Sphingolipid Trafficking and Purification in Chlamydia trachomatis–Infected Cells

Elizabeth R. Moore1

1 Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 11A.2
DOI:  10.1002/9780471729259.mc11a02s27
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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. Curr. Protoc. Microbiol. 27:11A.2.1‐11A.2.19. © 2012 by John Wiley & Sons, Inc.

Keywords: Chlamydia trachomatis; sphingomyelin; lipid extraction

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Labeling Cells with C6‐NBD‐Ceramide to Examine Trafficking of Golgi‐Derived Sphingolipids to the Chlamydial Inclusion
  • Alternate Protocol 1: Labeling Cells with 14C‐Ceramide
  • Support Protocol 1: Seeding of Nonpolarized Eukaryotic Cells
  • Support Protocol 2: Polarization of Epithelial Cells
  • Support Protocol 3: Reverse Transfection of siRNA Prior to Labeling Cells with Fluorescent Lipid
  • Support Protocol 4: Small‐Scale Chlamydial Purification for Lipid Extraction
  • Basic Protocol 2: Live‐Cell Imaging of Fluorescent (NBD‐) Lipids
  • Basic Protocol 3: Lipid Extraction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Labeling Cells with C6‐NBD‐Ceramide to Examine Trafficking of Golgi‐Derived Sphingolipids to the Chlamydial Inclusion

  Materials
  • Seeded, infected tissue culture cells (see Support Protocols protocol 31, protocol 42, or protocol 53)
  • 5 mM C 6‐NBD‐ceramide (see recipe)
  • EMEM supplemented with 0.035% dfBSA (see recipe)
  • Ice
  • EMEM supplemented with 0.7% defatted‐BSA (dfBSA; see recipe)
  • Eagle's minimum essential medium (EMEM; ATCC #30‐2003)
  • Low‐speed refrigerated benchtop centrifuge with tissue culture plate adaptors
  • Vortex mixer

Alternate Protocol 1: Labeling Cells with 14C‐Ceramide

  Materials
  • N‐1‐[14C]oleoy‐D‐sphingosine (50‐60 mCi/molar)
  • Normal growth medium supplemented with lipid‐free (defatted) fetal bovine serum albumin (dfFBS)
  • Seeded, infected tissue culture cells in two to four 150‐cm2 flasks/condition (see Support Protocols protocol 31, protocol 42, or protocol 53)
  • Seeded, uninfected tissue culture cells in two to four 150‐cm2 flasks for control

Support Protocol 1: Seeding of Nonpolarized Eukaryotic Cells

  Materials
  • Trypsinized eukaryotic cells (see Table 11.2.1)
  • Complete tissue culture medium (see Table 11.2.1)
  • 0.4% trypan blue solution or erythrocin B stain
  • Autoclaved glass coverslips, circles, 12‐mm diameter, 0.12‐mm thickness (required for live‐cell imaging only)
  • 0.5‐ml microcentrifuge tubes (does not have to be sterile)
  • Hemacytometer (Bright‐Line, Hausser Scientific)
  • Light microscope with a 20× objective
  • Tissue culture plate/dish (consult Table 11.2.1)

Support Protocol 2: Polarization of Epithelial Cells

  Materials
  • Tissue culture inserts coated with fibrillar collagen type I and pore size 0.4‐µm (BD Biosciences; see Table 11.2.1 for size)
  • Complete tissue culture medium (see Table 11.2.1)
  • Trypsinized eukaryotic cells (see Table 11.2.1)
  • 0.4% trypan blue solution or erythrocin B stain
  • Intestinal differentiation media pack (BD Biosciences) containing:
    • Basal seeding medium
    • Stimulating medium
  • 0.5‐ml microcentrifuge tubes (these do not have to be sterile)
  • Hemacytometer (Bright‐Line, Hausser Scientific)
  • Light microscope with a 20× objective
  • EVOM ohm meter (World Precision Instruments)
  • STX2, EVOM chopstick electrodes (World Precision Instruments)

Support Protocol 3: Reverse Transfection of siRNA Prior to Labeling Cells with Fluorescent Lipid

  Materials
  • Opti‐Mem + GlutaMAX (Life Technologies)
  • Experimental and control siRNA at 20µM stock concentration (Ambion)
  • Lipofectamine RNAiMAX (Life Technologies)
  • Trypsinized eukaryotic cells (see Table 11.2.1)
  • Antibiotic‐free, complete tissue culture medium (see Table 11.2.1)
  • Rocker or orbital shaker
  • Tissue culture plate/dish (see Table 11.2.1)

Support Protocol 4: Small‐Scale Chlamydial Purification for Lipid Extraction

  Materials
  • Seeded, labeled cells (refer to Table 11.2.1)
  • Hanks' balanced salt solution (HBSS)
  • Ice
  • SPG (see unit 11.1)
  • 30%, 40%, 44%, 54% Renografin
  • Large cell scraper (38 cm)
  • Polycarbonate centrifuge bottles with caps, 29 × 104 mm (Beckman Coulter, cat. no. 361693)
  • Sonicator
  • Avanti J‐30I high‐speed centrifuge (Beckman Coulter) or equivalent, see notes in protocol
  • JA‐25.50 fixed‐angle rotor (Beckman Coulter) or equivalent, see notes in protocol
  • 5‐ to 10‐ml syringes
  • Ultra‐clear centrifuge tubes, 14 × 89 mm tubes (Beckman Coulter, cat. no. 344059)
  • JS‐24.15 swinging buckets or equivalent, see notes in protocol
  • Harvard trip balance
  • JS‐24 rotor or equivalent, see notes in protocol
  • 4‐in. cannula

Basic Protocol 2: Live‐Cell Imaging of Fluorescent (NBD‐) Lipids

  Materials
  • Cells seeded on coverslips or in 6.5‐mm inserts (see Table 11.2.1) and labeled with C 6‐NBD‐ceramide (see protocol 1)
  • HCMF (see recipe)
  • Biosafety cabinet
  • 18‐G needle, 1.5‐in. long
  • Curved forceps, serrated, 4.5 in.
  • Kimwipes
  • Glass slides
  • Fluorescent microscope with 60× oil objective
  • Scalpel

Basic Protocol 3: Lipid Extraction

  Materials
  • Chloroform, chromatography grade
  • Methanol, chromatography grade
  • dH 2O, greater than 15 mΩ‐cm resistivity, autoclaved
  • Nitrogen
  • Standards (e.g., NBD‐sphingomyelin, Matreya)
  • 4‐, 12‐, ad 20‐ml chemically resistant glass vials with lids
  • Fume hood
  • Glass serological pipets
  • Vortex mixer with large/ampule tube attachment (optional)
  • Glass Pasteur pipets and bulb
  • Thermo Reacti‐Therm and Reacti‐Vap manifold (this is an apparatus that gently heats the glass vials and allows nitrogen gas to dry the lipid extracts)
  • Glass scoring pencil with tungsten carbonide tip
  • Ruler
  • Thin‐layer chromatography (TLC) plates, silica Gel 60 Å, 20 × 20 cm
  • Pencil
  • 65°C oven
  • Hamilton syringe
  • Drummond microcaps
  • TLC developing chamber (many of these chambers exist, a “latch lid” model, such as provided by Sigma or Camag will limit the evaporation of solvents used to develop TLC plates)
  • UV light source
  • −20°C freezer
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   AbdelRahman, Y.M. and Belland, R.J. 2005. The chlamydial developmental cycle. FEMS Microbiol. Rev. 29:949‐959.
   Bacallao, R., Antony, C., Dotti, C., Karsenti, E., Stelzer, E.H.K., and Simons, K. 1989. The subcellular organization of madin‐darby canine kidney cells during the formation of a polarized epithelium. J. Cell Biol. 109:2817‐2832.
   Beatty, W.L. 2006. Trafficking from CD63‐positive late endocytic multivesicular bodies is essential for intracellular development of Chlamydia trachomatis. J. Cell Sci. 119:350‐359.
   Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911‐917.
   Caldwell, H.D., Kromhout, J., and Schachter, J. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31:1161‐1176.
   Capmany, A. and Damiani, M.T. 2010. Chlamydia trachomatis intercepts Golgi‐derived sphingolipids through a rab 14‐mediated transport required for bacterial development and replication. PLoS One 5:e14084.
   Carabeo, R.A., Mead, D.J., and Hackstadt, T. 2003. Golgi‐dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc. Natl. Acad. Sci. U.S.A. 100:6771‐6776.
   Derre, I., Swiss, R., and Agaisse, H. 2011. The lipid transfer protein CERT interacts with the Chlamydia inclusion protein IncD and participates to ER‐Chlamydia inclusion membrane contact sites. PLoS Pathogens 7:e1002092.
   Elwell, C.A., Jiang, S., Kim, J.H., Lee, A., Wittmann, T., Hanada, K., Melancon, P., and Engel, J.N. 2011. Chlamydia trachomatis co‐opts GBF‐1 and CERT to acquire host sphingomyelin for distinct roles during intracellular development. PLoS Pathogens 7:e1002198.
   Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. 1987. Lipofection: a highly efficient, lipid‐mediated DNA‐transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:7413‐7417.
   Fried, B. and Sherma, J. 1999. Thin Layer Chromatography, Revised and Expanded 4th ed. Marcel Dekker, New York.
   Hackstadt, T., Scidmore, M.A., and Rockey, D.D. 1995. Lipid metabolism in Chlamydia trachomatis‐infected cells: Directed trafficking of Golgi‐derived sphingolipids to the chlamyidal inclusion. Proc. Natl. Acad. Sci. U.S.A. 92:4877‐4881.
   Hackstadt, T., Rockey, D.D., Heinzen, R.A., and Scidmore, M.A. 1996. Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. EMBO J. 15:964‐977.
   Hatch, G.M. and McClarty, G. 1998. Phospholipid composition of purified Chlamydia trachomatis mimics that of the eukaryotic host cell. Infect. Immun. 66:3727‐3735.
   Heuer, D., Lipinski, A.R., Machuy, N., Karlas, A., Wehrens, A., Siedler, F., Brinkmann, V., and Meyer, T.F. 2009. Chlamydia causes fragmentation of the Golgi compartment to ensure reproduction. Nature 457:731‐735.
   Lipinski, A.R., Heymann, J., Meissner, C., Karlas, A., Brinkmann, V., Meyer, T.F., and Heuer, D. 2009. Rab6 and Rab11 regulate Chlamydia trachomatis development and Golgin‐84‐dependent Golgi fragmentation. PLoS Pathog. 5:1‐12.
   Lipsky, N.G. and Pagano, R.E. 1983. Spingolipid metabolism in cultured fibroblasts: Microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc. Natl. Acad. Sci. U.S.A. 80:2608‐2612.
   Lipsky, N.G., and Pagano, R.E. 1985a. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the golgi apparatus en route to the plasma membrane. J. Cell Biol. 100:27‐34.
   Lipsky, N.G. and Pagano, R.E. 1985b. A vital stain for the golgi apparatus. Science 228:745‐747.
   Mital, J. and Hackstadt, T. 2011. Role for the SRC family kinase Fyn in sphingolipid acquisition by chlamydiae. Infect. Immun. 79:4559‐4568.
   Moore, E.R., Fischer, E.R., Mead, D.J., and Hackstadt, T. 2008. The chlamydial inclusion preferentially intercepts basolaterally directed sphingomyelin‐containing exocytic vacuoles. Traffic 9:2130‐2140.
   Moore, E.R., Mead, D.J., Dooley, C.A., Sager, J., and Hackstadt, T. 2011. The trans‐Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane. Microbiology 157:830‐838.
   Moorhead, A.R., Rzomp, K.A., and Scidmore, M.A. 2007. The Rab6 effector bicaudal D1 associates with Chlamydia trachomatis inclusions in a biovar‐specific manner. Infect. Immun. 75:781‐791.
   Newhall, W.J. 1988. Macromolecular and antigenic composition of chlamydiae. In Microbiology of Chlamydiae (A.L. Baron, ed.) pp. 47‐70. CRC Press, Boca Raton, Fla.
   Robertson, D.K., Gu, L., Rowe, R.K., and Beatty, W.L. 2009. Inclusion biogenesis and reactivation of persistent Chlamydia trachomatis requires host cell sphingolipid biosynthesis. PLoS Pathog. 5:e100664.
   Rockey, D.D., Fischer, E.R., and Hackstadt, T. 1996. Temporal analysis of the developing Chlamydia psittaci inclusion by use of fluorescence and electron microscopy. Infect. Immun. 64:4269‐4278.
   Rzomp, K.A., Scholtes, L.D., Briggs, B.J., Whittaker, G.R., and Scidmore, M.A. 2003. Rab GTPases are recruited to chlamydial inclusions in both a species‐dependent and species‐independent manner. Infect. Immun. 71:5855‐5870.
   Rzomp, K.A., Moorhead, A.R., and Scidmore, M.A. 2006. The GTPase Rab4 interacts with Chlamydia trachomatis inclusion membrane protein CT229. Infect. Immun. 74:5362‐5373.
   Simonetti, A.C., Melo, J.H., de Souza, P.R., Bruneska, D., and de Lima Filho, J.L. 2009. Immunological's host profile for HPV and Chlamydia trachomatis, a cervical cancer cofactor. Microbes Infect. 11:435‐442.
   van Ooij, C., Kalman, L., van Ijzendoorn, S., Nishijima, M., Hanada, K., Mostov, K., and Engel, J.N. 2000. Host cell‐derived sphingolipids are required for the intracellular growth of Chlamydia trachomatis. Cell. Microbiol. 2:627‐637.
   Wolf, K. and Hackstadt, T. 2001. Sphingomyelin trafficking in Chlamydia pneumoniae‐infected cells. Cell. Microbiol. 3:145‐152.
   Wylie, J.L., Hatch, G.M., and McClarty, G. 1997. Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis. J. Bacteriol. 179:7233‐7242.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library