Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane.

Abstract

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.

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

“…Routine propagation was carried out in HeLa 229 epithelial cells (CCL 1.2; American Type Culture Collection, Manassas, VA) maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 10 g of gentamicin ml Ϫ1 (Mediatech, Herndon, VA). Cultures were incubated at 37°C in an atmosphere of 5% CO 2 and 95% humidified air, and infections were carried out as previously described (4,22,65) in Hanks balanced salt solution (HBSS; Invitrogen, Carlsbad, CA), for 1 h at 37°C. When appropriate, EBs were purified by centrifugation through MD-76R (Mallinckrodt, Inc., St. Louis, MO) density gradients as described previously (4).…”

Rottlerin Inhibits Chlamydial Intracellular Growth and Blocks Chlamydial Acquisition of Sphingolipids from Host Cells

“…It is known that Chlamydia has to import lipids from host cells to maintain its growth (3,5,15,25). For example, the chlamydial acquisition of sphingolipids from host cells was elegantly demonstrated by monitoring the trafficking of sphingomyelin synthesized from fluorochrome-labeled ceramide in the Golgi apparatus into chlamydial inclusions (14,15).…”

“…This is probably due to the rottlerin-mediated inhibition of the shared requirement of all chlamydiae to acquire lipids from host cells to maintain chlamydial growth. The rottlerin antimicrobial effect may also be very specific to the genus Chlamydia since chlamydiae seem to be the only type of microbial pathogens that are known to acquire host lipids by intercepting the Golgi apparatus lipid exocytic pathway (14). Finally, rottlerin can selectively block sphingolipid trafficking from the Golgi apparatus to chlamydial inclusions without affecting ceramide trafficking to and sphingomyelin synthesis in the Golgi apparatus, as shown by the normal accumulation of fluorochromelabeled sphingomyelin in the Golgi apparatus of rottlerintreated cells regardless of infection.…”

“…in rottlerin-treated cells. This is because inhibition of ceramide trafficking into inclusions by BFA is not sufficient to block production of chlamydial infectious progeny (14). Rottlerin may alter multiple host pathways required for intracellular chlamydial growth.…”

Bacterial subversion of the host secretory pathway

“…To import nutrients and expand this vacuole, it is becoming increasingly clear that vacuoles must retain the ability to receive membrane and cargo from vesicles traveling through the host cell, which sometimes involves intercepting host secretory vesicles. For example, maintenance of vacuoles containing Chlamydia trachomatis has been shown to involve transport of lipids from the trans-Golgi network (6). Recent data show that Salmonella enterica has the ability to interact with the Golgi apparatus and that this interaction is important for intracellular replication (7).…”