Chlamydia trachomatis acquires C6‐NBD‐sphingomyelin endogenously synthesized from C6‐NBD‐ceramide and transported to the vesicle (inclusion) in which they multiply. Here we explore the mechanisms of this unusual trafficking and further characterize the association of the chlamydial inclusion with the Golgi apparatus. Endocytosed chlamydiae are trafficked to the Golgi region and begin to acquire sphingolipids from the host within a few hours following infection. The transport of NBD‐sphingolipid to the inclusion is energy‐ and temperature‐dependent with the characteristics of an active, vesicle‐mediated process. Photo‐oxidation of C5‐DMB‐ceramide, in the presence of diaminobenzidine, identified DMB‐lipids in vesicles in the process of fusing to the chlamydial inclusion membrane. C6‐NBD‐sphingomyelin incorporated into the plasma membrane is not trafficked to the inclusion to a significant degree, suggesting the pathway for sphingomyelin trafficking is direct from the Golgi apparatus to the chlamydial inclusion. Lectins and antibody probes for Golgi‐specific glycoproteins demonstrate the close association of the chlamydial inclusion with the Golgi apparatus but do not detect these markers in the inclusion membrane. Collectively, the data are consistent with a model in which C.trachomatis inhabits a unique vesicle which interrupts an exocytic pathway to intercept host sphingolipids in transit from the Golgi apparatus to the plasma membrane.

Chlamydia trachomatis undergoes its entire life cycle within an uncharacterized intracellular vesicle that does not fuse with lysosomes. We used a fluorescent Golgispecific probe, {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]}ami-nocaproylsphingosine (C6-NBD-Cer), in conjunction with conventional fluorescence or confocal microscopy to identify interactions between the Golgi apparatus and the chlamydial inclusion. We observed not only a close physical association between the Golgi apparatus and the chlamydial inclusion but the eventual presence of a metabolite of this fluorescent probe associated with the chlamydiae themselves. Sphingomyelin, endogenously synthesized from C6-NBD-Cer, was specifically transported to the inclusion and incorporated into the cell wall of the intracellular chlamydiae. Incorporation of the fluorescent sphingolipid by chlamydiae was inhibited by brefeldin A. Chlamydiae therefore occupy a vesicle distal to the Golgi apparatus that receives anterograde vesicular traffic from the Golgi normally bound for the plasma membrane. Collectively, the data suggest that the chlamydial inclusion may represent a unique compartment within the trans-Golgi network Chlamydia trachomatis is the causative agent of several significant human diseases including trachoma, the leading cause of infectious blindness worldwide, and is the most common cause of sexually transmitted disease in the United States and in developed countries (1). Chlamydiae are obligate intracellular bacteria with a biphasic life cycle characterized by functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication. This developmental cycle takes place entirely within an intracellular vesicle (inclusion) that is not believed to be acidified and does not fuse with lysosomes (2). Infection is initiated by a small, metabolically dormant cell type called the elementary body (EB). After endocytosis, an EB differentiates into a larger, pleomorphic, and metabolically active cell type called the reticulate body. The reticulate bodies divide by binary fission throughout the remainder of the infection until the cell lyses at 40-44 hr after infection. However, at -18 hr after infection the developmental cycle becomes asynchronous, as increasing numbers of reticulate bodies differentiate back to EBs that accumulate within the inclusion until cell lysis occurs. The environmental signals that regulate this developmental cycle are unknown. There are many fundamental questions regarding the nature of the chlamydial inclusion-including its composition, permeability properties, biosynthetic origin, and lumenal contents. The chlamydial inclusion is isolated from established routes of intracellular trafficking; with the exception of vacuoles containing other chlamydiae, no cellular vesicles are known to fuse with the chlamydial inclusion (3). Although chlamydiae obviously acquire essential nutrients from the host cell, the mech-The publication costs of this article were defrayed in part by page charg...

The obligate intracellular bacterium Chlamydia trachomatis has a unique developmental cycle that involves functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication. Infection is initiated by an environmentally resistant cell type called an elementary body (EB). Over the first several hours of infection, EBs differentiate into a larger replicative form, termed the reticulate body (RB). Late in the infectious process, RBs asynchronously begin to differentiate back to EBs, which accumulate within the lumen of the inclusion until released from the host cell for subsequent rounds of infection. In an effort to characterize temporal gene expression in relation to the chlamydial developmental cycle, we have used quantitative–competitive polymerase chain reaction (QC‐PCR) and reverse transcription (RT)‐PCR techniques. These analyses demonstrate that C. trachomatis double their DNA content every 2–3 h, with synthesis beginning between 2 and 4 h after infection. We determined the onset of transcription of specific temporal classes of developmentally expressed genes. RT‐PCR analysis was performed on several genes encoding key enzymes or components of essential biochemical pathways and functions. This comparison encompassed approximately 8% of open reading frames on the C. trachomatis genome. In analysis of total RNA samples harvested at 2, 6, 12 and 20 h after infection, using conditions under which a single chlamydial transcript per infected cell is detected, three major temporal classes of gene expression were resolved. Initiation of transcription appears to occur in three temporal classes which we have operationally defined as: early, which are detected by 2 h after infection during the germination of EBs to RBs; mid‐cycle, which appear between 6 and 12 h after infection and represent transcripts expressed during the growth and multiplication of RBs; or late, which appear between 12 and 20 h after infection and represent those genes transcribed during the terminal differentiation of RBs to EBs. Collectively, the data suggest that chlamydial early gene functions are weighted toward initiation of macromolecular synthesis and the establishment of their intracellular niche by modification of the inclusion membrane. Surprisingly, representative enzymes of intermediary metabolism and structural proteins do not appear to be transcribed until 10–12 h after infection; coinciding with the onset of observed binary fission of RBs. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs back to EBs.

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the periGolgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFPRab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFPRab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.Chlamydiae are major bacterial pathogens of ocular, urogenital, and pulmonary mucosal surfaces (51). Infections caused by Chlamydia trachomatis are the leading cause of bacterially acquired sexually transmitted disease (10), as well as of preventable blindness worldwide (64). In addition, Chlamydia pneumoniae infections are major causes of upper respiratory tract infections and have recently been linked to chronic heart disease (24, 25). Chlamydiae are obligate intracellular bacteria that replicate within a nonacidified vacuole termed an inclusion (26). Within the inclusion, chlamydiae undergo a biphasic developmental cycle that alternates between the infectious metabolically inactive elementary body (EB) and the noninfectious metabolically active reticulate body (40). Although chlamydiae enter nonprofessional phagocytes by multiple mechanisms (reviewed in reference 26), once the chlamydiae are internalized, they actively modify the properties of the nascent vacuole during the first 2 h postinfection, resulting in trafficking of the inclusion to the peri-Golgi region, fusion of the inclusion with a subset of Golgi-derived exocytic vesicles, and avoi...