Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

Duke University, United States of America
PLoS Pathogens (Impact Factor: 7.56). 09/2011; 7(9):e1002198. DOI: 10.1371/journal.ppat.1002198
Source: PubMed


The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.

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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
    • "C. trachomatis growing and replication heavily rely upon the acquisition of certain lipids, mainly sphingolipids and cholesterol, from eukaryotic host cells (Saka and Valdivia, 2010). Interestingly, C. trachomatis acquire lipids through non-vesicular and vesicular mechanisms: i) by recruiting transporters and synthases from host cytosolic sphingolipid factories at the boundaries of inclusions (Derré et al., 2011; Elwell et al., 2011) and ii) from Golgi-derived exocytic vesicles and multivesicular bodies (MVBs) (Beatty, 2006; Cocchiaro et al., 2008; Hackstadt et al., 1996; Moore et al., 2008). "
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    ABSTRACT: Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure their access to multiple host sources of essential lipids by interfering vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi apparatus. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating MVBs-inclusion interaction remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles -mainly MVBs- that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process and depending on its GTP/GDP binding state. Interestingly, Rab39a participates in the delivery of MVB and host sphingolipids to maturing chlamydial inclusions thereby promoting inclusion growth and bacterial development. Altogether, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing a late endocytic Rab GTPase involved in chlamydial infection development. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 07/2015; 128(16). DOI:10.1242/jcs.170092 · 5.43 Impact Factor
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    • "Sphingomyelin (SM) is at least in part acquired by C. trachomatis in a post-GA-vesicular trafficking pathway [11] although some SM can also be transported through direct transfer from the ER [25], [26]. When fluorescent ceramide is added to infected cells, SM-transport to first the inclusion membrane and then into the bacterial membranes can be visualized. "
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    ABSTRACT: Chlamydia grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how Chlamydia organizes this transport. One model posits that the Chlamydia-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with Chlamydia trachomatis to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to C. trachomatis still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.
    PLoS ONE 07/2014; 9(7):e103220. DOI:10.1371/journal.pone.0103220 · 3.23 Impact Factor
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