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Originally published In Press as doi:10.1074/jbc.M407227200 on August 16, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46896-46906, November 5, 2004
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Conservation of the Biochemical Properties of IncA from Chlamydia trachomatis and Chlamydia caviae

OLIGOMERIZATION OF IncA MEDIATES INTERACTION BETWEEN FACING MEMBRANES*{boxs}

Cédric Delevoye{ddagger}§, Michael Nilges¶, Alice Dautry-Varsat{ddagger}, and Agathe Subtil{ddagger}||

From the {ddagger}Unité de Biologie des Interactions Cellulaires and the Unité de Bio-Informatique Structurale, Institut Pasteur, CNRS URA 2582 and 2185, 25 rue du Docteur Roux, 75015 Paris, France

Received for publication, June 28, 2004 , and in revised form, August 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlamydiaceae are obligate intracellular parasites that develop in a host cell within a membrane-bound compartment, termed an inclusion. The membrane of the inclusion is initially formed by the invagination of the plasma membrane, and pinching off of a vesicle containing the infectious form of the bacterium, the elementary body (EB).1 Thereafter, EBs differentiate into noninfectious but metabolically active reticulate bodies, which proliferate within the expanding inclusion, giving rise to 1000 or more progeny per host cell. The infectious cycle ends after 2–3 days depending on the strain, when bacteria that have differentiated back to EBs are released in the extracellular medium.

The composition of the inclusion and the origin of its constituents is not yet fully understood. EB-containing vesicles seem to become unable to fuse with early endosomes soon after entry (1). They also fail to fuse with late endocytic compartments and lysosomes, thus escaping degradation by the host cell (2). The early separation between the chlamydial inclusion and the endocytic trafficking distinguish the inclusion from the parasitophorous vacuoles of several pathogens (reviewed in Ref. 3). Acquisition of lipids necessary for the growth of the inclusion membrane, as well as for incorporation into bacteria (4), must originate from other sources. One origin of the bacterial lipid content is the Golgi apparatus, as sphingomyelin, synthesized in the Golgi apparatus from a fluorescent precursor, is transported to the inclusion and accumulates into the bacteria (5). This process is dependent on bacterial protein synthesis: inhibition of chlamydial early transcription or translation prevents the incorporation of sphingomyelin, and the inclusion eventually fuses with lysosomes (6). The bacteria also appear to acquire host cell cholesterol by the same Golgi-dependent pathway as sphingomyelin (7). Surprisingly, no host cell protein has been found inserted into the inclusion (2). The only host protein known to interact directly with the inclusion is 14-3-3, for which interaction with a bacterial component of the inclusion has been demonstrated (8). Host proteins recruited to the inclusion include dynein, {beta}-catenin, and specific Rab GTPases, but the mechanisms of their association with the inclusion are not known (9–11).

In contrast with the apparent absence of eukaryotic proteins in the inclusion, a large family of bacterial proteins, termed Inc proteins, are known to be inserted in this compartment (12). These proteins are unique to Chlamydiaceae, and members of the family share little primary sequence identity with each other within one species. Some of the members are somewhat conserved between different Chlamydiaceae species, but in that case the conservation is usually low. They share, however, one remarkable feature that allows the prediction of when a given protein probably belongs to the family: they possess a very large (50–80 amino acids) bilobed hydrophobic domain. Confirmation that a protein that contains such a domain is located in the inclusion requires specific antibodies against this protein (13). So far about 10 proteins have been shown to localize to the inclusion membrane, and between 40 and 90 proteins are predicted to belong to the family, from C. trachomatis and C. pneumoniae genome analysis, respectively (13, 14). The topology of the insertion of Inc proteins in the inclusion has not been directly addressed, but microinjection of antibodies against four of these proteins demonstrated that at least the C-terminal domain is exposed to the cytosol (15, 16). The large hydrophobic domain is probably required for insertion in the inclusion membrane, and may be compatible with a hairpin insertion, with both extremities of the protein facing the cytosol. The mechanism by which Inc proteins are secreted out of the Chlamydiaceae for insertion in the inclusion membrane has been identified. Chlamydiaceae possess a type III secretion apparatus, which is found in several Gram-negative pathogenic bacteria, and which allows for the translocation of bacterial proteins through the bacterial membranes and across a eukaryotic membrane (17). Using heterologous secretion, it was shown that Inc proteins are recognized by type III secretion machineries of other pathogens, strongly suggesting that it is the mechanism used by Chlamydiaceae to secrete these proteins into the inclusion membrane (18, 19).

The first member of the Inc family to be identified, IncA, is also the one that attracted most of the attention. First cloned from C. caviae (CCA00550, it has homologs in a similar genetic environment in all sequenced genomes (CT119 in C. trachomatis serovar D and CPn0186 in C. pneumoniae CWL029). The level of sequence identity between homologs is low, and antibodies against IncA do not cross-react between different species. Antibodies against IncA from each of these species have been obtained, and have revealed an important accumulation of the protein on the membrane of the inclusion of all species (13, 20, 21), as well as on fibers emanating from the inclusion that are particularly enriched in some species (22). IncA from C. caviae was shown to be exposed on the cytoplasmic face of the inclusion, and to be phosphorylated by the host cell (15). Finally, IncA is expressed rather late compared with most Inc proteins, the transcript being detected 12 h post-infection with the C. trachomatis serovar L2 strain and 16 h post-infection with the C. trachomatis serovar D strain (23, 24).

IncA is the only member of the Inc family for which a function has been proposed, namely a role in the homotypic fusion of inclusions in C. trachomatis. Typical C. trachomatis isolates occupy inclusions that fuse with each other when the cells are infected at high multiplicities of infection. This fusion is inhibited at low temperature (32 °C) and requires bacterial protein synthesis (25). Evidence for the involvement of IncA in this process came from two independent studies. First, microinjection of anti-IncA antibodies blocked the fusion of inclusions in cells infected at high multiplicities of infection (16). Second, a minority (1.5%) of C. trachomatis clinical isolates form multiple non-fusogenic inclusions and do not express IncA (26). Careful analysis of this collection of variants later showed that if most of these variants (24/27) do not express IncA, three non-fusogenic strains do express a normal protein at the inclusion membrane, suggesting that other elements of the fusion machinery are missing in these strains (27). Consistent with the implication of IncA in inclusion fusion is the observation that the majority of inclusion fusions occurs between 10 and 16 h post-infection with the serovar L2 (25, 28). This correlates with the time when IncA can be detected in the strain (16). Moreover, the inhibition of inclusion fusion at low temperature correlated with an inhibition of IncA export to the inclusion membrane in these conditions (29). However, the temperature block is likely to affect the export of several proteins, which could also account for the inhibition of fusion.

Altogether, these data argue for a role of IncA in the fusion of inclusions observed with C. trachomatis strains. However, several questions remain unsolved. In cells infected at low multiplicity of infection, microinjection of anti-IncA antibodies leads to the septation of the inclusion (16). One explanation is that inclusions, like other eukaryotic organelles, are dynamic entities that can fuse and septate, and that in the presence of antibodies against IncA, fusion is slowed down, resulting in multiple inclusions. However, clinical isolates of non-fusogenic phenotypes contain only a single inclusion at low multiplicity of infection, implying that the absence of IncA does not result in multiple inclusions in this case. Microinjection of whole anti-IncA antibody was shown to induce the aggregation of IncA on the surface of the inclusion, while the protein remained homogenously distributed when Fab fragments of the same antibody were injected (15). Therefore, microinjection of anti-IncA antibody may induce a more general disorganization of the inclusion proteins involved in fusion/septation and some of the consequences of microinjection may be indirect. Even if IncA plays a direct role in C. trachomatis inclusion fusion, other roles may be envisioned, especially in other species such as C. caviae and C. pneumoniae, which appear to be less fusogenic than C. trachomatis.

In this report, we investigated the biochemical properties of IncA from C. caviae GPIC strain (CcaIncA) and from C. trachomatis serovar L2 (CtrIncA). We showed that IncA from both species can self-interact. Dynamic modeling on membrane-proximal domains of CcaIncA and CtrIncA showed that tetramers of IncA are compatible with a structure similar to the SNARE complex, which is a conserved complex involved in the fusion of vesicles with their target membrane. We used heterologous expression of IncA by HeLa cells to further investigate the biochemical properties of IncA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Bacteria, Antibodies, and Other Reagents—The human cervical adenocarcinoma cell line, HeLa 229, was from the American Type Culture Collection (ATCC) and was grown in Dulbecco's modified Eagle's medium with glutamax (Invitrogen Life Technologies) supplemented with 10% fetal calf serum (complete medium). The GPIC strain of C. caviae was obtained from Roger Rank (University of Arkansas). C. trachomatis serovar L2 strain 434 (VR-902B) and C. trachomatis serotype D (27F0734) were from ATCC. C. trachomatis serotype D(s)5058 is a clinical isolate, which does not express IncA, and was kindly given by Drs. Dan Rockey and Walter Stamm (27). Chlamydiaceae were prepared as described (30). Bovine serum albumin (BSA), saponin, dimethyl sulfoxide (Me2SO), and 1,4-diazabicyclo-[2.2.2] octane (DABCO) were purchased from Sigma, paraformaldehyde was from Merck, Mowiol was from Calbiochem. Ni-nitrilotriacetic acid (NTA)-agarose beads were purchased from Qiagen, dithio-bis-succinimidyl propionate (DSP) was from Interchim (Montluçon, France). The mouse anti-Chlamydia antibodies were purchased from Argene, Biosoft (12-114, Varilhes, France). Rabbit anti-histidine (sc-803) and rabbit anti-green fluorescent protein (GFP) (sc-8334) antibodies were from Santa Cruz Biotechnology, mouse anti-histidine from Invitrogen (46-0693), and rabbit anti-calnexin antibodies were from Stressgen (SPA-860). Horseradish peroxidase-linked (HRP) anti-rabbit and CyTM-3-conjugated goat anti-mouse antibodies were from Amersham Biosciences, alkaline phosphatase-linked secondary antibodies were obtained from Pierce, goat anti-rabbit or mouse Alexa Fluor-488 antibodies were from Molecular Probes, and goat anti-rabbit TRITC antibodies were from Immunotech (France). Rabbit anti-C. caviae IncA antibodies were prepared as described (30).

Plasmids—Genomic DNA from C. caviae strain GPIC and C. trachomatis serovar L2 were prepared from bacteria using the RapidPrep Micro Genomic DNA isolation kit (Amersham Biosciences). The incA genes were amplified by PCR, and cloned in NcoI-KpnI sites of pQE-TriSystem vector for His-tagged proteins (Qiagen) and in EcoRI-KpnI sites in pEGFP-C1 vector for GFP-tagged proteins (Clontech). Sequences of the primers used are listed in Table I. In some cases, introduction of the NcoI cloning site necessitated a modification of the second amino acid of the protein. To construct {Delta}118CcaIncA-NS5B, the last 714 nucleotides of CcaIncA were amplified by PCR and cloned into pCMVGFPNS5BconC26 digested by NheI and BsrGI, a generous gift from Dr. D. Moradpour (Freiburg Hospital University).


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  		TABLE I
Primers used for plasmid construction by PCR

 
Infection—HeLa cells, grown in 10-cm dishes and less than 50% confluent, were washed twice in warmed phosphate-buffered saline (PBS). For infection with C. caviae strain GPIC and with C. trachomatis serovar L2, the cells were incubated in complete medium with bacteria (GPIC) for 90 min at 37 °C, washed twice in PBS, and incubated for the indicated time at 37 °C. For infection with C. trachomatis serovar D, a centrifugation step of 30 min at 3,000 x g was added before incubating the cells at 37 °C.

Cross-linking Experiments—Cells (6 x 106 cells per condition) were washed three times in PBS, incubated with 0.5 mM DSP in 1% Me2SO or in 1% Me2SO alone in cross-linking buffer (150 mM NaCl, 0.2 mM CaCl2, 0.2 mM MgCl2, 10 mM Hepes, pH 7.3) for 30 min at 4 °C, and neutralized with three washes in 150 mM NaCl, 20 mM Tris, pH 7.3. Cells were lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, pH 7.5) supplemented with 5 mM phenylmethylsulfonyl fluoride (Sigma) and protease inhibitor mixture (Sigma) for 30 min at 4 °C before scraping and centrifugation at 18,000 x g for 20 min at 4 °C. In the case of infected cells, the supernatant fractions were boiled in SDS-PAGE sample buffer (62 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromphenol blue) with or without {beta}-mercaptoethanol and resolved by electrophoresis in polyacrylamide gels using SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane (Millipore), and the membrane was used for blotting with anti-CcaIncA or anti-histidine antibodies followed with HRP- or alkaline phosphatase-conjugated antibodies, and identified by enhanced chemiluminescence (ECL) or chemifluorescence, respectively, according to the manufacturer's instructions (Amersham Biosciences).

Co-purification of GFP-IncA and IncA-His on Histidine Affinity Columns—HeLa cells (6 x 106 per point) were transfected by electroporation with 20 µg of indicated plasmid in 40 mM NaCl solution at 900 µfarad, 200 V (EasyJet, Eurogentec). Cells were seeded in 10-cm dishes for 24 h and lysed on ice in lysis buffer. Cell lysates were centrifuged at 18,000 x g for 20 min at 4 °C. Equal volumes of supernatant were incubated for 2 h with 20 µl of Ni-NTA beads at 4 °C with gentle agitation. The beads were washed extensively with lysis buffer at 4 °C, boiled in SDS-PAGE sample buffer, and proteins bound to the beads were analyzed by SDS-PAGE followed with transfer to a polyvinylidene difluoride membrane and Western blotting. The same membrane was first probed with anti-histidine and HRP-conjugated anti-rabbit antibodies, and revealed by ECL. The membrane was then stripped by a 30-min incubation at 50 °C in 0.7% {beta}-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.8, and probed again, using anti-GFP and HRP-conjugated anti-rabbit antibodies, and revealed by ECL.

Modeling of IncA Structure—For both CcaIncA and CtrIncA, a stretch of 39 amino acids 23 residues after the end of the second predicted transmembrane helix was modeled. The sequences were aligned by hand to the four helices in the endosomal SNARE complex (31), such that the hydrophobic a and d positions matched. This naturally aligned a central hydrophilic residue (glutamine for CcaIncA, threonine for CtrIncA) with the central glutamine residues in the SNARE complex, in the d position. The side chains of CcaIncA and CtrIncA were built onto the coordinates of the central part of the endosomal SNARE complex (31) by a molecular dynamics-based strategy essentially as published (32). In this method, missing side chain atoms are initially placed in random positions and the resulting structure is then minimized in a three-stage protocol: first, simulated annealing with covalent and packing interactions only; second, a short molecular dynamics run with a full molecular dynamics force field in vacuo, followed by conjugate gradient minimization; and third, a molecular dynamics run in explicit solvent. We used the program X-plor (33) for stages 1 and 2, and GROMACS 3.2 (34) for stage 3. We extended the third stage to a molecular dynamics trajectory in explicit solvent for 1 ns. The structures were embedded in a box of SPC water molecules with minimum distance between the solute and the box boundary of 1 nm. The system consisting of protein, water, and sodium ions to neutralize the total charge was slowly heated from 50 K to the simulation temperature (300 or 350 K) with positional restraints on the solute during 300 ps. The electrostatic interactions were treated with the Particle-Mesh-Ewald method (35) for interactions beyond 1 nm; weak temperature coupling with a relaxation time of 5 ps, using the Berendsen method, was employed (36).

Immunofluorescence Microscopy—HeLa cells grown in 6-well plates were transfected with the indicated plasmids using FuGENE 6 reagent (Roche Applied Science). Twenty-four hours later, the cells were washed twice in PBS and fixed with 4% paraformaldehyde, 120 mM sucrose in PBS for 30 min at room temperature. The cells were washed in PBS, incubated for 10 min in 50 mM NH4Cl in PBS at room temperature, saturated in 1 mg/ml BSA in PBS and permeabilized in 0.05% saponin, 1 mg/ml BSA in PBS. To observe histidine-tagged proteins, the cells were first labeled with anti-histidine antibody before being incubated with anti-rabbit Alexa Fluor-488 antibody. The endoplasmic reticulum and the chlamydial inclusion were labeled with anti-calnexin and anti-Chlamydia antibodies, respectively, followed by incubation with CyTM-3-conjugated goat antimouse antibodies. To quantify bacterial entry in cells transfected with CcaIncA-His, the cells were fixed 3 h after infection. Extracellular and intracellular bacteria and transfected cells were labeled as described (40), except that transfected cells were identified using anti-His antibodies. Coverslips were mounted in Mowiol with 100 mg/ml DABCO and examined under an epifluorescence microscope (Axiophot, Zeiss, Germany) attached to a cooled CDD-camera (Photometrics, Tucson, AZ), using an x63 Apochromat lens. Images were acquired and analyzed using Metamorph software (Universal Imaging Corporation).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IncA Proteins Are Found in High Molecular Mass Complexes—IncA proteins share the same general organization: a short N-terminal domain, a large bilobed hydrophobic domain, and a C-terminal cytoplasmic domain (Fig. 1). This domain was shown to be exposed on the cytosolic face of the inclusion membrane with CcaIncA and CtrIncA (15, 16). The level of identity between IncA of different species is low (around 20% of identical residues, which are distributed along the whole molecule). Using tools designed to calculate the propensity of a given sequence to form coils, we noticed that all IncA proteins are predicted to engage in coiled-coil structures over the whole length of their C-terminal domain with a high probability. This prediction is strengthened by the presence of one or more leucine zipper domains in these regions.



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  		FIG. 1.
A schematic representation of IncA from different species. The scale refers to amino acid numbering.

 
The potential of IncA to interact with other molecules via coiled-coil interactions prompted us to look for its partner(s). First, we performed cross-linking experiments on cells infected for 24 h with C. caviae. Infected cells were incubated for 30 min at 4 °C with the cross-linking reagent DSP before cell lysis and analysis of whole cell extracts by SDS-PAGE, followed by Western blot analysis using anti-CcaIncA antibodies (Fig. 2A). In control cells, IncA migrated around 37 kDa, which corresponds to its expected molecular mass. In cells incubated with the cross-linker, IncA also migrated as a higher molecular mass complex, around 75 kDa. This band disappeared when the sample was treated with {beta}-mercaptoethanol, which cleaves the disulfide bridge present in the cross-linker and separates IncA from its partner. A faint upper band (around 150 kDa) was also observed in cells treated with DSP, suggesting that IncA may participate in the formation of complexes of molecular mass even higher than 75 kDa that may consist of more than 2 molecules.



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  		FIG. 2.
IncA participates in high molecular mass complexes by self-interaction. A, HeLa cells were infected with C. caviae for 24 h before cross-linking, cell lysis, and centrifugation. The supernatant fractions of control cells or cells treated with 0.5 mM DSP were treated with or without 5% {beta}-mercaptoethanol ({beta}-me) before SDS-PAGE and Western blot analysis using anti-CcaIncA antibody. The three forms of IncA around 40 kDa correspond to different phosphorylation states (P0, P1, P2) of IncA (15). B, HeLa cells untransfected (left) or transiently expressing CcaIncA-His (middle) or CtrIncA-His (right) were treated with DSP, lysed, and centrifuged. The supernatant fractions were incubated with Ni-NTA-agarose beads, and the bound fractions were treated with or without 5% {beta}-mercaptoethanol, before analysis by SDS-PAGE and Western blot using anti-His antibody. In addition to the specific signal, the anti-His antibody gives a nonspecific signal (ns) around 70 kDa on cell extracts. C, HeLa cells were co-transfected with plasmids coding for CcaIncA-His and GFP-CcaIncA (lane 1, left and right), or for CtrIncA-His and GFP-CtrIncA (lane 2, left and right). The following day, the cells were lysed, centrifuged, and the supernatant fractions were incubated with Ni-NTA beads. Ni-NTA-bound molecules were analyzed by SDS-PAGE and Western blot. His-tagged proteins were first detected by blotting with anti-His antibody (left), then the membrane was stripped and re-used for blotting with anti-GFP antibody (right).

 
Because IncA is found on the cytosolic face of the membrane of the inclusion, which contains several chlamydial proteins, its partners may be of eukaryote or prokaryote origin. To assess if the presence of other chlamydial proteins were necessary for the association of IncA with its partners, we performed cross-linking experiments in cells heterologously expressing IncA. For this purpose, incA was cloned in a vector that provided a promoter for expression in eukaryotic cells as well as a C-terminal histidine tag.

Cells transfected with CcaIncA-His were treated with DSP, lysed, and the affinity of the histidine tag for Ni2+ was used to purify the His-tagged protein from cell extracts using Ni-NTA beads. Ni-NTA-associated proteins were analyzed by SDS-PAGE and Western blot. In cells incubated with DSP, in addition to the band corresponding to CcaIncA-His monomers (42 kDa), CcaIncA-His migrated in intermediate (around 90 kDa) and high (around 180 kDa) molecular mass complexes, which disappeared when the samples were prepared under reducing conditions (Fig. 2B). These results are similar to what we observed in cells infected with C. caviae; the histidine tag accounting for the shift in the migration of the different bands. This result shows that the participation of CcaIncA to high molecular mass complexes does not require the presence of other chlamydial proteins, and that the CcaIncA partner is either itself or a protein from the host cell.

To see whether the formation of high molecular mass complexes involving CcaIncA was specific to this protein we performed the same cross-linking experiments in cells expressing CtrIncA-His. In cells incubated with DSP, in addition to the band corresponding to CtrIncA-His monomers (35 kDa), CtrIncA-His migrated in intermediate (around 70 kDa) and high (around 100 kDa) molecular mass complexes, which disappeared when the samples were prepared in reducing conditions (Fig. 2B). Therefore, the property that induces IncA to participate in high molecular mass complexes is common to at least two species of Chlamydiaceae.

Altogether, these experiments show that CcaIncA-His and CtrIncA-His participate in high molecular mass complexes. Considering the fact that cross-linking is never complete, the relative abundance of intermediate and even high molecular mass complexes suggests that most of the IncA molecules are engaged in these complexes.

IncA Interacts with Itself—Because the size of the intermediate molecular mass complexes were compatible with the formation of dimers of IncA, we investigated further whether this protein was able to form multimers. There is no useful gene manipulation technique to manipulate the genome of Chlamydiaceae, and to address reporter molecules to the membrane of the inclusion. Therefore, to determine whether CcaIncA was able to self-interact, we expressed CcaIncA in HeLa cells with two different tags: a C-terminal histidine tag (CcaIncA-His), and an N-terminal GFP CcaIncA. The affinity of the histidine tag for Ni2+ was used to purify the His-tagged protein from cell extracts using Ni-NTA beads. The beads were washed, and Ni-NTA-associated proteins were analyzed by SDS-PAGE and Western blot. Probing of the membrane with anti-histidine antibody showed that, as expected, CcaIncA-His was retained on the beads (Fig. 2C, left lane 1). The membrane was then stripped and re-used for probing with anti-GFP antibody, which revealed the presence of GFP-CcaIncA in the Ni-NTA-associated fraction (Fig. 2C, right lane 1). In control experiments, when the cells had been transfected with GFP and CcaIncA-His, or with GFP-CcaIncA alone, no signal was seen after the anti-GFP blotting, indicating that neither GFP nor CcaIncA interacts directly with the NiNTA beads (data not shown).

An identical experiment was performed using cells co-expressing CtrIncA-His and GFP-CtrIncA. Probing with anti-GFP antibody showed that GFP-CtrIncA was retained with CtrIncA-His on the Ni-NTA beads, demonstrating that CtrIncA is also able to form dimers (Fig. 2C, left and right lane 2).

These experiments show that both CcaIncA and CtrIncA were able to form dimers that resisted the detergent (0.5% Triton) present in the cell extracts. It is therefore very likely that the intermediate molecular mass complexes, revealed by cross-linking in the previous experiments, correspond to IncA dimers.

Modeling of IncA Tetrameric Interactions Is Compatible with a Four Helix Bundle Structure Similar to That of the SNARE Complex—Upon closer examination of IncA sequences, we noticed that CcaIncA showed sequence similarity with a well characterized domain of the eukaryotic fusion machinery, the SNARE domain. In eukaryotic cells, fusion of compartments is preceded by assembly of the fusion machinery, which involves mainly proteins of the SNARE superfamily. The hallmark of all SNARE proteins is that they contain conserved heptad repeat sequences that form coiled-coil structures called the SNARE complex, with a highly conserved glutamine or arginine residue at the center of the complex (37). Importantly, the SNARE domain is close to the transmembrane domain of the SNARE proteins (or brought in proximity of the membrane during SNARE complex formation). This position brings opposite membranes together during SNARE complex formation and is essential for membrane fusion. Strikingly, CcaIncA first leucine zipper (amino acids 140–178) consists of six heptad repeats with leucine residues in positions a and other hydrophobic residues in positions d, except for the glutamine residue present in d in the center of the domain (Fig. 3A). This similarity with SNARE domains, together with the fact that we showed that IncA dimerizes and probably tetramerizes, suggested to us a parallel organization of four CcaIncA helices similar to that found in the SNARE complex. We modeled a 39-residue stretch of the CcaIncA and CtrIncA sequences, 23 residues after the predicted transmembrane domain, as a parallel tetrameric coiled-coil. These sequences are shown in Fig. 3A, aligned with the four helices of the SNARE domain of the endosomal SNARE complex (31). Fig. 3B shows helical wheel representation of the CcaIncA tetramer, with some key interactions indicated by lines. Fig. 3, C and D show the tridimensional structures of CcaIncA and CtrIncA models, respectively. In these models, the helices are connected by layers of hydrophobic amino acids with the exception of the hydrophilic central layer, like in the SNARE complex. The association of neighboring helices is favored by numerous polar interactions. We next analyzed the stability of the modeled tetramer over time in aqueous solution. The simulations were performed at two temperatures, 27 and 77 °C. Both simulations showed that the modeled tetramers were very stable, presenting only small fluctuations around the average structure. Indeed, during the whole simulation, we observed neither separation of the monomers nor modification of the secondary structure. For comparison, we performed the same simulation on the SNARE complex. We observed that the molecular dynamics trajectory of the IncA models were very similar to those of the SNARE complex itself. The only difference was that the four central polar residues of the IncA tetramers exchanged conformations several times during the simulations whereas those of the SNARE complex did not. It is noteworthy that such exchanges of conformations were observed experimentally in the case of a dimeric leucine zipper (38). Movies of the simulations are available as supplementary material (Supplemental Fig. 3Csup, 3Dsup, and 3Esup). Altogether, our modeling shows that CcaIncA and CtrIncA sequences are compatible with the formation of very stable four parallel helix bundles, resulting in a structure similar to the SNARE complex.



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  		FIG. 3.
Modeling of IncA structure. A, alignment of the modeled regions of the sequences of CtrIncA and CcaIncA, with the sequences forming the central part of the endosomal SNARE complex (31). The modeled region starts 23 residues after the end of the second predicted transmembrane helix. The alignment with the SNARE complex was done manually based on predicted hydrophobic a and d positions. The central polar residue is threonine in the case of CtrIncA and Glutamine in the case of CcaIncA. Hydrophobic residues on a and d positions are colored in blue, mismatches in yellow; the central polar residue is marked in green, or in red (for the arginine in the SNARE complex). B, helical wheel representation of CcaIncA as a parallel tetramer. The polar interactions observed in the model are indicated. C and D, side view of the CcaIncA (C) and CtrIncA (D) tetramers, showing one monomer (left), one monomer with the backbone of the other 3 helixes (middle) and the tetramer (right). The backbone is shown as a yellow ribbon. Lysine, arginine, and histidine residues are shown in blue, aspartate and glutamate in red, other polar residues in green, hydrophobic residues in white. The figure was generated with VMD (47).

 
IncA-His Expressed by HeLa Cells Localizes to the Endoplasmic Reticulum—Using biochemical approaches, we showed that IncA molecules interact similarly when they localize in the inclusion, during normal infection, or when they are artificially expressed by a cell in the absence of inclusion. To determine the localization of IncA in the latter case, we labeled cells transfected with IncA-His with anti-His antibody. Both CcaIncAHis and CtrIncA-His were found in a reticulate compartment extending throughout the cell. This compartment was identified as the endoplasmic reticulum (ER) using antibodies to calnexin, a well characterized ER marker (39) (Fig. 4A). This distribution was not due to the His tag as it was also observed in cells transfected with GFP-tagged IncA constructs (see Fig. 7A).



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  		FIG. 4.
Distribution of IncA and of CcaIncA mutants. A, HeLa cells expressing CcaIncA-His or CtrIncA-His were fixed, permeabilized and probed with mouse anti-His and rabbit anti-calnexin antibodies, followed with Alexa Flu-or-488-coupled anti-mouse (left) and TRITC-coupled anti-rabbit (center) antibodies. The superimposition of the two images is shown on the right. B, cells transiently expressing the indicated construct were fixed, permeabilized, and stained with anti-His antibody. Numbers on the scheme refer to the first and last amino acids from CcaIncA incorporated in the construct.

 



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  		FIG. 7.
Disruption of the infectious cycle is mediated by direct interactions between IncA molecules on the inclusion and on the ER membranes. A, cells transfected with GFP-CtrIncA were infected with the IncA-negative C. trachomatis serovar D(s)5058 strain 24 h after transfection, and fixed 1 day later. Bacteria were labeled using anti-Chlamydia (red). Note that this IncA-negative strain is non-fusogenic and forms multiple inclusions. B, percentage of infected cells was quantified in the non-transfected (open bars) and transfected (black bars) populations. The experiment is representative of three.

 
To determine the domains of IncA that are responsible for the cellular localization of the protein, we constructed several truncated forms of CcaIncA-His (Fig. 4B). Deletion of the N-terminal domain ({Delta}53CcaIncA-His) had no affect on the distribution of the protein. Further deletion of the hydrophobic domain ({Delta}118CcaIncA-His) resulted in a cytosolic distribution, showing that this domain is necessary for membrane anchoring of the protein. Finally, truncation of the last 135 amino acids of CcaIncA (CcaIncA{Delta}135His) resulted in redistribution of the protein, which was enriched at the plasma membrane, suggesting that this mutant was able to exit from the ER. Altogether, these experiments show that the large hydrophobic domain of CcaIncA is necessary to anchor the protein in a membrane, but is not sufficient for ER retention, for which part of the C-terminal domain is necessary.

Heterologous Expression of IncA Disrupts the Infectious Cycle—We have shown that IncA molecules interact with each other when they localize in a cellular membrane, either the inclusion, during normal infection, or the ER, when experimentally expressed in eukaryotic cells. In both cases, IncA can form dimers and higher molecular mass complexes. We also showed that IncA could form a tetrameric structure similar to that of the four helix bundle of the SNARE complex. In the latter case, the complex involves SNARE proteins localized on two different membrane compartments. To test the relevance of our structural model in vivo, we asked whether IncA molecules expressed on two different compartments were able to interact with each other. To that end, cells were first transfected with CcaIncA-His for 24 h, or with GFP as a control, then infected with C. caviae GPIC for another 24 h before fixation and labeling of the inclusion and of CcaIncA-His by immunofluorescence. Unexpectedly, we observed that none of the transfected cells were infected, although in GFP-transfected cells infection had occurred normally (Fig. 5A). Similarly, C. trachomatis serovar L2 or D were not able to develop in cells transfected with CtrIncA-His. The same result was observed when GFP-IncA was used instead of IncA-His (see below). This suggested that some step(s) in the infectious cycle was inhibited in cells expressing IncA. Inhibition on chlamydial development was complete only when IncA corresponding to the strain used for the infection was expressed. However, in cells transfected with GFP-CcaIncA, C. trachomatis serovar L2 development was inhibited by 50%, and in cells transfected with GFP-CtrIncA, C. caviae development was inhibited by 20% (data not shown). Using an assay that allows to measure bacteria entry (40), we quantified C. caviae GPIC entry 3 h after infection in cells transfected with CcaIncA-His and in nontransfected cells. We observed no difference in the efficiency of bacteria entry between the two cell types, indicating that the expression of IncA-His did not affect bacterial attachment or entry (Fig. 5B). To obtain further insights into the mechanism by which IncA inhibits chlamydial development, we performed the experiment in the reverse order: cells were first infected for 24 h, then transfected with GFP-IncA, and fixed 8 h after transfection when expression of GFP-IncA becomes detectable by immunofluorescence. In both infected and non-infected cells, GFP-IncA distributed in a reticulate compartment. However, in infected cells, this compartment was distorted (Fig. 5C). The inclusion itself showed large changes in morphology, as the bacteria scattered in the cell, and it was not clear whether the integrity of the inclusion membrane was intact. When cells were observed later after transfection (16 h), very few transfected cells that contained bacteria were visible, and many dead cells were present in the culture dishes, suggesting that heterologous IncA expression eventually resulted in the death of infected cells. Altogether, these experiments show that experimental IncA expression in the host cell ER inhibits inclusion development.



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  		FIG. 5.
Expression of IncA in the ER disrupts inclusion development. A, cells transfected with the indicated constructs were infected with C. caviae or C. trachomatis serovar L2 or D 24 h after transfection, and fixed 1 day later. Infected cells were labeled using anti-Chlamydia antibodies, and cells transfected with IncA constructs were identified using anti-His antibodies. The percentage of infected cells was quantified in the GFP-transfected (open bars) and IncA-transfected (black bars) populations. The experiment is representative of three experiments. B, HeLa cells were transfected with CcaIncA-His and infected with C. caviae the following day. Three hours later, extracellular and intracellular bacteria, as well as transfected cells, were labeled as described under "Experimental Procedures." The number of intracellular bacteria was quantified in the transfected and non-transfected populations (n > 30 cells). The average of two experiments is represented. C, HeLa cells were infected with C. caviae (two top panels) or with C. trachomatis serovar L2 (two bottom panels) for 24 h before transfection with the indicated constructs. Eight hours later, the cells were fixed and IncA-transfected cells were stained with rabbit anti-His antibody followed by Alexa Fluor-488-coupled anti-rabbit antibodies (left) whereas bacteria were labeled with mouse anti-Chlamydia antibodies and CyTM-3-coupled anti-mouse antibodies (middle). On the superimposition of the images, IncA and GFP appear in green and the bacteria in red (right).

 
Expression of IncA C-terminal Cytoplasmic Domain on the ER Is Sufficient to Inhibit the Inclusion Development—We tested the CcaIncA-His mutants for their ability to disrupt C. caviae infection to determine the regions of IncA that are responsible for this effect. Cells expressing {Delta}53CcaIncA-His could not be infected by C. caviae, indicating that even in the absence of the IncA N-terminal domain the disruptive effect was total (Fig. 6A). To determine whether the CcaIncA hydrophobic domain played a role in this effect, we created a chimera between hepatitis C virus nonstructural protein NS5B and CcaIncA C-terminal domain. The hydrophobic C-terminal domain of NS5B forms a transmembrane {alpha}-helix that contains a signal for retention in the ER. It has been shown that this domain was sufficient to target a protein on the cytosolic side of the ER (41). Indeed, fusion of the last 26 amino acids from NS5B with CcaIncA C-terminal cytoplasmic domain resulted in a chimera that had a distribution characteristic of the ER (Fig. 6B). HeLa cells expressing this chimera presented the same defect in the development of bacteria as HeLa cells expressing CcaIncaA-His, indicating that the IncA hydrophobic domain is not required for the inhibition of chlamydial development (Fig. 6A). Finally, transfection of the CcaIncA C-terminal cytoplasmic domain ({Delta}118CcaIncA-His), which has a cytosolic distribution, had no effect on subsequent infection by C. caviae, which developed into similar inclusions in transfected and non-transfected cells. Altogether, these data show that, when anchored to the ER, the CcaIncA C-terminal cytoplasmic domain is sufficient to disrupt the infection.



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  		FIG. 6.
Expression of IncA C-terminal cytoplasmic domain on the ER is sufficient to disrupt the development of the inclusions. A, cells transfected with the indicated constructs were infected with C. caviae 24 h after transfection, and fixed 1 day later. Infected and transfected cells were identified using anti-Chlamydia and anti-His antibodies, respectively. The percentage of infected cells was quantified in the non-transfected (open bars) and transfected (black bars) populations. The experiment is representative of two. B, a schematic representation of {Delta}118CcaIncA-NS5B. Cells transfected with {Delta}118CcaIncA-NS5B were fixed, permeabilized, and stained with anti-IncA antibodies. Note that in addition to the reticulate distribution characteristic of the ER, {Delta}118CcaIncA-NS5B associates with a perinuclear compartment that may be the Golgi apparatus.

 
IncA-His Interacts with Endogenous IncA at the Membrane of the Inclusion and Disrupts Bacterial Development—The effect of ER-located IncA on the development of the inclusion might be caused by interactions with endogenous IncA on the membrane of the inclusion, or with any other inclusion-associated protein. To test these hypotheses, we used a strain of C. trachomatis that does not express IncA, D(s)5058 (27). This strain develops forming multiple inclusions that are non-fusogenic. When HeLa cells transfected with GFP-CtrIncA for 24 h were infected with D(s)5058, inclusions developed in transfected cells as efficiently as in non-transfected cells, and the ER had a normal appearance (Fig. 7). This experiment demonstrates that in the absence of IncA on the membrane of the inclusion there was no disruptive effect of heterologous IncA expression on inclusion development. This result, together with the biochemical experiments demonstrating that IncA self-interacts, show that the disruption of the inclusion development is mediated by interactions between IncA proteins on the inclusion and on the ER. Therefore, we concluded that IncA molecules anchored in facing membranes associate to form multimeric structures.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this article we show that IncA from two species, C. trachomatis and C. caviae, self-associate with themselves. First, in infected cells, we showed using a cross-linker that CcaIncA participated in high molecular mass complexes whose sizes were compatible with the cross-linking of two or more CcaIncA molecules. High molecular mass complexes were also present in cells transfected with CcaIncA and CtrIncA treated with a cross-linker, showing that no other bacterial protein than IncA was necessary for their formation. Finally, we showed, using cells transfected with two IncA constructs linked to different tags, that IncA molecules interacted with each other in cell extracts. Our results are in partial agreement with a previous report in which a yeast two-hybrid system was used to show interactions between CtrIncA molecules (16). However, interactions between CcaIncA molecules were hardly detected with this technique, supporting the authors' conclusion that the function of IncA proteins may differ between species. Here we show that the ability of IncA to form multimers is common to the two species, and we propose that this property underlies similar function(s) during infection.

When artificially expressed in HeLa cells, IncA was localized in the ER, as shown by colocalization with an ER marker, calnexin. The same distribution was observed for other Inc proteins heterologously expressed by the host cell, although some were detected on the plasma membrane (data not shown). Inc proteins are normally synthesized by the bacteria and are transported through the bacterial and inclusion membranes via a type III mechanism. Their insertion in the inclusion membrane may be coupled to the translocation process. In the case of heterologous expression, Inc proteins may be recognized by the cellular machinery as a substrate for ER-associated ribosomes and may be inserted into the ER membrane cotranslationally. Alternatively, the association might be post-translational, because of a better affinity for the ER membrane lipid composition than for other cellular membranes, or via interactions with ER membrane proteins. The large bilobed hydrophobic domain, which is common to all Inc proteins, is probably essential for their interaction with membranes, and deletion of this domain in CcaIncA ({Delta}118CcaIncA-His) resulted in a cytosolic distribution. However the hydrophobic domain is not sufficient to ensure ER location, because deletion of part of CcaIncA C-terminal cytoplasmic domain (CcaIncA{Delta}135His), preserving the entire hydrophobic domain, resulted in preferential localization to the plasma membrane. We did not directly address the topology of IncA insertion into the ER membrane. However, we observed, as described previously, that heterologously expressed CcaIncA-His showed the same migration profile in SDS-PAGE as endogenous CcaIncA (Fig. 2B), with three bands corresponding to three phosphorylation states of the protein (15). This finding strongly supports the hypothesis that heterologously expressed CcaIncA-His is inserted in the ER membrane with the same topology as in the inclusion membrane, with at least the C-terminal domain exposed to the cytosol (16).

Expression of GFP-IncA in infected cells had a very striking effect. Soon after the appearance of IncA, the morphology of the inclusion was modified, with a scattering of the bacteria and simultaneous distortion of the ER. Later after transfection, all infected cells had disappeared from the transfected population, and many dead cells had detached from the culture plate, strongly suggesting that heterologous expression of IncA in the ER of infected cells resulted in cell death. When the experiment was performed in the reverse order, by infecting cells, which had been transfected prior to infection, no inclusion developed in the transfected population. This was not caused by an inhibition of bacteria entry, as we determined that bacteria entry was not affected by IncA expression. In fact, infection with C. caviae seems to proceed normally during the first 10 h. Fifteen hours after infection, the inclusions were disrupted. This correlates with the kinetics of incA expression, as its transcript was first detected by RT-PCR at 10 h post-infection in C. caviae-infected cells (not shown)

To determine which domain of IncA was involved in the inhibition of chlamydial development when expressed by transfection, we measured infection in cells transfected with truncated forms of IncA. Deletion of the N-terminal domain did not suppress the deleterious effect of IncA expression, showing that the IncA N-terminal domain was not required. In fact, the C-terminal cytoplasmic domain alone, targeted to the ER using a transmembrane ER-targeting signal from hepatitis C virus non-structural protein NS5B, was sufficient to produce the inhibitory effect observed with full-length IncA. Finally, expression of CcaIncA C-terminal cytoplasmic domain ({Delta}118CcaIncA-His), which has a cytosolic distribution, had no effect on the infection. These data suggest that to disrupt the infection, CcaIncA C-terminal cytoplasmic domain needs to be anchored to a cellular compartment. We do not know at this stage whether this compartment needs to be the ER, which is naturally distributed in the whole cytosol and therefore in proximity to the inclusion. It may be that expression of IncA at another intracellular compartment, such as the Golgi apparatus, also found in close proximity to the inclusion, would similarly disturb inclusion development. However this hypothesis would be difficult to test, as the only known signals for targeting proteins to the Golgi apparatus are in the cytosolic portions of these proteins, which would need to be replaced with the IncA C-terminal domain.

The deleterious effect on chlamydial development was observed only with IncA expression, not with C. caviae IncB, which is also expressed on the ER by transfection, showing that the effect was specific of this Inc protein (data not shown). Using a strain that does not express IncA on the surface of its inclusions, we were able to demonstrate that the deleterious effect of IncA expression on chlamydial development required the presence of IncA on the membrane of the inclusion. This result, together with our demonstration that IncA molecules interact with each other, show that the disruption of the inclusion development is mediated by direct interactions between IncA molecules from the two compartments in which they localize. Inhibition of chlamydial development was complete only when IncA, corresponding to the strain used for the infection, was expressed, showing that the effect is very specific. However partial interspecies inhibition was also observed, suggesting that, although very different in terms of sequence, C. caviae and C. trachomatis IncA biochemical properties are sufficiently close that they can interact with each other to some extent. This supports the idea that IncA may fulfill similar roles during infection by different species.

In infected cells, expression of IncA in the ER leads to the disruption of the inclusion and to changes in the morphology of the ER. Our microscopy observations are consistent with the hypothesis that these interactions lead to fusion events between the inclusion and the ER. Observation at a better resolution would be necessary to support this hypothesis. Unfortunately, the process is not easy to observe because infected cells are transfected with a low efficiency (independent of the transfected gene), and the few infected and transfected cells die rapidly after IncA expression. Our data therefore support previous reports indicating that IncA may play a role in the fusion of inclusions. The aim of the modeling and molecular dynamics studies was to get insights into possible mechanisms of IncA-induced vesicle fusion. Amphipathic helices alone can be sufficient to induce membrane fusion in vitro (42). However, a fusion mechanism similar to the one involving the formation of the SNARE complex is attractive because of the similarity of the sequences and their properties: there is strong coiled-coil or leucine zipper propensity; the coiled-coil sequence starts shortly after a putative transmembrane region. There is a conserved hydrophilic residue in the interface that may serve to organize the helices, infer specificity, or to bias the equilibrium between multiple possible oligomeric states. Finally, the tetramer proved very stable in molecular dynamics calculations at 27 and 77 °C.

The model is based on the capability of the IncA sequences to form stable oligomers. Whereas the stability in molecular dynamics calculations is an indication that the model is energetically favorable, we cannot directly compare the stability to that of a different oligomeric state (such as a dimer). For mutants of GCN4, Harbury et al. (43) investigated which properties of coiled-coil sequences favor tetramerization. These properties are met in our structural model. (i) {beta}-Branched residues at a positions, which could disfavor tetramers due to rotamer preferences of these residues, are absent in both modeled sequences. (ii) A polar residue in the interface, common in coiled-coils and which may serve to organize the four helices such that the four hydrophilic residues form one layer, is found in both modeled sequences. (iii) Residues on the surface of the tetramers form interactions; as an example, the polar interhelical interactions involving b, c, g, and e positions are shown in Fig. 3B. (iv) Interactions involving b and c positions are more likely in tetrameric than in dimeric coiled-coils because of the different relative orientations of the helices in tetramers. (v) There are no repulsions between residues of like charge in neighboring helices. For CcaIncA, an antiparallel orientation of the helices, on the other hand, would bring the residues on the g position on one helix into contact with those on the neighboring helix; thus leading six arginine residues into close proximity. We conclude that a parallel orientation of the helices is more likely.

It is generally agreed that these membrane-bridging SNARE complexes mediate membrane fusion directly (44, 45). Our IncA model proposes a parallel tetramer formed from helices from two different vesicles. The transmembrane regions of the four monomers are all on the same side of this rod-like structure, similar to proteins involved in viral fusion and in membrane fusion involving the SNARE complex. The formation of the tetramer would therefore induce close approach of the membranes, and possibly destabilize the membranes locally close to the transmembrane domains (46). In that case, one could expect expression of IncA to disturb the ER structure, although this compartment has homotypic fusion abilities of its own. Indeed, we noted that expression of CtrIncA slightly modified the ER, whose network had a somewhat rougher appearance compared with that in non-transfected cells (see Fig. 4A). Whether formation of IncA tetramers by itself is sufficient to promote fusion remains to be examined. The difference in the fusogenicity of C. trachomatis and C. caviae inclusions may indicate that IncA by itself is not sufficient to trigger fusion between inclusions, and that accessory molecules, absent from C. caviae, participate in the fusion of C. trachomatis inclusions. This hypothesis is supported by the observation that some of the non-fusogenic isolates do express IncA, suggesting that other factors necessary for the fusion to occur are missing from these strains (27).

Interestingly, we found, using BLAST analyses, that the genome of the chlamydial-related symbiont of free-living amoebae (UWE25) encodes an open reading frame annotated pc0399, which presents some sequence similarity with IncA (48). Moreover, it shares the characteristic organization of IncA proteins, with a short N-terminal domain, a large bilobed hydrophobic domain, and a C-terminal domain that is predicted to engage in coiled-coils interactions. If this molecule is present on the surface of the inclusion, it suggests that IncA has coevolved with its host cells since the divergence of the pathogenic and symbiotic Chlamydiae, more than 700 million years ago.

Finally, we would like to speculate that, in addition to its role in the fusion of inclusions, IncA may participate in the fusion of cellular vesicles with the inclusion membrane. The fact that IncA structure is compatible with the participation to a SNARE-like complex suggests that this molecule may have evolved to interact not only with itself but also with cellular SNAREs to form fusion-competent SNARE complexes. One possibility is that IncA mimicry with host SNARE proteins could enable the bacteria to hijack part of the cellular traffic by allowing fusion of vesicles with the inclusion.


   FOOTNOTES
 
* This work was supported by Action Concertée Incitative Biologie Cellulaire, Moléculaire et Structurale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplementary videos. Back

§ Supported by an Allocation de Recherche du Ministère de l'Enseignement Supérieur et de la Recherche. Back

|| To whom correspondence should be addressed. Tel.: 33-1-40-61-30-49; Fax: 33-1-40-61-32-38; E-mail: asubtil{at}pasteur.fr.

1 The abbreviations used are: EB, elementary body; BSA, bovine serum albumin; DSP, dithio-bis-succinimidyl propionate; ECL, enhanced chemiluminescence; ER, endoplasmic reticulum; GFP, green fluorescent protein; HRP, horseradish peroxidase; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline; SNARE, soluble NSF attachment protein receptors; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank Stéphanie Perrinet for bacterial preparation. We thank Drs. Dan Rockey and Walter Stamm for the kind gift of the D(s)5058 strain from the University of Washington Chlamydia Repository, and Dr. Dan Rockey for critical reading of the article.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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