Sphingolipid Trafficking and Purification in Chlamydia trachomatis–Infected Cells

Abstract

Chlamydia trachomatis is an obligate intracellular human pathogen, which lacks a system that allows genetic manipulation. Therefore, chlamydial researchers must manipulate the host cell to better understand chlamydial biology. Host-derived lipid acquisition is critical for chlamydial survival within the host. Hence, the ability to track and purify sphingolipids in/from chlamydial infected cells has become an integral part of pivotal studies in chlamydial biology. This unit outlines protocols that provide details about labeling eukaryotic cells with exogenous lipids to examine Golgi-derived lipid trafficking to the chlamydial inclusion and then performing imaging studies or lipid extractions for quantification. Details are provided to allow these protocols to be applied to subconfluent, polarized, or siRNA knockdown cells. In addition, one will find important experimental design considerations and techniques. These methods are powerful tools to aid in the understanding of mechanisms, which allow C. trachomatis to manipulate and usurp host cell trafficking pathways.

Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

“…To visualize the retention of fluorescent lipid by the chlamydial inclusion, HeLa cells were reverse transfected with either NT, VAMP4, or syntaxin 6 siRNA and incubated for 48 h prior to infection with C. trachomatis serovar L2 (MOI of 2). Following 18 h of infection, the cells were labeled with 5 M fluorescent lipid 6-{[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}sphingosine (C 6 -NBD-ceramide [also called NBD-lipid here]) (Life Technologies) as described previously (21,39) (the C 6 -NBD-ceramide metabolites NBD-glucosylceramide and NBD-sphingomyelin are described in Results). Briefly, infected monolayers were incubated for 15 min at 12°C and then labeled with 5 M C 6 -NBD-ceramide in cold Eagle's modified essential medium (EMEM) plus 0.035% defatted BSA (Sigma) for 30 min at 12°C.…”

“…To determine the amount of NBD-sphingomyelin retained by the chlamydial organisms recovered from syntaxin 6 or VAMP4 siRNA-treated cells, EBs were isolated and the lipids were extracted (39). Briefly, cells were reverse transfected with siRNA and infected with C. trachomatis serovar L2 (MOI of 3) as described above.…”

“…After 24 h of infection, cells were labeled with 5 M C 6 -NBD-ceramide (Life Technologies), and unincorporated NBD-lipid was back-exchanged for 20 h as described above. The EBs were purified after 40 to 44 h of infection by using a Renografin (Mallinckrodt, Inc., St. Louis, MO) gradient as previously described (39). To control for lipid extraction efficiency, 0.06 g/ml of NBD-lactosylceramide (Matreya, Pleasant Gap, PA) was added to each sample and then lipids were extracted by a modified Bligh and Dyer chloroform-methanol extraction (24,39,40).…”

“…The EBs were purified after 40 to 44 h of infection by using a Renografin (Mallinckrodt, Inc., St. Louis, MO) gradient as previously described (39). To control for lipid extraction efficiency, 0.06 g/ml of NBD-lactosylceramide (Matreya, Pleasant Gap, PA) was added to each sample and then lipids were extracted by a modified Bligh and Dyer chloroform-methanol extraction (24,39,40). The lipids were resuspended in a 2:1 chloroform-methanol solution, spotted onto TLC plates precoated with 60Å-pore-size silica gel (Whatman/GE Healthcare, Piscataway, NJ), and resolved using a 40:11.5:1.5 chloroformmethanol-distilled water mixture.…”

“…Therefore, the absence of syntaxin 6 or VAMP4 would hinder chlamydial nutrient acquisition and, thus, development, resulting in a decrease of infectious progeny. A well-established technique to examine Golgi apparatus-derived lipid trafficking to the chlamydial inclusion utilizes the fluorescent lipid, C 6 -NBDceramide (21,39). C 6 -NBD-ceramide is a vital stain for the Golgi apparatus and, within the Golgi apparatus, is metabolized into two major metabolites: NBD-glucosylceramide and NBD-sphingomyelin (42)(43)(44).…”