Role of cholesterol of membrane microdomain in the uptake of Francisella tularensis by mouse macrophages

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1 223,,,,,,, : LVS pfnltp6 gro-gfp LVS ;, -1 Alexa 594 ; Z LVS ; - -,,,, -1 ;, -1 : ; ; ; -1; Role of cholesterol of membrane microdomain in the uptake of Francisella tularensis by mouse macrophages PAN Xin, LI Guang-Bo, QU Min, ZHAO Zi-Ye, LI Han, QI Zhong-Tian Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai , China Abstract: The purpose of the current study was to evaluate the role of cholesterol of the membrane microdomain in the uptake of Francisella tularensis ( F. tularensis) LVS by mouse macrophages. A shuttle plasmid, pfnltp6 gro-gfp, was transformed by electroporation to F. tularensis LVS. Cell cholesterol was stained with Filipin, and caveolin-1 was detected with monoclonal antibody and visualized with Alexa 594 conjugated goat anti-mouse antibodies. F. tularensis LVS infection was analyzed using a fluorescence microscope equipped with a motorized Z-focus. In order to evaluate the effect of depletion of the membrane microdomain on F. tularensis entry into macrophages, interference with lipid-rich plasma membrane through the depletion of cholesterol was performed by methyl- -cyclodextrin. The cholesterol-binding agent, Filipin III, was used to detect the effect of cholesterol depletion. The results showed that cell cholesterol was co-localized with F. tularensis live vaccine strain in the early uptake stage, and both had close contact with the membrane microdomain-associated components, such as caveolin-1. F. tularensis requires cholesterol-rich membrane microdomains for entry into macrophages. These findings suggest that cholesterol-rich membrane microdomains and caveolin-1 play an important role in F. tularensis early entry into macrophages. Key words: Macrophage; Francisella tularensis; Membrane microdomain; Caveolin-1; Cholesterol ( Francisella tularensis), ( ) [ 1] Schu, 1 4 : ( ) ; ( ) ; ( 2008 ZXJ ) ; ( 08A) ; ( B901) :, xinpanpx@ yahoo. com;, qizt@ smmu. edu. cn Corresponding author: PAN Xin, xinpanpx@ yahoo. com; QI Zhong-Tian, qizt@ smmu. edu. cn

2 Journal of Microbes and Infection, December 2009, Vol. 4, No. 4 50% [ 2 ] [ 3] LVS, [ 3] LVS, LVS J774, h, 2 3 log [ 4 ],,16 h [ 5], 1( early endosome antigen 1, EEA1), 1 ( lysosome-associated membrane protein-1, LAMP-1) D, [ 5, 6] [ 7] ( membrane microdomain) [ 8] J774, RNA ( small interference RNA, sirna) J774, - - ( methyl- - cyclodextrin, M CD), sirna, ;,, LVS( Francisella tularensis subsp. holarctica LVS, FTLVS) Eric Hanson ( ) 6- J774A. 1 ATCC groe GFP ( pfnltp6 gro-gfp) IsoVitaleX ( BD Biosciences) 37 C 72 h [ 4 ] Mueller-Hinton IsoVitaleX, DMEM( Sigma) 10% ( fetal bovine serum, FBS; HyClone, ) , 1 ml [ 9 ] 2, 10% 2, 200 l 10% 50 l 10% 1 l pfnltp6 gro-gfp,, 5 min Bio-Rad Gene Pulser, 0. 2 cm, V, 25 F, 200, -DNA 500 l MHB Eppendorf, 37 C 30 min 250 l FTLVS- ( green fluorescent protein, GFP) ( FTLVS-GFP) FTLVS-GFP GFP FTLVS, 10% FBS DMEM g 10 min 10% FBS DMEM 2, 10% FBS DMEM A 6 00 / ( multiplicity of infection, MOI) 50 J774A. 1 DMEM ( 10% FBS) ( MOI =50), 37 5% CO J774A. 1 FTLVS-GFP 5 g/ml ( Filipin III; Sigma), [ 10 ] 3. 7% ( Polysciences) 30 min, ( phosphate-buffered saline, PBS) 1, 0. 5% Triton X-100 ( IBI Scientific ) PBS 30 min, PBS 3, Image-iTFX ( Invitrogen) 30 min PBS, 60 min ( Invitrogen) GFP LVS, -1 ( Cav1, Abcam) 1 500, Alexa 594 IgG ( Invitrogen) DAPI FITC, Z 30 ( 30

3 225 ), Volocity 4. 1( Improvision) M CD,, 10% FBS CO2 M CD [ 11 ] 10 mmol/l M CD( Sigma-Aldrich) 60 min MOI 50 FTLVS-GFP, 5 g/ml Z 40, x-y 10 min ( Invitrogen),, 10, 200, , IsoVitaleX,, 15 min, Olympus IX81, ( 1) A: Caveolin-1 was visualized with Cav-1 mab and Alexa 594-conjugated secondary antibodies ( red fluorescence). For red fluorescence, the excitation filter was 575 nm and the emission filter was 624 nm. B: Plasmids pfnltp6 gro-gfp were transfected into Francisella tularensis LVS. For green fluorescence, the excitation filter was 494 nm and the emission filter was 531 nm. C: Filipin III binding with cell cholesterol displays blue fluorescence. For blue fluorescence, the excitation filter was 387 nm and the emission filter was 422 nm. D: Consecutive images were obtained for each fluorescence channel at focal planes 0. 2 m apart and deconvolved. Flurescence channels were then merged. Purple areas indicate overlay of blue cholesterol and red caveolin-1, or tight connection of them. White-blue areas indicate overlay of blue cholesterol and green GFP-bacteria, with caveolin-1 contacting tightly with them. 1 Fig 1. Involvement of membrane microdomain in the uptake of Francisella tularensis in macrophages 2. 2 M CD M CD, ( MOI = 50) M CD, 30 min, ( ) % ; 10 mmol/l M CD 60 min, 30 min, ( ) % ( 2), J774A mmol/l M CD 60 min,,, ( 3), Cell cholesterol was visualized with Filipin ( blue fluorescence). 2 Fig 2. Uptake of fluorescent zymosan A particles was not impaired by cholesterol depletion

4 Journal of Microbes and Infection, December 2009, Vol. 4, No. 4 ( 1) 10 mmol /L M CD 60 min, GFP,, M CD ( 4A) M CD,, 10% M CD, ( 4B) Macrophages were treated with 10 mmol /L M CD for 60 min. Cell cholesterol was visualized with filipin III. Infection was conducted with Francisella constitutively expressing GFP in CO 2 -independent medium in glassbottom petri dishes on a heated microscopy stage at 37 C. Images were acquired in four dimensions ( xyzt) at 10-minute intervals. Representative deconvolved images at each time point are shown Fig 3. Methyl- -cyclodextrin inhibited Francisellatularensis entry into macrophages A: J774 A. 1 cells were treated with 10 mmol /L M CD for 60 min or left untreated ( control). Filipin III ( blue fluorescence) and FTLVS-GFP ( green fluorescence) were added into the cell dishes with CO 2 -independent medium at the same time on a heated microscopy stage at 37 C. Images were acquired in four dimensions ( xyzt) at 30 -minute intervals. Representative deconvolved images at each time point are shown. B: Ten independent visual fields were quantified at each time point under the microscope and expressed as the percentage of macrophages infected Fig 4. Effect of methyl- -cyclodextrin on depletion of plasma membrane cholesterol 3,,, [ 12], [ 13] LVS, [ 14],, PubMed, LVS, [ 15 ] LVS,, 2 :,, ;, [ 16]

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