The obligate intracellular bacterium Chlamydia trachomatis rapidly induces its own entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the host cell, followed by irreversible binding to an unknown secondary receptor. This secondary binding leads to the recruitment of actin to the site of attachment, formation of an actin-rich, pedestallike structure, and finally internalization of the bacteria. How chlamydiae induce this process is unknown. We have identified a high-molecular-mass tyrosine-phosphorylated protein that is rapidly phosphorylated on attachment to the host cell. Immunoelectron microscopy studies revealed that this tyrosine-phosphorylated protein is localized to the cytoplasmic face of the plasma membrane at the site of attachment of surface-associated chlamydiae. The phosphoprotein was isolated by immunoprecipitation with the antiphosphotyrosine antibody 4G10 and identified as the chlamydial protein CT456, a hypothetical protein with unknown function. The chlamydial protein (Tarp) appears to be translocated into the host cell by type III secretion because it is exported in a Yersinia heterologous expression assay. Phosphotyrosine signaling across the plasma membrane preceded the recruitment of actin to the site of chlamydial attachment and may represent the initial signal transduced from pathogen to the host cell. These results suggest that C. trachomatis internalization is mediated by a chlamydial type III-secreted effector protein. Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that is a leading cause of sexually transmitted diseases and blindness worldwide (1). Chlamydiae have a biphasic developmental cycle characterized by an infectious but metabolically inactive extracellular form, called the elementary body (EB), that initiates infection by attaching to and inducing uptake by the host cell. Once internalized, chlamydiae remain within a membrane-bound vacuole termed an inclusion, where the organism differentiates into the larger, metabolically active reticulate body. Reticulate bodies replicate and differentiate back to EBs before release at the end of the developmental cycle.EBs attach to and enter cultured eukaryotic cells so efficiently that the process has been termed parasite-specified phagocytosis (2). Despite the importance of this event to chlamydial pathogenesis, little consensus exists regarding the identity of the chlamydial ligands and respective host receptors (for a review, see ref.3). Considerable evidence suggests that electrostatic interactions mediate attachment with heparan sulfate-like proteoglycans involved in an initial, reversible interaction with the eukaryotic host cell for many, but not all, strains and species of chlamydiae (4 -9). Recent studies using chemically mutagenized cell lines distinguished a subsequent, irreversible secondary binding step in the entry process, although the receptor was not identified (8, 10). Entry of C. trachomatis requires participation of the actin cy...

did not display such microvillar hypertrophy following exposure to L2 EBs, which is in contrast to infection with serovar D, to which it is susceptible. We propose that C. trachomatis entry is facilitated by an active actin remodeling process that is induced by the attachment of this pathogen, resulting in distinct microvillar reorganization throughout the cell surface and the formation of a pedestal-like structure at the immediate site of attachment and entry.Chlamydia trachomatis is a gram-negative bacterium that absolutely requires an intracellular niche for its replication (42-44). Because of their obligate intracellular nature, chlamydiae have evolved very efficient means of entering host eukaryotic cells, a process which has been described as parasite-directed entry (11,12). Chlamydiae have a biphasic developmental cycle consisting of infectious and replicative forms. Infection of eukaryotic host cells is initiated by elementary bodies (EBs). EBs can superficially be considered spore-like, in that they are metabolically inactive and relatively stable in the extracellular environment so as to promote their survival for sufficient time to encounter a susceptible host cell. Through largely unknown mechanisms, EBs attach to and induce their internalization by host cells. Once internalized, EBs transform into a larger and more pleomorphic form called the reticulate body (RB) within the first few hours postinfection. RBs are metabolically active, and they replicate; however, they are noninfectious. Eighteen hours following infection with C. trachomatis L2, increasing proportions of the dividing RBs revert to EBs until the cell lyses at 40 to 44 h postinfection. Non-lymphogranuloma venereum (LGV) strains (serovars A to K) typically have a somewhat longer developmental cycle.The precise molecular mechanisms of chlamydial attachment and entry have not been defined. However, chlamydiae, like other pathogens such as Toxoplasma gondii (15) and varicella-zoster virus (62), attach to host cells via a relatively weak and reversible electrostatic interaction with heparan sulfate proteoglycans (53, 59) and a stronger, more specific binding to an as yet unknown secondary receptor (14). Once attached, a majority of the EBs are internalized.Actin is a critical component of receptor-mediated endocytosis and phagocytosis in a variety of cell types (3, 31, 47), and a number of studies have demonstrated that the cytoskeleton can be manipulated by microbial pathogens to facilitate productive infection (6,19,20,24). For example, enteropathogenic Escherichia coli has the ability to aggregate actin to form its pedestal structures, a trademark of attaching and effacing lesions (4, 26), and Salmonella induces membrane ruffles for internalization (24,27,30).The role of actin-dependent mechanisms in chlamydial internalization has long been debated. Early studies using cytochalasin B as an inhibitor of microfilament function found no effect on chlamydial internalization (32, 51). Subsequent studies using the more efficient agent cytochala...

Chlamydiae are pathogenic obligate intracellular bacteria with a biphasic developmental cycle that involves cell types adapted for extracellular survival (elementary bodies, EBs) and intracellular multiplication (reticulate bodies, RBs). The intracellular development of chlamydiae occurs entirely within a membrane-bound vacuole termed an inclusion. Within 2 hours after entry into host cells, Chlamydia trachomatis EBs are trafficked to the perinuclear region of the host cell and remain in close proximity to the Golgi apparatus, where they begin to fuse with a subset of host vesicles containing sphingomyelin. Here, we provide evidence that chlamydial migration from the cell periphery to the peri-Golgi region resembles host cell vesicular trafficking. Chlamydiae move towards the minus end of microtubules and aggregate at the microtubule-organizing center (MTOC). In mammalian cells the most important minus-end-directed microtubule motor is cytoplasmic dynein. Microinjection of antibodies to a subunit of cytoplasmic dynein inhibited movement of chlamydiae to the MTOC, whereas microinjection of antibodies to the plus-directed microtubule motor, kinesin, had no effect. Surprisingly, overexpression of the protein p50 dynamitin, a subunit of the dynactin complex that links vesicular cargo to the dynein motor in minus directed vesicle trafficking, did not abrogate chlamydial migration even though host vesicle transport was inhibited. Nascent chlamydial inclusions did, however, colocalize with the p150(Glued) dynactin subunit, which suggests that p150(Glued) may be required for dynein activation or processivity but that the cargo-binding activity of dynactin, supplied by p50 dynamitin subunits and possibly other subunits, is not. Because chlamydial transcription and translation were required for this intracellular trafficking, chlamydial proteins modifying the cytoplasmic face of the inclusion membrane are probable candidates for proteins fulfilling this function.

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Chlamydia trachomatis attachment to cells induces the secretion of the elementary body–associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C. trachomatis invades non-phagocytic cells.