Hackstadt, T., Scidmore, M. A. & Rockey, D. D. Lipid metabolism in Chlamydia trachomatis-infected cells: Directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc. Natl Acad. Sci. USA 92, 4877-4881

Host-Parasite Interactions Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/1995; 92(11):4877-81. DOI: 10.1073/pnas.92.11.4877
Source: PubMed


Chlamydia trachomatis undergoes its entire life cycle within an uncharacterized intracellular vesicle that does not fuse with lysosomes. We used a fluorescent Golgi-specific probe, (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]) aminocaproylsphingosine (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.

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Available from: Marci A Scidmore, May 21, 2014
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    • "Chlamydia trachomatis is an obligate intracellular Gram-negative bacterium infecting human ocular and genital epithelial cells, whose dissemination into the female upper genital can cause pelvic inflammatory disease and infertility [reviewed in Chavez et al., 2011]. Similar to other Chlamydia spp., C. trachomatis forms parasitophorous vacuoles ( " chlamydial inclusions " ) in infected cells, and needs to acquire host lipids from post-Golgi vesicles to support inclusion growth and bacterial replication (Hackstadt et al., 1995; Boleti et al., 2000; Wolf and Hackstadt, 2001; Heuer et al., 2009; Chen et al., 2012). Exploiting the requirement of Chlamydia to acquire lipids to replicate, a prior study utilized a 2–3 h in vitro incubation with fluorescent, Golgi-specific lipid to subsequently distinguish between uninfected and Chlamydia-infected McCoy cells by flow cytometry (Alzhanov et al., 2007). "
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    ABSTRACT: Chlamydia replication requires host lipid acquisition, allowing flow cytometry to identify C. trachomatis-infected cells that accumulated fluorescent Golgi-specific lipid. Herein, we describe modifications to currently available methods that allow precise differentiation between uninfected and C. trachomatis-infected human endometrial cells and rapidly and accurately quantify chlamydial inclusion forming units.
    Journal of microbiological methods 10/2015; 119. DOI:10.1016/j.mimet.2015.10.003 · 2.03 Impact Factor
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    • "The growth of C. trachomatis is dependent upon its ability to acquire lipids from the host (Hackstadt et al., 1995, Wylie et al., 1997, Carabeo et al., 2003, Beatty, 2006, Derre et al., 2011, Elwell et al., 2011), and multiple redundant pathways contribute to the acquisition of glycosphingolipids (Derre et al., 2011, Elwell et al., 2011) and cholesterol (Carabeo et al., 2003). We have focused on PC because it cannot be made by C. trachomatis and it is one of the most abundant phospholipids associated with isolated bacteria (Wylie et al., 1997, Stephens et al., 1998). "
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    ABSTRACT: Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analog to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labeled fluorescent phosphatidylcholine analog and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.
    Cellular Microbiology 09/2015; DOI:10.1111/cmi.12523 · 4.92 Impact Factor
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    • "The cells were then incubated for an additional 12 h. This step allows for the transfer of the fluorescent dye from the Golgi apparatus to the Chlamydia within the infected cells, while allowing for the efflux of the dye from the uninfected cells through exocytosis (Alzhanov et al., 2007; Hackstadt et al., 1995; Boleti et al., 2000). Before microdissection, the Chlamydia-infected cells were washed two times in 2 ml of HBSS. "
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    ABSTRACT: Chlamydia are obligate intracellular parasites of humans and animals that cause a wide range of acute and chronic infections. To elucidate the genetic basis of chlamydial parasitism, several approaches for making genetic modifications to Chlamydia have recently been reported. However, the lack of the available methods for the fast and effective selection of genetic modified bacteria restricts the application of genetic tools. We suggest the use of laser microdissection to isolate of single live Chlamydia-infected cells for the re-cultivation and whole-genome sequencing of single inclusion-derived Chlamydia. To visualise individual infected cells, we made use of the vital labelling of inclusions with the fluorescent Golgi-specific dye BODIPY® FL C5-ceramide. We demonstrated that single Chlamydia-infected cells isolated by laser microdissection and placed onto a host cell monolayer resulted in new cycles of infection. We also demonstrated the successful use of whole-genome sequencing to study the genomic variability of Chlamydia derived from a single inclusion. Our work provides the first evidence of the successful use of laser microdissection for the isolation of single live Chlamydia-infected cells, thus demonstrating that this method can help overcome the barriers to the fast and effective selection of Chlamydia. Copyright © 2014. Published by Elsevier B.V.
    Journal of Microbiological Methods 12/2014; 109. DOI:10.1016/j.mimet.2014.12.018 · 2.03 Impact Factor
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