamydia
species. mCherry-syntaxin 6 was recruited to inclusions
formed by C. trachomatis serovar L2, C. muridarum,
C. pneumoniae and C. caviae (Fig. 2). Syntaxin 6 localized
to the inclusion membrane of C. muridarum and
C. pneumoniae inclusions in a similar manner as seen for
C. trachomatis serovar L2 (Fig. 2). Syntaxin 6 clustered
amongst the multiple lobed inclusions formed by C. caviae;
however, the morphology was distinct from the ring-like
pattern of syntaxin 6 surrounding the inclusions of
chlamydial species that form a single inclusion within the
host cell. Syntaxin 6 was not recruited to the parasitophor-
ous vacuole formed by the unrelated intracellular bac-
terium, Coxiella burnetii Nine Mile Phase II (Fig. 2). The
interaction of syntaxin 6 with the chlamydial inclusion
therefore appears to be chlamydia-specific and is conserved
across chlamydial species.
Requirement of chlamydial protein synthesis for
syntaxin 6 colocalization
Because sphingomyelin trafficking to the chlamydial inclu-
sion requires chlamydial protein synthesis (Hackstadt et al.,
1996; Scidmore et al., 1996b, 2003), we examined whether
chlamydial protein synthesis wasalso required for syntaxin 6
colocalization. C2BBe1 cells were infected with C. tracho-
matis and allowed to develop for 18 h, then treated with
chloramphenicol for an additional 24 h (Fig. 3). C2BBe1
Fig. 2. Localization of syntaxin 6 to the
inclusions of multiple chlamydial species.
C2BBe1 cells were seeded onto coverslips
and transfected with mCherry-syntaxin 6 or
with eGFP-syntaxin 6, then infected with C.
trachomatis serovar L2 (m.o.i. 6:1), C. mur-
idarum (m.o.i. 0.1:1), C. caviae (m.o.i. 0.2:1),
C. pneumoniae (m.o.i. 13:1) or Coxiella
burnetii Nile Mile phase II (m.o.i. 50:1) as
described in Methods. To terminate the infec-
tions, cells were fixed in methanol and
processed for indirect immunofluorescence to
detect the organisms (green). Samples were
visualized with an LSM 510 Laser Module
Zeiss Axiovert 200M confocal microscope.
Bar, 10 mm.
Syntaxin 6 localizes to the chlamydial inclusion
cells fixed at 18 h post-infection displayed the characteristic
ring-like pattern of syntaxin 6 localization to the inclusion
membrane. However, no inclusion-membrane-associated
staining of syntaxin 6 wasvisible in chloramphenicol-treated
cells (Fig. 3). These results suggest that syntaxin 6 locali-
zation to the inclusion and retention at the inclusion
membrane are likely mediated by a chlamydial protein.
Treatment of C. trachomatis-infected cells at 18 h post-
infection with brefeldin A, which collapses the Golgi
(Lippincott-Schwartz et al., 1989), or nocodozole, which
disrupts microtubules and fragments the Golgi apparatus
(Cheung & Terry, 1980; Tassin et al., 1985), did not inhibit
syntaxin 6 retention or recruitment to the inclusion
membrane (data not shown).
Characterization of the syntaxin 6 domain
responsible for localization to the chlamydial
Syntaxin 6 contains two helical domains (H1 and H2) and
a plasma membrane retrieval signal (PMRS) which are
required for syntaxin 6 cycling and function in eukaryotic
cells (Fig. 4a). The H1 domain resides within the N-
terminus of syntaxin 6 and is responsible for protein–
protein interactions as characterized by its interaction with
a-SNAP (Bock et al., 2001). The H2 domain resides
proximal to the C-terminal transmembrane domain and
comprises the Q-SNARE activity, which is similar to the
SNARE activity of SNAP25 (Bock et al., 2001; Watson &
Pessin, 2000). Within the C-terminal portion of syntaxin 6
is a 10 amino acid domain, which is characterized as the
PMRS, with residues YGRL being absolutely required for
activity (Watson & Pessin, 2000). Because syntaxin 6 is
trafficked within vesicles from the Golgi apparatus to the
plasma membrane, the YGRL signal sequence is required
for the recycling of syntaxin 6 from the plasma membrane
to the trans-Golgi region. Deletion of this domain causes
an accumulation of syntaxin 6 in the plasma membrane
(Watson & Pessin, 2000). Both the H2 and PMRS domains
contribute to the trans-Golgi localization of syntaxin 6 . To
understand which signalling or protein–protein binding
domains facilitate the trafficking of syntaxin 6 to the
chlamydial inclusion, we constructed a series of syntaxin 6
mutants from which the H1, H2 or PMRS domains from
syntaxin 6 were deleted. Subsequently, C2BBe1 cells were
transfected with 3XFLAG wild-type and mutant syntaxin 6
constructs and their subcellular localization relative to C.
trachomatis serovar L2 inclusions was examined (Fig. 4b).
Deletion of either the H1 or H2 domain did not have any
appreciable effect on syntaxin 6 localization to the
chlamydial inclusion. However, deletion of the PMRS
domain resulted in loss of recruitment of syntaxin 6 to the
chlamydial inclusion membrane (Fig. 4b). These results
suggest that the PMRS may be acting as signal sequence
which targets eukaryotic proteins to the chlamydial
inclusion, and/or that the chlamydial inclusion membrane
may be mimicking the trans-Golgi membrane.
Recent studies have indicated that the chlamydial inclusion
preferentially intercepts sphingomyelin from a basolaterally
directed pathway (Moore et al., 2008). The recognition of a
specific pathway targeted by chlamydiae stimulated a
search for host proteins that may serve to regulate this
pathway. Syntaxin 6 is recruited to the chlamydial inclu-
sion in a process that requires chlamydial protein synthesis
and is conserved across chlamydial species. Interestingly,
the PMRS signal of syntaxin 6 is required for syntaxin 6
colocalization to the inclusion.
Vesicle fusion in eukaryotic cells is of necessity a highly
regulated process designed to maintain the integrity and
distinction of intracellular compartments. Specificity in
vesicle fusion with target membranes is conferred by
integral membrane proteins
receptors, respectively (Rothman & Wieland, 1996). Bind-
ing of t-SNAREs with v-SNAREs on opposing membrane
faces causes the vesicles to dock with the acceptor mem-
brane. v-SNAREs and t-SNAREs have more recently been
termed Q-SNAREs and R-SNAREs, respectively, based on
conserved amino acids within the SNARE protein fusion
termed v-SNAREs and
Fig. 3. Chlamydial protein synthesis requirement for syntaxin 6
localization to the inclusion. C2BBe1 cells were seeded onto glass
coverslips in 24-well plates 48 h prior to infection with C.
trachomatis serovar L2 (m.o.i. 9:1). After 18 h, cells were either
fixed in absolute ethanol (”Chlor) or treated with 200 mg ml”1for
an additional 24 h (+Chlor), then fixed in absolute ethanol and
processed forindirect immunofluorescence
described in Methods. IncG staining was used to identify the
inclusion. Samples were visualized with an LSM 510 Laser Module
Zeiss Axiovert 200M confocal microscope. Bar, 10 mm.
E. R. Moore and others
complex. Once the complex is formed, two soluble proteins,
N-ethylmaleimide-sensitive factor (NSF) and SNAP, bind
the complex. Subsequent ATP hydrolysis by NSF promotes
actual membrane fusion. Several other host proteins are also
involved in regulation of vesicle trafficking. Specific small
GTPases of the Rab family are localized to the surface of the
various compartments of the endocytic and exocytic path-
ways where, depending upon the concentration of the GTP-
bound state, they positively or negatively regulate the rates
of SNARE complex assembly and membrane fusion (Novick
& Zerial, 1997; Schimmo ¨ller et al., 1998).
Microbial manipulation of host SNARE machinery is
an emerging theme in cellular microbiology. Myco-
bacterium tuberculosis-containing vacuoles (MCVs) tran-
siently acquire syntaxin 3, acquire and retain syntaxins 4
and 8, but exclude syntaxin 6 (Fratti et al., 2003; Parlati
et al., 2002; Perskvist et al., 2002). Each step of syntaxin
acquisition or exclusion marks a critical step in the MCV
maturation from a plasma-membrane-derived vacuole.
Simlarly, syntaxin 13 appears to play a role in maturation
of the Salmonella-containing vacuole (Smith et al., 2005).
Virulent Legionella pneumophila require syntaxins 2, 3 and
Fig. 4. Identification of the syntaxin 6 protein
domain mediating localization to the chlamydial
inclusion. (a) Functional domains of syntaxin 6.
These include the 24 amino acid H1 domain,
the 10 amino acid plasma membrane retrieval
signal (PMRS), including the YGRL tyrosine
motif and the 59 amino acid H2 domain. Also
depicted is the C-terminal transmembrane
domain (TM); this domain anchors syntaxin 6
in vesicular membranes (Wendler & Tooze,
2001). (b) Examination of the involvement of
syntaxin 6 functional domains in localization to
the chlamydial inclusion. C2BBe1 cells were
transfected with the indicated 3XFLAG-syn-
taxin 6 (syn6) constructs, followed by infection
with C. trachomatis serovar L2 (m.o.i. 4:1). At
18 h post-infection, cells were fixed in abso-
lute ethanol and processed for indirect immu-
nofluorescence. Chlamydial inclusions were
labelled with an antibody that recognizes the
inclusion membrane (IncG), and the syntaxin 6
constructs were detected with an anti-M2
FLAG tag antibody (Sigma-Aldrich). Slides
were visualized with an LSM 510 Laser
Module Zeiss Axiovert 200M confocal micro-
scope. Bar, 10 mm.
Syntaxin 6 localizes to the chlamydial inclusion
4 for proper vacuolar biogenesis and fusion with
endoplasmic-reticulum-derived vesicles (Arasaki & Roy,
2010). As shown here, C. trachomatis excludes syntaxins 4
and 16, but recruits syntaxin 6, a trans-Golgi SNARE
protein. While the SNARE domain of syntaxin 6 is not
involved in localizing the protein to the chlamydial
inclusion, we hypothesize that the SNARE domain plays
an important role at the inclusion membrane and may
mediate specific vesicle fusion events. The role(s) of
syntaxin 6, however, remain undefined as siRNA depletion
did not dramatically diminish inclusion development or
trafficking of sphingomyelin to the inclusion (data not
Chlamydiae are known to extensively modify the inclusion
membrane very early in infection by the insertion of type
III secreted intrinsic membrane proteins collectively known
as Incs. The Inc proteins show little similarity to known
host proteins but display a predicted, bi-lobed hydro-
phobic domain approximately 40 amino acids in length. C.
trachomatis encodes up to 50 Inc proteins (Rockey et al.,
2002; Shaw et al., 2000). In addition to recruiting
eukaryotic SNARE proteins to the inclusion membrane,
Inc proteins may mimic eukaryotic SNAREs. Computer
modelling has identified two chlamydial proteins, IncA and
CT813, as having SNARE-like domains (Delevoye et al.,
2004, 2008). Additionally, in vitro studies utilizing recon-
stituted liposomes with recombinant proteins have found
that IncA can bind the SNARE protein vamp 3 in vitro
(Delevoye et al., 2008). Interestingly, vamp 3 operates
along a basolateral trafficking pathway in polarized
epithelial cells (Pocard et al., 2007).
C. trachomatis IncA is required for homotypic vesicle
fusion of multiple C. trachomatis inclusions within the
same cell. Other chlamydia species also carry genes
annotated as IncA; however, C. caviae IncA does not
mediate fusion with other inclusions formed by C. caviae
or other chlamydial species. It is likely that host factors are
also required for C. trachomatis homotypic vesicle fusion
(Delevoye et al., 2008). In addition to promoting fusion of
C. trachomatis inclusions, IncA has also been proposed to
act as an inhibitory SNARE by blocking specific SNARE-
mediated membrane fusion events in vitro (Paumet et al.,
2009). Interestingly, C. trachomatis IncA had no inhibitory
effect on exocytic complexes examined but was specific
for endocytic SNAREs (Paumet et al., 2009). This is
particularly relevant since chlamydial inclusions are non-
fusogenic with endocytic compartments but are believed
to intercept sphingolipids and cholesterol from exocytic
Several Rab-family GTPases are also recruited to the
chlamydial inclusion membrane but not in patterns
common throughout the genus. For example, Rab1,
Rab4, Rab11 and Rab14 are recruited to C. trachomatis,
C. muridarum and C. pneumoniae inclusions. Rab 6 is
recruited to C. trachomatis exclusively, however, and Rab10
is recruited only to C. muridarum and C. pneumoniae
inclusions (Brumell & Scidmore, 2007; Rzomp et al., 2003).
The endocytic Rabs, Rab5, Rab7 and Rab9, are excluded
from the chlamydial inclusion. Rab4 recruitment to C.
trachomatis inclusions is mediated by Inc229 (Rzomp et al.,
2006) whereas C. pneumoniae Cpn585 interacts with Rabs
1, 10 and 11, but not Rab 4 (Cortes et al., 2007). How this
combination of Rab proteins associated with different
compartments and pathways affects inclusion membrane
fusion events remains to be fully defined, although recent
studies have implicated a role in mediating the phos-
phoinositide composition of the inclusion membrane
(Moorhead et al., 2010).
Syntaxin 6 is recruited to the chlamydial inclusion
membrane protein by a mechanism that requires both an
unknown chlamydial protein, presumably one localized to
the inclusion membrane, and a plasma membrane retrieval
signal on syntaxin 6. SNARE complexes consist of three
proteins, which overall contribute three Q-SNARE motifs
and one R-SNARE motif (Fasshauer et al., 1998). In a
typical SNARE complex, a syntaxin supplies one Q-SNARE
motif, a vamp supplies the single R-SNARE motif, and
cytosolic SNAP 23 supplies the additional two Q-SNARE
motifs (Sutton et al., 1998). SNARE proteins, such as
vamps and syntaxins, remain membrane bound whether or
not they are found within a complex, and control fusion
events within distinct subcellular compartments (Teng
et al., 2001). An improved understanding of the chlamydial
proteins involved in conferring specificity to the cellular
interactions of the chlamydial inclusion is critical to
elucidating chlamydial pathogenesis.
This work was supported by the Intramural Research Program of the
NIAID/NIH. We thank Dr Jeffrey Pessin (Albert Einstein College of
Medicine, Bronx, NY) for the eGFP-syntaxin 6 construct and Dr
Harlan Caldwell (NIAID Rocky Mountain Laboratories, Hamilton,
MT) for the antibody against C. pneumoniae. We would also like to
thank Tina Clark for excellent technical assistance.
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Edited by: P. C. F. Oyston
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838 Microbiology 157