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...

Cholesterol, a lipid not normally found in prokaryotes, was identified in purified Chlamydia trachomatis elementary bodies and in the chlamydial parasitophorous vacuole (inclusion) membrane of infected HeLa cells. Chlamydiae obtained eukaryotic host cell cholesterol both from de novo synthesis or low-density lipoprotein. Acquisition of either de novo-synthesized cholesterol or low-density lipoprotein-derived cholesterol was microtubule-dependent and brefeldin A-sensitive, indicating a requirement for the Golgi apparatus. Transport also required chlamydial protein synthesis, indicative of a pathogen-directed process. The cholesterol trafficking pathway appears to coincide with a previously characterized delivery of sphingomyelin to the inclusion in that similar pharmacological treatments inhibited transport of both sphingomyelin and cholesterol. These results support the hypothesis that sphingomyelin and cholesterol may be cotransported via a Golgidependent pathway and that the chlamydial inclusion receives cholesterol preferentially from a brefeldin A-sensitive pathway of cholesterol trafficking from the Golgi apparatus to the plasma membrane.C hlamydia trachomatis is the etiologic agent of the most common form of sexually transmitted disease in the United States and preventable blindness worldwide (1). Chlamydiae are obligate intracellular bacteria with a biphasic life cycle consisting of an extracellular infectious form and an intracellular replicative form. Infection is initiated by the metabolically inert elementary body (EB), which develops into the noninfectious but metabolically active reticulate body (RB). RBs differentiate back to EBs before release from the infected cell (2). Chlamydiae replicate within a specialized parasitophorous vacuole, termed an inclusion, that is neither acidified nor fusogenic with lysosomes (3, 4). Rather than interacting with the endocytic pathway, the chlamydial inclusion is fusogenic with exocytic vesicles containing sphingomyelin en route from the Golgi apparatus to the plasma membrane (5, 6). Acquisition of sphingomyelin requires chlamydial de novo transcription and translation of proteins that are thought to modify the inclusion membrane (7).Because sphingomyelin is delivered to the chlamydial inclusion and EBs contain both cholesterol and sphingomyelin (5, 8, 9), we asked whether cholesterol may be similarly regulated and transported to the inclusion. Cholesterol is an essential component of eukaryotic membranes and is enriched in the plasma membrane. Although synthesis occurs in the endoplasmic reticulum (ER), the role of the Golgi apparatus in the transport of cholesterol to the plasma membrane has been controversial. Dissection of intracellular cholesterol transport is complicated by the fact that there are a number of possible sources of this lipid. Eukaryotic cells acquire cholesterol from either de novo synthesis or from the extracellular media via the low-density lipoprotein (LDL) pathway (10-12). We find that C. trachomatis has the ability to obtain host cholest...

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...

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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.

Chlamydiae are gram‐negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.

Chlamydiae are obligate intracellular pathogens that are sensitive to pro-inflammatory cytokine interferon-γ. IFN-γ-inducible murine p47 GTPases have been demonstrated to function in resistance to chlamydia infection in vivo and in vitro. Because the human genome does not encode IFN-γ-inducible homologues of these proteins, the significance of the p47 GTPase findings to chlamydia pathogenesis in humans is unclear. Here we report a pair of IFN-γ-inducible proteins, the human guanylate binding proteins (hGBPs) 1 and 2 that potentiate the anti-chlamydial properties of IFN-γ. hGBP1 and 2 localize to the inclusion membrane, and their anti-chlamydial functions required the GTPase domain. Alone, hGBP1 or 2 have mild, but statistically significant and reproducible negative effects on the growth of Chlamydia trachomatis, whilst having potent anti-chlamydial activity in conjunction with treatment with a sub-inhibitory concentration of IFN-γ. Thus, hGBPs appear to potentiate the anti-chlamydial effects of IFN-γ. Indeed, depletion of hGBP1 and 2 in cells treated with IFN-γ led to an increase in inclusion size, indicative of better growth. Interestingly, chlamydia species/strains harboring the full-length version of the putative cytotoxin gene, which has been suggested to confer resistance to IFN-γ was not affected by hGBP overexpression. These findings identify the guanylate binding proteins as potentiators of IFN-γ inhibition of C. trachomatis growth, and may be the targets of the chlamydial cytotoxin.

SummaryChlamydiae are Gram-negative obligate intracellular pathogens to which access to an intracellular environment is fundamental to their development.