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...

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H ؉) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A 1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid-phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C 6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C 6-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.

Many strains of Chlamydia suis, a pathogen of pigs, express a stable tetracycline resistance phenotype. We demonstrate that this resistance pattern is associated with a resistance gene, tet(C), in the chlamydial chromosome. Four related genomic islands were identified in seven tetracycline-resistant C. suis strains. All resistant isolates carry the structural gene tet(C) and the tetracycline repressor gene tetR(C). The islands share significant nucleotide sequence identity with resistance plasmids carried by a variety of different bacterial species. Three of the four tet(C) islands also carry a novel insertion sequence that is homologous to the IS605 family of insertion sequences. In each strain, the resistance gene and associated sequences are recombined into an identical position in a gene homologous to the inv gene of the yersiniae. These genomic islands represent the first examples of horizontally acquired DNA integrated into a natural isolate of chlamydiae or within any other obligate intracellular bacterium.