Domain Analyses Reveal That Chlamydia trachomatis CT694 Protein Belongs to the Membrane-localized Family of Type III Effector Proteins*

Background: The Chlamydia trachomatis secreted effector CT694 is deployed during invasion and exerts multiple effects on host cells. Results: Residues 40–80 of CT694 contain a domain necessary and sufficient for peripheral localization to eukaryotic membranes. Conclusion: CT694 employs membrane association to manifest effects on host cells. Significance: Elucidating functional protein domains is essential to understand molecular mechanisms of infection employed by the pathogen C. trachomatis. The Chlamydia trachomatis type three-secreted effector protein CT694 is expressed during late-cycle development yet is secreted by infectious particles during the invasion process. We have previously described the presence of at least two functional domains within CT694. CT694 was found to interact with the human protein Ahnak through a C-terminal domain and affect formation of host-cell actin stress fibers. Immunolocalization analyses of ectopically expressed pEGFP-CT694 also revealed plasma membrane localization for CT694 that was independent of Ahnak binding. Here we provide evidence that CT694 contains multiple functional domains. Plasma membrane localization and CT694-induced alterations in host cell morphology are dependent on an N-terminal domain. We demonstrate that membrane association of CT694 is dependent on a domain resembling a membrane localization domain (MLD) found in anti-host proteins from Yersinia, Pseudomonas, and Salmonella spp. This domain is necessary and sufficient for localization and morphology changes but is not required for Ahnak binding. Further, the CT694 MLD is able to complement ExoS ΔMLD when ectopically expressed. Taken together, our data indicate that CT694 is a multidomain protein with the potential to modulate multiple host cell processes.

The Chlamydia trachomatis type three-secreted effector protein CT694 is expressed during late-cycle development yet is secreted by infectious particles during the invasion process. We have previously described the presence of at least two functional domains within CT694. CT694 was found to interact with the human protein Ahnak through a C-terminal domain and affect formation of host-cell actin stress fibers. Immunolocalization analyses of ectopically expressed pEGFP-CT694 also revealed plasma membrane localization for CT694 that was independent of Ahnak binding. Here we provide evidence that CT694 contains multiple functional domains. Plasma membrane localization and CT694-induced alterations in host cell morphology are dependent on an N-terminal domain. We demonstrate that membrane association of CT694 is dependent on a domain resembling a membrane localization domain (MLD) found in anti-host proteins from Yersinia, Pseudomonas, and Salmonella spp. This domain is necessary and sufficient for localization and morphology changes but is not required for Ahnak binding. Further, the CT694 MLD is able to complement ExoS ⌬MLD when ectopically expressed. Taken together, our data indicate that CT694 is a multidomain protein with the potential to modulate multiple host cell processes.
Chlamydia trachomatis infection has been the most reported sexually transmitted disease in the United States because1994, with over 1.2 million cases reported in 2009 (1). However, it is believed that the true number of cases is much higher because of the potential for asymptomatic infections, particularly in males (1). Sequelae resulting from untreated or repeated C. trachomatis serovar D-K infections can include infertility, pelvic inflammatory disease, ectopic pregnancy, or pelvic pain (2).
Additionally, ocular infection with C. trachomatis serovars A-C causes blinding trachoma, the leading cause of preventable blindness worldwide, particularly in developing countries (3).
An obligate intracellular bacterium, C. trachomatis exhibits a biphasic developmental cycle consisting of an extracellular, non-metabolic elementary body (EB) 3 and an intracellular, replicative reticulate body (RB) (4). Both particle types posses a functional type-III secretion system (T3SS), which is essential for bacterial development (5). Contact with the host cell surface triggers secretion of effectors through the T3SS into the host cell (6). In Chlamydia, these immediate early effectors are prepackaged during the reticulate body-to-EB transition to be readily available immediately upon host cell contact (7,8). One such effector is the conserved translocated actin recruiting phosphoprotein (TarP), which is translocated into host cells within minutes of host cell contact and induces actin cytoskeletal reorganization that is important for invasion (9). The ability to dynamically reorganize the host cytoskeleton is a common general theme among bacteria expressing T3SS, including Yersinia spp., Salmonella, Shigella spp., and Pseudomonas aeruginosa (10 -12).
To achieve efficient anti-host function, some T3SS effectors must be targeted to the correct subcellular compartment (12). The presence of a membrane localization domain (MLD) is one mechanism employed to accomplish this goal. For example, discrete MLD domains within YopE of Yersinia spp. or ExoS of P. aeruginosa mediate association with host membranes (12). These MLDs lack the characteristic predicted hydrophobic ␣ helix of a transmembrane domain (13) but contain a leucine-rich region that is essential for membrane association (12,14). It has been proposed that interactions with membranes could be direct (15). Alternatively, there is evidence that these membrane-localized effectors do * This work was supported by Public Health Service Grant AI065530 from so through interactions with membrane-associated proteins rather than direct interactions with the host cell membrane (14,16,17). Regardless of the mechanism, localization of effectors to cellular membranes allows a targeted response in which respective effector proteins manifest activities in a constrained microenvironment. CT694 is a recently described C. trachomatis-specific T3SS effector (18). Aside from a predicted coiled-coil domain from resides 285-305, the primary sequence of CT694 does not contain additional domains identifiable via similarity searches or domain predictions (18). Like TarP (19), this protein is translocated during host cell invasion (18). CT694 protein levels decrease slightly around 1 h post-infection (hpi) but are then maintained until a robust increase during de novo CT694 synthesis at 18 -24 hpi during late-cycle development (7,18). Previous work (18) demonstrated that a GFP-CT694 chimera localizes to the plasma membrane, where it interacts with Ahnak, a large human protein involved in cytoskeleton maintenance and cell signaling (20,21). Ectopic expression studies also revealed that deletion of the C terminus of CT694 precludes the interaction with Ahnak but does not affect membrane localization (18). Herein, we test the possibility that the N terminus of CT694 expresses an MLD that is necessary for localization of CT694 to host membranes.
DNA Methods-C. trachomatis open reading frames were amplified from C. trachomatis serovar L2 genomic DNA using EconoTaq PLUS Green Master Mix (Lucigen, Middleton, WI) according to the guidelines of the manufacturer and using custom oligonucleotide primers containing engineered restriction sites, synthesized by Integrated DNA Technologies (Coralville, IA). Cloning was performed according to standard protocols (24). PCR products were ligated into vectors utilizing the appropriate restriction enzymes, unless otherwise noted. Primer sequences with restriction sites are listed in supplemental Table S1. Transformations with plasmid DNAs were performed using chemically competent Escherichia coli DH5␣ strains (Invitrogen). CT694 truncations were cloned into pEGFP-C3 (Clontech, Mountain View, CA). Primers SHCT694-1A and SHCT694-6B were used for CT694-⌬39, SHCT694-1A and SHCT694 -8B for CT694-⌬192, SHCT694 -1A and SHCT694-9B for CT694-⌬243, and SHCT694-10A and SHCT694-4B2 for ⌬42-CT694. CT694ϩ 40-80 was generated using primers CT694 MLD F and CT694 MLD R. The resulting PCR products were digested with the appropriate restriction enzymes and ligated into pEGFP-C3.
Transfection and Microscopy-Semiconfluent HeLa monolayers were grown on 12-mm-diameter glass coverslips for immunofluorescence analysis. Monolayers were directly infected with C. trachomatis or transfected with endotoxinfree plasmid DNAs. Transfections were accomplished using Lipofectamine 2000 according to the directions of the manufacturer (Invitrogen). For indirect immunofluorescence, samples were fixed with methanol, blocked in 5% BSA (Sigma) in Tris-buffered saline plus Tween 20 (TBST) (137 mM NaCl, 2.68 mM KCl, 2.48 mM Tris base (pH 7.4), supplemented with 0.5% (v/v) Tween 20), and appropriate antibodies were diluted in 5% BSA in TBST. Proteins were visualized by direct fluorescence of GFP-containing proteins or, where indicated, with ␣-GFP (Sigma), and the appropriate secondary antibody conjugated to Alexa Fluor 488 (Invitrogen). Nuclear staining was achieved by staining with DAPI (Invitrogen). Images were acquired by epi-fluorescence microscopy using a ϫ60 apochromat objective plus ϫ1.5 intermediate magnification on a TE2000U inverted photomicroscope (Nikon, Melville, NY) equipped with a Retiga EXi 1394, 12-bit monochrome CCD camera (QImaging, Surrey, BC, Canada) and MetaMorph imaging software version 6.3r2 (Molecular Devices, Downington, PA). Images were processed using Adobe Photoshop CS2 version 9.0 (Adobe Systems, San Jose, CA).
Yeast Two-hybrid Assay-Yeast two-hybrid assays were performed as described (18). Briefly, specific plasmid constructs were transformed into Saccharomyces cerevisiae AH109 using the S. cerevisiae Easycomp transformation kit (Invitrogen) followed by selection on S.D. agar plates deficient in leucine (Leu) and tryptophan (Trp) (Clontech). CT694 (full-length and truncations) were cloned in-frame downstream of the gal4 binding domain (BD) and used as bait in the MATCHMAKER Two Hybrid System 3 (Clontech). Primers used are listed in supplemental Table S1. Interaction studies with Ahnak were performed with engineered Ahnak constructs cloned downstream of the gal4 activating domain (AD) of pGADT7 (Clontech). Colonies expressing interacting proteins were selected for on QDO x-␣-gal. To rule out growth because of auto-activation on selective media, BD/CT694 was cotransformed with AD/T or pGADT7 (AD/Empty). In the same way, Ahnak constructs were cotransformed with BD/53 or pGBKT7 (BD/Empty). Yeast protein expression was evaluated by immunoblot with ␣-AD, ␣-BD, ␣-HA (Santa Cruz Biotechnology, Santa Cruz, CA), ␣-c-myc (BD Biosciences), or ␣-CT694.
Membrane Fractionation-Semiconfluent HeLa monolayers were mock-infected or inoculated with C. trachomatis at an MOI of 100 for 2 or 24 h, as indicated. The cultures were then scraped into 10 ml of ice-cold 0.25 M sucrose buffer (10 mM Tris (pH 7.5), 1 mM EDTA, 0.25 M sucrose) supplemented with protease inhibitors (complete mixture, Roche). Whole-culture material was homogenized for 20 -25 strokes in a Dounce homogenizer followed by centrifugation to remove cell debris. The resulting supernatant was layered over a 24 -44% sucrose gradient, centrifuged, and processed as described (25).
Triton X-114 extractions were performed as described previously (26). HeLa monolayers were transfected or infected with C. trachomatis serovar L2. Cultures were lysed in 1.5 ml of ice-cold 1% Triton lysis buffer (1% Triton X-114 (Sigma), 100 mM KCl, 50 mM Tris-HCl (pH 7.4)), rotated for 30 min at 4°C, and clarified by centrifugation for 35 min (14,000 rpm at 4°C) using an Eppendorf 5810 R centrifuge (Eppendorf AG, Hamburg, Germany). The resulting supernatant was incubated at 37°C for 10 min, followed by centrifugation for 12 min (14,000 rpm at room temperature) to separate the detergent phase (bottom fraction) from the aqueous phase (top fraction). The aqueous phase was removed to a fresh tube containing 200 l of 10% Triton X-114 buffer (10% Triton X-114, 100 mM KCl, 50 mM Tris-HCL (pH 7.4)), and the detergent phase was mixed with 1 ml of buffer 1 (50 mM Tris (pH 7.4), 100 mM KCl). Both phases were incubated on ice for 10 min, followed by 10 min of incubation at 37°C, and finally centrifugation for 12 min (14,000 rpm at room temperature). The top fraction of the detergent phase was removed, and the detergent-rich phase was mixed with 1 ml of buffer 1. The detergent-depleted aqueous phase was again mixed with 200 l of 10% Triton X-114 buffer, and each tube was subjected to the same incubations on ice, followed by 37°C, followed by centrifugation, repeated a total of four times. The final detergent and aqueous phases were precipitated in 50% acetone (v/v) at Ϫ20°C overnight.
Statistical Analyses-All experiments were repeated a minimum of three times, and representative data are shown. Statistical analysis was evaluated using a two-tailed Student's t test where appropriate. In all figures, p values were labeled as follows: *, p Ͻ 0.01 and **, p Ͻ 0.001. Statistical analysis was performed using GraphPad Prism software.

Endogenous CT694 Is Membrane-associated During Early
Cycle Development-We have demonstrated previously that ectopically expressed CT694 is capable of colocalization with the plasma membrane of transfected HeLa cells (18). These data raised the possibility that CT694 membrane localization correlates with effector function(s). We first sought to investigate the subcellular partitioning of endogenous CT694 to confirm the relevance of interaction with host cell membranes. HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 100, disrupted 2 hpi, and lysates were subjected to a sucrose gradient fractionation. Mock-infected parallel cultures were similarly processed as an immunoblot control for antibody specificity (data not shown). Proteins in subsequent gradient fractions were probed in immunoblot analyses with antibodies specific for CT694 or TarP. Immunoblot analyses were probed with C. trachomatis major outer membrane protein (MOMP) antibodies or human Caveolin-1 as controls for intact bacteria and host membranes, respectively (Fig. 1). Both CT694 and TarP were detected in fractions 1-5 as well as fraction 14. Conversely, MOMP was detected only in the lower column fractions, indicating migration of intact chlamydiae. As expected, the host transmembrane protein Caveolin-1 was present in the top fractions, indicating that these fractions contained the majority of host cell membrane material (Fig. 1). These results are consistent with membrane association of both CT694 and TarP during C. trachomatis infections. This localization was specific for early infection time points because CT694 was detected solely in fractions containing intact bacteria when cultures were harvested during late-cycle development (supplemental Fig. S1).
We examined phase separation of CT694 in detergent extracts to further assess the subcellular localization of endog- enous CT694 during C. trachomatis infection. HeLa cells were infected with C. trachomatis at an MOI of 100 and subjected to Triton X-114 extraction at 1, 3, and 6 hpi. At each time point, CT694 was detected in both the detergent and aqueous phases of infected cells ( Fig. 2A). GAPDH, Caveolin-1, and MOMP were used as controls and fractionated as expected. Unlike CT694, TarP was detected only in aqueous fractions, indicating that any membrane association of TarP is fundamentally different from and perhaps less direct than that of CT694. Triton X-114 extraction was also performed on equivalent amounts of density gradient-purified EBs. In these experiments, CT694 was detected solely in aqueous material, indicating that Chlamydia-localized CT694 is not associated with membranes (Fig.  2B). In aggregate, these data are consistent with secreted CT694 stably associating with eukaryotic membranes during invasion and early-cycle development.
Membrane Localization Domain of CT694-Transmembrane prediction tools such as TMPred (27) and Kyte Doolittle (13) analysis do not indicate the presence of typical transmembrane domains within the primary sequence of CT694 (data not shown). Therefore, a systematic series of GFP-tagged CT694 truncations were engineered and expressed in HeLa cells to delineate the minimal domain necessary for membrane localization (Fig. 3A). Equivalent truncations were generated in the yeast two-hybrid expression system to assess the impact on CT694 interaction with human Ahnak. As reported previously (18), concentrations of full-length CT694 (694-FL, Fig. 3C) could be detected in both cytosolic aggregates and plasma membranes. The cytosolic aggregates are not present in all cells, and we cannot rule out the possibility that they are a product of CT694 overexpression. Curiously, the accumulation of aggregates seems positively correlated with membrane localization. Deletion of the C-terminal 39 (694-⌬39), 101 (694-⌬101), or 192 residues (694-⌬192) did not affect CT694 localization but did disrupt CT694 association with Ahnak in yeast two-hybrid studies. Expression of CT694 residues 43-322 (⌬42-694) did not interfere with Ahnak interaction but did result in less overt localization of CT694 to plasma membranes. Finally, in-frame deletion of residues 40 -80 (694-⌬40 -80) completely abolished membrane localization of CT694, but did not affect the interaction with Ahnak. These data indicate that a domain within residues 40 -80 is necessary for membrane localization but not for interaction with Ahnak. Consistent with previous studies, CT694-transfected HeLa cells also routinely displayed aberrant cell morphology in which cellular edges had a feathered appearance (Fig. 3B). This phenotype correlated with membrane localization because versions of CT694 (694-⌬243, ⌬42-694) that showed decreased abundance in membrane localization also mediated less overt alterations in HeLa morphology. In addition, HeLa cells transfected with 694-⌬40 -80 were comparable with the GFP control with respect to gross morphology (Fig. 3C).
Residues 40 -80 of CT694 were expressed as a GFP-fusion protein to further assess the ability of this segment to act as a membrane localization domain (Fig. 4A). Similar to full-length 694, the signal for GFPϩ40 -80 was detectible in the plasma membrane of transfected HeLa cells. Interestingly, the cellular morphology appeared comparable with the GFP-only control. Triton X-114 extraction was also performed on HeLa cells expressing GFP, 694-FL, 694-⌬40 -80, or GFPϩ40 -80 to confirm and quantify membrane localization of chimeric proteins. Immunoblot analyses confirmed that ectopically expressed CT694, like endogenous CT694, partitioned into both the detergent and aqueous fractions (Fig. 4B). Densitometry analysis of immunoblots revealed nearly equal distribution of protein (Fig. 4C). Partitioning of 694-⌬40 -80 appeared similar to the GFP-only control and was detected predominantly in the aqueous fraction. Conversely, GFPϩ40 -80 was detected most abundantly in the detergent phase, indicating that these residues were both necessary and sufficient for membrane localization of CT694.
Residues 40 -80 Constitute a Functional Membrane Localization Domain-The mechanism for membrane association was unclear given the lack of a predicted transmembrane domain in CT694. Interestingly, T3SS effector proteins in other systems have been shown to possess a functional MLD that is essential for these proteins to express respective anti-host functions. The best characterized effectors of this family include the Yersinia T3SS effector YopE and ExoS of P. aeruginosa (12). Both YopE and ExoS are targeted to the host cell plasma membrane upon translocation via a discrete MLD domain, and such localization is necessary to exert RhoGAP activity (12, 28 -30). Consistent with the divergent nature of amino acids among the MLD family of proteins, there was no significant similarity between CT694 and the MLD domains of either YopE or ExoS. Likewise, there is no significant overall sequence similarity between full-length CT694 and either YopE or ExoS. This is in contrast to direct comparisons of YopE and ExoS, which share significant N-terminal homology (12,31). However, all three domains are predicted to contain an ␣ helix of approximately 20 residues (residues 51-72 of ExoS and 54 -75 of YopE) (data not shown, 12). Because the YopE and ExoS MLD domains can be functionally exchanged (12), we tested whether replacing the MLD of ExoS with amino acids 40 -80 of CT694 would result in functional complementation.
Transient expression of GFP-ExoS in mammalian cells results in cell rounding, and mutation of the MLD is sufficient to abrogate this phenotype because maintained membrane localization is required for optimal RhoGAP activity of ExoS A, fractions were probed in immunoblot for CT694 as well as TarP and MOMP as bacterial aqueous and detergent controls, respectively, or GAPDH and Caveolin-1 as respective host cell aqueous and detergent controls. B, detergent phase and aqueous phase proteins from EBs were probed in immunoblot for CT694, TarP, or MOMP. FIGURE 3. CT694 truncation analyses. A, CT694 truncations were engineered and expressed either in HeLa cells as GFP fusion proteins to examine membrane localization or in S. cerevisiae for yeast-two hybrid (Y2H) screens to assess interaction with Ahnak. A "ϩ" for Ahnak-binding indicates growth on stringent selection medium and is indicative of interaction between Ahnak and the respective version of CT694. A "ϩ" for membrane localization indicates fluorescence microscopy detection of the recombinant protein in a pattern consistent with plasma membrane localization. B, representative images demonstrating aberrant cell morphology of HeLa cells expressing GFP-694-FL compared with cells expressing GFP empty vector. Insets represent digitally zoomed areas indicated by arrows. C, representative images for membrane localization and morphology changes for the indicated GFP-CT694 mutants. Scale bar ϭ 10 m. (14,28,32). We tested whether complementation of ExoS ⌬MLD with the CT694 MLD (ExoS/694MLD) would restore ExoS-mediated HeLa cell rounding. HeLa cells were transfected with 400 ng of the indicated DNA, and GFP or DAPI staining were subsequently used to assess subcellular localization and cell rounding (Fig. 5). As published previously, GFP-ExoS was detected in perinuclear concentrations, whereas GFP-ExoS ⌬MLD was localized diffusely in the cytosol (15). CT694 MLD was able to complement both the perinuclear localization and the rounded cell phenotype in cells expressing GFP-ExoS/694MLD (Fig. 5A). In addition, rounded cells were quantified for each construct, with GFP-ExoS/694MLD displaying a similar percentage of rounded cells compared with GFP-ExoS WT, whereas vector-only and GFP-ExoS ⌬MLD had significantly fewer rounded cells (Fig. 5B). These data are therefore consistent with residues 40 -80 containing a functional MLD that targets the chlamydial effector to the plasma membrane of infected host cells, similar to the MLD family of T3S effector proteins.

DISCUSSION
The data presented here support a model in which the C. trachomatis T3SS effector CT694 contains multiple functional domains. These domains include the previously described Ahnak binding domain (18), an undefined toxicity domain that manifests as altered cell morphology in transfected HeLa cells, and a membrane localization domain residing within amino acids 40 to 80. The CT694 MLD is necessary to target CT694 to host cell membranes. Our transfection studies show that this localization is independent of the Ahnak binding domain, and the MLD alone is sufficient to localize GFP to the plasma membrane. However, the MLD alone is not sufficient to induce host cell morphology changes, suggesting that although the Ahnak-binding activity is independent of localization, toxicity is not. In the absence of a tractable genetic system, we cannot directly establish the role of this domain during C. trachomatis infection. However, endogenous CT694 also localizes to cellular membranes as early as immediately post-infection and stays associated with membranes for up to 6 h post-infection. These data provide evidence that secreted CT694 does associate with host membranes and is consistent with our model in which CT694 plays a role in host cell invasion or establishment of a subcellular niche for C. trachomatis development. Immediate deployment of effectors concomitant with host cell contact is a common theme among bacteria expressing a T3SS. For example, Shigella spp., Salmonella typhimurium, Yersinia spp., and P. aeruginosa each express unique sets of T3SS effectors that are translocated upon host cell contact (33). In C. trachomatis, the immediate-early T3SS effectors that have thus far been identified are the effectors TarP (19) and CT694 (18) and the translocator proteins CopB and CopB2 (25, 34). Of the described T3SS effectors in other systems, several associate with host cell membranes after translocation. Shigella effector IpaC integrates into host cell membranes, where it is involved in inducing actin polymerization to mediate invasion (9). Yersinia spp. encode genes for at least two effectors that are targeted to host cell membranes after translocation. YpkA/YopO is targeted to the plasma membrane, whereas YopE localizes to the perinuclear region (12,35). Salmonella SptP (16) and Pseu-  (14) also have established roles that require association with host membranes. AexT and AexU of Aeromonas spp. likely represent additional family members on the basis of sequence similarity with the MLD of YopE (29). Although the MLD of YopE can functionally complement loss of the ExoS MLD (33), the precise nature of the membrane association remains unclear. Barbieri and colleagues (12) and Zhang and Barbieri (14) proposed that a leucine-rich motif arranged on one side of a predicted ␣ helix within the MLD of these proteins mediates membrane association. More hydrophilic residues within the domain are proposed to have a membrane-stabilizing role (28).
The data presented here demonstrate that CT694 may associate with host membranes in a similar manner as ExoS and YopE. CT694 is translocated through the T3SS upon host cell contact and is then targeted to the host cell membrane. This membrane localization is dependent on a discrete domain, similar to YopE and ExoS, that also lacks a predicted transmembrane structure (13). However, CT694 appears to lack an apparent leucine-rich domain. Therefore, we cannot rule out the possibility of distinct molecular mechanisms manifesting as stable interaction with host membranes. Interestingly, ectopically expressed ExoS also partitioned to the TritonX114 phase of fractionated HeLa cells similar to CT694 (data not shown). Coupled with our complementation data, we believe that, regardless of mechanism, the MLD of CT694 accomplishes a functional role similar to that of other MLD-containing effectors.
Although this class of membrane-localized effector proteins displays some similarities, the role each effector plays varies depending on the system. YopE and ExoS each contain GTPase activating protein (GAP) domains targeting Rho family GTPases to prevent host cell phagocytosis of bacteria (30,36). On the opposite end of the spectrum, the Shigella effector IpaC plays an important role in cytoskeleton rearrangement to promote bacterial invasion and uptake by host cells (37). SptP, a Salmonella T3SS effector, also localizes to host cell plasma membranes, where it is hypothesized to participate in repairing the host cell membrane after bacterial invasion through the activity of the SptP N-terminal GAP domain (11,16,38). On the basis of our collective observations, we believe that CT694 functions at the host cell membrane during C. trachomatis host cell invasion and early cycle development. Later functions have not been ruled out but would presumably not require membrane association because endogenous CT694 did not fractionate with the membrane during late-cycle development. In transfected cells, CT694 is able to disrupt actin stress fibers (18). Moreover, our domain analyses indicate an activity residing between the MLD and Ahnak binding domain that mediates altered cellular morphology in transfected HeLa cells. It is formally possible that CT694 expresses RhoGAP activity, yet the morphological phenotype induced by CT694 differs significantly from those induced by YopE (39), ExoS (40), and SptP (11). TarP-mediated arrangement of actin and recruitment to the invasion site is a necessary step to C. trachomatis invasion (9), and Rac1, but not RhoA or Cdc42, is recruited and activated during C. trachomatis invasion (41). We have been unable to detect Rac1-specific GAP activity for CT694 (data not shown) but cannot exclude the possibility of GAP activity for CT694 directed toward alternative host targets. Interestingly, Salmonella utilize a careful balance of effectors to promote actin nucleation through SopE GEF activity and revert and repair the host cell cytoskeleton and membrane through SptP GAP activity (42). It is possible that CT694 may have a similar general role during C. trachomatis infection and that one function of CT694 is to repair cytoskeletal insult because of TarP activity. We are currently pursuing work to address this intriguing possibility.