Microbes and Infection 16 (2014) 962e966 www.elsevier.com/locate/micinf
Short communication
The intracellular pathogen Orientia tsutsugamushi responsible for scrub typhus induces lipid droplet formation in mouse fibroblasts Motohiko Ogawa a,*, Masayoshi Fukasawa b,**, Masaaki Satoh a, Kentaro Hanada b, Masayuki Saijo a, Tsuneo Uchiyama c, Shuji Ando a a Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan c Department of Microbiology, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan b
Received 19 May 2014; accepted 10 September 2014 Available online 22 September 2014
Abstract Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogeninfection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Infection; Orientia tsutsugamushi; Lipid droplets; Triglycerides; Lipid metabolism
1. Introduction Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium growing only in eukaryotic cells including mammalian, insect and tick cells [1]. A trombiculid mite transmits the microorganism, which grows at the biting site of the mite and causes a lesion named eschar. Then, the microorganism spreads through the entire body via blood vessels and lymphatic ducts, infecting endothelial cells, hepatocytes, and splenocytes [2]. The microorganism enters the cells and multiplies in the cytosol until the cells are ruptured. This lytic infection results in developing systemic symptoms: fever, rash, and high levels
* Corresponding author. Tel.: þ81 3 5285 1111x2563; fax: þ81 3 5285 1208. ** Corresponding author. Tel.: þ81 3 5285 1111x2127; fax: þ81 3 5285 1117. E-mail addresses: [email protected], [email protected] (M. Ogawa), [email protected] (M. Fukasawa).
of C-reactive protein and liver enzymes [3]. A delay in effective antibiotic treatment could lead to severe morbidity and even death. Fatty acids constitute a major source of energy, but they can be toxic when in excess. Therefore, mammalian cells have developed the capacity to store fatty acids as neutral lipids [i.e., triglycerides (TGs) and/or sterol esters] within lipid droplets (LDs). These unique organelles consist of a core of neutral lipids (i.e., TGs and sterol esters) surrounded by a monolayer of phospholipids and proteins [4]. The main function of LDs is lipid storage and mobilization to maintain lipid homeostasis inside the cells [5,6]. For example, the storage of TGs in adipocytes increases in obese animals, including humans [7]. In obese animals, the enhanced lipolysis of adipocytes maintains higher circulating levels of fatty acids, resulting in the accumulation of LDs in skeletal muscles, the liver, and some other tissues. In contrast, when the supply of fatty acids is scarce, TGs in LDs are degraded to fatty acids and glycerol and used as energy sources.
http://dx.doi.org/10.1016/j.micinf.2014.09.004 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
M. Ogawa et al. / Microbes and Infection 16 (2014) 962e966
In the present study, we characterized LD-like organelles in L-929 fibroblasts infected with the intracellular bacterium O. tsutsugamushi. Furthermore we analyzed the relationship between the infection of O. tsutsugamushi and the formation of these LD-like organelles. 2. Methods 2.1. Cell culture L-929 cells (a mouse fibroblast cell line, JCRB9003) [8] were grown in Eagle's minimum essential medium (MEM, Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 5% heat-inactivated fetal calf serum (FCS, SigmaeAldrich Japan Co. LCC., Tokyo, Japan) at 37 C in 5% CO2.
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and the cells were permeabilized with 0.2% Triton X-100 (Wako Pure Chemical Industries). Fluorescent staining of O. tsutsugamushi was performed with rabbit hyperimmunized serum against O. tsutsugamushi and anti-rabbit IgG conjugated with AlexaFluor®488 (Life Technologies Japan, Ltd., Tokyo, Japan), as previously reported [10]. A commercially available rabbit monoclonal antibody against adipose differentiationrelated protein (ADRP, Abcam, Ltd., Tokyo, Japan) was used as a specific marker of LDs. Two fluorescent staining reagents, Nile Red [11] (Life Technologies Japan) and DAPI (Wako Pure Chemical Industries), were used to stain the LDs and nuclei, respectively. Images were captured using a confocal microscope (LSM510; Carl Zeiss Japan Co., Tokyo, Japan) or a fluorescent microscope (Axioskop2 Plus; Carl Zeiss Japan Co.).
2.2. Propagation of O. tsutsugamushi
2.4. Cellular lipid content of cells infected with O. tsutsugamushi
The mycoplasma-free, highly virulent Ikeda strain of O. tsutsugamushi [9] was used in this study. For the inoculation, the Ikeda strain was deposited on monolayers of L-929 cells for 1 h. The inoculated cells were washed twice with Dulbecco's phosphate buffered saline (PBS, SigmaeAldrich Japan Co. LCC.), then incubated in MEM supplemented without (non-lipid condition) or with 2% FCS (normal lipid condition). In each assay, uninfected cells were used as the control.
Cellular lipids were extracted from infected and uninfected L-929 cells according to previous reports [12,13]. Cholesterol content was determined by the cholesterol oxidase method [14], and total phospholipid content by the method by Rouser et al. [15]. TG content was determined using a commercially available kit according to the instruction manual (Adipogenesis Assay Kit, BioVision, CA, USA). Statistical significance was evaluated by Welch's t-test. P < 0.01 was considered significant.
2.3. Immunofluorescent staining
3. Results
The L-929 cells were seeded on 24-well plates with round cover slips of 14-mm diameter. The cover slips were removed and washed twice with PBS. After fixation with 4% paraformaldehyde, the cover slips were washed twice with PBS,
3.1. Immunolocalization of LDs in L-929 cells Mouse fibroblast L-929 cells were infected with O. tsutsugamushi and then cultured in the present or absence of 2%
Fig. 1. Immunolocalization of LDs in cells infected with O. tsutsugamushi. The LD-like organelles were detected under (A) lipid-rich and (B) non-lipid conditions by double staining. Nile Red stained neutral lipids (LDs core content; red staining). A rabbit monoclonal anti-ADRP antibodies, followed by AlexaFluor®488 conjugated goat anti-rabbit IgG, were used to stain LDs (green staining). ADRP: adipose differentiation-related protein, OTS: O. tsutsugamushi.
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FCS. The cultures were examined by immunofluorescence microscopy for the presence of LDs, on the basis of the markers of the neutral lipid content (Nile Red) and surface membrane (anti-ADRP antibodies). This specific double staining technique detected LDs throughout the cytosol of the host cells under normal lipid (2% FCS) (Fig. 1A) and nonlipid (without FCS) (Fig. 1B) conditions. 3.2. Time-course of LD formation during O. tsutsugamushi infection The infection of L-929 cells with O. tsutsugamushi under normal lipid condition (2% FCS) induced the gradual accumulation of LDs in the cytosol over 7 days (Fig. 2A). Furthermore, the size and number of LDs increased as O. tsutsugamushi multiplied in the cells. In contrast, uninfected cultures did not show any LDs in L-929 cells during this entire period (Fig. 2A). Both in the infected cultures of high and low multiplicity of infection (MOI ¼ 0.060 vs. 0.015), LDs were detected mainly in infected cells (Fig. 2A and B).
post-inoculation. In uninfected cells, the presence of FCS, a major source of fatty acids did not affect TG content (major LDs component) (Fig. 3A). By contrast, infected cells cultured even in non-lipid condition (without FCS) exhibited baseline TG level significantly higher than uninfected cells (P < 0.01). Furthermore, normal lipid condition (2% FCS) led to a 4-fold increase in TG content in infected cultures (Fig. 3A). Therefore, combination of FCS and O. tsutsugamushi infection had an additive effect on the increase of TG content in L-929 cells. In contrast, the cellular content in cholesterol was not affected by the infection (Fig. 3B). The only significant differences between uninfected cells maintained under non-lipid or normal lipid conditions was observed in cholesterol content, which was higher in the presence of 2% FCS (Fig. 3B). Finally, total phospholipids content was not affected by infection of the culture conditions (Fig. 3C). In summary, these data show that O. tsutsugamushi selectively induced the accumulation of the lipids concentrated in LDs, namely TGs. 4. Discussion
3.3. Lipid composition of cells infected with O. tsutsugamushi The impact of infection and culture cells condition on the lipid composition of L-929 cells was determined on day 8
The present study demonstrates that the LD-like organelles detected in mouse fibroblast L-929 cells infected with O. tsutsugamushi are typical LDs, because they present a core of neutral lipids surrounded by a membrane expressing ADRP.
Fig. 2. Induction of LD formation during O. tsutsugamushi infection. O. tsutsugamushi was stained with rabbit O. tsutsugamushi antiserum and AlexaFluor®488 conjugated goat anti-rabbit IgG (green), LDs with Nile Red (red), and host cell nucleus with DAPI (blue). (A) Induction of LDs during the time-course of infection (MOI ¼ 0.06). (B) Comparison of LDs formation in two different areas: highly infected area and less infected area (MOI ¼ 0.015). High-magnification view of the two areas are shown in Fig. 2B x and y, respectively. OTS: O. tsutsugamushi.
M. Ogawa et al. / Microbes and Infection 16 (2014) 962e966
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Other reports have shown that other pathogens affect cellular lipid metabolism and/or LD formation [17e19], and that they use LDs as a source of energy for growth or survival in host cells [20e22]. In fact, recent studies demonstrated that LDs are necessary for the growth of the intracellular bacterium Chlamydia trachomatis [23,24]. In a similar manner, LD formation may play important roles in the growth and/or survival of O. tsutsugamushi in host cells. Future studies should address molecular mechanisms of alteration in lipid metabolism, and critical LD roles in O. tsutsugamushi infection. Conflict of interest There is No conflict. Acknowledgments This study was supported by a grant from the Ministry of Health, Labour and Welfare, Japan (H23-Shinkou-Ippan-007). References Fig. 3. Cellular lipid content of infected and uninfected cells with O. tsutsugamushi. Cellular contents in (A) TG, (B) cholesterol and (C) total phospholipid were extracted from infected and uninfected cells. Each assay was performed on cells cultured in medium with (lipid-rich condition) or without FCS (non-lipid condition). Statistical significance was evaluated by Welch ttest (n ¼ 3; P < 0.01). ns: no statistical difference, OTS: O. tsutsugamushi.
Time-course analysis demonstrated that the accumulation of LDs occurred in parallel with the multiplication of O. tsutsugamushi in L-929 cells. Finally, lipid composition analysis indicated that the pathogen selectively induced accumulation of TG content (not phospholipids or cholesterol contents) both in the presence and absence of fatty acids. The most remarkable finding is that the increase of TG content was not substantiated only by the addition of a source of fatty acids in the culture medium, however it was extremely substantiated by combination of O. tsutsugamushi infection and the fatty acids source. It is widely believed that mammalian cells store excess fatty acids as neutral lipids (i.e., TGs) within LDs to prevent cytotoxicity of free fatty acids, as in obese animals [7]. The present study suggests that, regardless of the presence or absence of externally supplied fatty acids, O. tsutsugamushi altered the cellular metabolism of fatty acids to increase TG content and finally induced the accumulation of LDs in host mammalian cells (mouse fibroblast L-929 cells). Nevertheless, the extracellular supply of fatty acids dramatically upregulated the TG accumulation and LD formation in response to O. tsutsugamushi infection. We interpreted these observations to suggest the existence of, at least, two pathways that additively or synergetically contribute to the LD-relevant response in host cells: one is dependent on extracellular fatty acids, whereas another is independent of them. Recent reports revealed interesting functions for LDs, including cellular signaling and membrane trafficking [4,16].
[1] Tamura A, Ohashi N, Urakami H, Miyamura S. Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov. Int J Syst Bacteriol 1995;45:589e91. [2] Seong SY, Choi MS, Kim IS. Orientia tsutsugamushi infection: overview and immune responses. Microbes Infect 2011;3:11e21. [3] Ogawa M, Hagiwara T, Kishimoto T, Shiga S, Yoshida Y, Furuya Y, et al. Scrub typhus in Japan: epidemiology and clinical features of cases reported in 1998. Am J Trop Med Hyg 2002;67:162e5. [4] van der Meer-Janssen YP, van Galen J, Batenburg JJ, Helms JB. Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome. Prog Lipid Res 2010;49:1e26. [5] Greenberg AS, Coleman RA, Kraemer FB, McManaman JL, Obin MS, Puri V, et al. The role of lipid droplets in metabolic disease in rodents and humans. J Clin Invest 2011;121:2102e10. [6] Krahmer N, Farese Jr RV, Walther TC. Balancing the fat: lipid droplets and human disease. EMBO Mol Med 2013;5:905e15. [7] Brasaemle DL, Wolins NE. Packaging of fat: an evolving model of lipid droplet assembly and expansion. J Biol Chem 2012;287:2273e9. [8] Earle WR. Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J Natl Cancer Inst 1943;4:165e212. [9] Ogawa M, Uchiyama T, Satoh M, Ando S. Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics. BMC Microbiol 2013;13:32. [10] Ogawa M, Matsumoto K, Parola P, Raoult D, Brouqui P. Expression of rOmpA and rOmpB protein in Rickettsia massiliae during the Rhipicephalus turanicus life cycle. Ann N Y Acad Sci 2006;1078:352e6. [11] Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 1985;100:965e73. [12] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911e7. [13] Fukasawa M, Nishijima M, Itabe H, Takano T, Hanada K. Reduction of sphingomyelin level without accumulation of ceramide in Chinese hamster ovary cells affects detergent-resistant membrane domains and enhances cellular cholesterol efflux to methyl-beta -cyclodextrin. J Biol Chem 2000;275:34028e34. [14] Heider JG, Boyett RL. The picomole determination of free and total cholesterol in cells in culture. J Lipid Res 1978;19:514e8. [15] Rouser G, Siakotos AN, Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1966;1:85e6.
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[16] Aflaki E, Radovic B, Chandak PG, Kolb D, Eisenberg T, Ring J, et al. Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages. J Biol Chem 2011;286:7418e28. [17] Arena ET, Auweter SD, Antunes LC, Vogl AW, Han J, Guttman JA, et al. The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets. Infect Immun 2011;79:4392e400. [18] D'Avila H, Melo RC, Parreira GG, Werneck-Barroso E, Castro-FariaNeto HC, Bozza PT. Mycobacterium bovis bacillus calmette-guerin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo. J Immunol 2006;176:3087e97. [19] Daniel J, Maamar H, Deb C, Sirakova TD, Kolattukudy PE. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog 2011;7:e1002093. [20] Hanses F, Kopp A, Bala M, Buechler C, Falk W, Salzberger B, et al. Intracellular survival of Staphylococcus aureus in adipocyte-like
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differentiated 3T3-L1 cells is glucose dependent and alters cytokine, chemokine, and adipokine secretion. Endocrinology 2011;152:4148e57. Mattos KA, Lara FA, Oliveira VG, Rodrigues LS, D'Avila H, Melo RC, et al. Modulation of lipid droplets by Mycobacterium leprae in schwann cells: a putative mechanism for host lipid acquisition and bacterial survival in phagosomes. Cell Microbiol 2011;13:259e73. Melo RC, Dvorak AM. Lipid bodyephagosome interaction in macrophages during infectious diseases: host defense or pathogen survival strategy? PLoS Pathog 2012;8:e1002729. Cocchiaro JL, Kumar Y, Fischer ER, Hackstadt T, Valdivia RH. Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proc Natl Acad Sci U S A 2008;105:9379e84. Kumar Y, Cocchiaro J, Valdivia RH. The obligate intracellular pathogen Chlamydia trachomatis targets host lipid droplets. Curr Biol 2006;16:1646e51.
Short communication
The intracellular pathogen Orientia tsutsugamushi responsible for scrub typhus induces lipid droplet formation in mouse fibroblasts Motohiko Ogawa a,*, Masayoshi Fukasawa b,**, Masaaki Satoh a, Kentaro Hanada b, Masayuki Saijo a, Tsuneo Uchiyama c, Shuji Ando a a Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan c Department of Microbiology, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan b
Received 19 May 2014; accepted 10 September 2014 Available online 22 September 2014
Abstract Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogeninfection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Infection; Orientia tsutsugamushi; Lipid droplets; Triglycerides; Lipid metabolism
1. Introduction Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium growing only in eukaryotic cells including mammalian, insect and tick cells [1]. A trombiculid mite transmits the microorganism, which grows at the biting site of the mite and causes a lesion named eschar. Then, the microorganism spreads through the entire body via blood vessels and lymphatic ducts, infecting endothelial cells, hepatocytes, and splenocytes [2]. The microorganism enters the cells and multiplies in the cytosol until the cells are ruptured. This lytic infection results in developing systemic symptoms: fever, rash, and high levels
* Corresponding author. Tel.: þ81 3 5285 1111x2563; fax: þ81 3 5285 1208. ** Corresponding author. Tel.: þ81 3 5285 1111x2127; fax: þ81 3 5285 1117. E-mail addresses: [email protected], [email protected] (M. Ogawa), [email protected] (M. Fukasawa).
of C-reactive protein and liver enzymes [3]. A delay in effective antibiotic treatment could lead to severe morbidity and even death. Fatty acids constitute a major source of energy, but they can be toxic when in excess. Therefore, mammalian cells have developed the capacity to store fatty acids as neutral lipids [i.e., triglycerides (TGs) and/or sterol esters] within lipid droplets (LDs). These unique organelles consist of a core of neutral lipids (i.e., TGs and sterol esters) surrounded by a monolayer of phospholipids and proteins [4]. The main function of LDs is lipid storage and mobilization to maintain lipid homeostasis inside the cells [5,6]. For example, the storage of TGs in adipocytes increases in obese animals, including humans [7]. In obese animals, the enhanced lipolysis of adipocytes maintains higher circulating levels of fatty acids, resulting in the accumulation of LDs in skeletal muscles, the liver, and some other tissues. In contrast, when the supply of fatty acids is scarce, TGs in LDs are degraded to fatty acids and glycerol and used as energy sources.
http://dx.doi.org/10.1016/j.micinf.2014.09.004 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
M. Ogawa et al. / Microbes and Infection 16 (2014) 962e966
In the present study, we characterized LD-like organelles in L-929 fibroblasts infected with the intracellular bacterium O. tsutsugamushi. Furthermore we analyzed the relationship between the infection of O. tsutsugamushi and the formation of these LD-like organelles. 2. Methods 2.1. Cell culture L-929 cells (a mouse fibroblast cell line, JCRB9003) [8] were grown in Eagle's minimum essential medium (MEM, Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 5% heat-inactivated fetal calf serum (FCS, SigmaeAldrich Japan Co. LCC., Tokyo, Japan) at 37 C in 5% CO2.
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and the cells were permeabilized with 0.2% Triton X-100 (Wako Pure Chemical Industries). Fluorescent staining of O. tsutsugamushi was performed with rabbit hyperimmunized serum against O. tsutsugamushi and anti-rabbit IgG conjugated with AlexaFluor®488 (Life Technologies Japan, Ltd., Tokyo, Japan), as previously reported [10]. A commercially available rabbit monoclonal antibody against adipose differentiationrelated protein (ADRP, Abcam, Ltd., Tokyo, Japan) was used as a specific marker of LDs. Two fluorescent staining reagents, Nile Red [11] (Life Technologies Japan) and DAPI (Wako Pure Chemical Industries), were used to stain the LDs and nuclei, respectively. Images were captured using a confocal microscope (LSM510; Carl Zeiss Japan Co., Tokyo, Japan) or a fluorescent microscope (Axioskop2 Plus; Carl Zeiss Japan Co.).
2.2. Propagation of O. tsutsugamushi
2.4. Cellular lipid content of cells infected with O. tsutsugamushi
The mycoplasma-free, highly virulent Ikeda strain of O. tsutsugamushi [9] was used in this study. For the inoculation, the Ikeda strain was deposited on monolayers of L-929 cells for 1 h. The inoculated cells were washed twice with Dulbecco's phosphate buffered saline (PBS, SigmaeAldrich Japan Co. LCC.), then incubated in MEM supplemented without (non-lipid condition) or with 2% FCS (normal lipid condition). In each assay, uninfected cells were used as the control.
Cellular lipids were extracted from infected and uninfected L-929 cells according to previous reports [12,13]. Cholesterol content was determined by the cholesterol oxidase method [14], and total phospholipid content by the method by Rouser et al. [15]. TG content was determined using a commercially available kit according to the instruction manual (Adipogenesis Assay Kit, BioVision, CA, USA). Statistical significance was evaluated by Welch's t-test. P < 0.01 was considered significant.
2.3. Immunofluorescent staining
3. Results
The L-929 cells were seeded on 24-well plates with round cover slips of 14-mm diameter. The cover slips were removed and washed twice with PBS. After fixation with 4% paraformaldehyde, the cover slips were washed twice with PBS,
3.1. Immunolocalization of LDs in L-929 cells Mouse fibroblast L-929 cells were infected with O. tsutsugamushi and then cultured in the present or absence of 2%
Fig. 1. Immunolocalization of LDs in cells infected with O. tsutsugamushi. The LD-like organelles were detected under (A) lipid-rich and (B) non-lipid conditions by double staining. Nile Red stained neutral lipids (LDs core content; red staining). A rabbit monoclonal anti-ADRP antibodies, followed by AlexaFluor®488 conjugated goat anti-rabbit IgG, were used to stain LDs (green staining). ADRP: adipose differentiation-related protein, OTS: O. tsutsugamushi.
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FCS. The cultures were examined by immunofluorescence microscopy for the presence of LDs, on the basis of the markers of the neutral lipid content (Nile Red) and surface membrane (anti-ADRP antibodies). This specific double staining technique detected LDs throughout the cytosol of the host cells under normal lipid (2% FCS) (Fig. 1A) and nonlipid (without FCS) (Fig. 1B) conditions. 3.2. Time-course of LD formation during O. tsutsugamushi infection The infection of L-929 cells with O. tsutsugamushi under normal lipid condition (2% FCS) induced the gradual accumulation of LDs in the cytosol over 7 days (Fig. 2A). Furthermore, the size and number of LDs increased as O. tsutsugamushi multiplied in the cells. In contrast, uninfected cultures did not show any LDs in L-929 cells during this entire period (Fig. 2A). Both in the infected cultures of high and low multiplicity of infection (MOI ¼ 0.060 vs. 0.015), LDs were detected mainly in infected cells (Fig. 2A and B).
post-inoculation. In uninfected cells, the presence of FCS, a major source of fatty acids did not affect TG content (major LDs component) (Fig. 3A). By contrast, infected cells cultured even in non-lipid condition (without FCS) exhibited baseline TG level significantly higher than uninfected cells (P < 0.01). Furthermore, normal lipid condition (2% FCS) led to a 4-fold increase in TG content in infected cultures (Fig. 3A). Therefore, combination of FCS and O. tsutsugamushi infection had an additive effect on the increase of TG content in L-929 cells. In contrast, the cellular content in cholesterol was not affected by the infection (Fig. 3B). The only significant differences between uninfected cells maintained under non-lipid or normal lipid conditions was observed in cholesterol content, which was higher in the presence of 2% FCS (Fig. 3B). Finally, total phospholipids content was not affected by infection of the culture conditions (Fig. 3C). In summary, these data show that O. tsutsugamushi selectively induced the accumulation of the lipids concentrated in LDs, namely TGs. 4. Discussion
3.3. Lipid composition of cells infected with O. tsutsugamushi The impact of infection and culture cells condition on the lipid composition of L-929 cells was determined on day 8
The present study demonstrates that the LD-like organelles detected in mouse fibroblast L-929 cells infected with O. tsutsugamushi are typical LDs, because they present a core of neutral lipids surrounded by a membrane expressing ADRP.
Fig. 2. Induction of LD formation during O. tsutsugamushi infection. O. tsutsugamushi was stained with rabbit O. tsutsugamushi antiserum and AlexaFluor®488 conjugated goat anti-rabbit IgG (green), LDs with Nile Red (red), and host cell nucleus with DAPI (blue). (A) Induction of LDs during the time-course of infection (MOI ¼ 0.06). (B) Comparison of LDs formation in two different areas: highly infected area and less infected area (MOI ¼ 0.015). High-magnification view of the two areas are shown in Fig. 2B x and y, respectively. OTS: O. tsutsugamushi.
M. Ogawa et al. / Microbes and Infection 16 (2014) 962e966
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Other reports have shown that other pathogens affect cellular lipid metabolism and/or LD formation [17e19], and that they use LDs as a source of energy for growth or survival in host cells [20e22]. In fact, recent studies demonstrated that LDs are necessary for the growth of the intracellular bacterium Chlamydia trachomatis [23,24]. In a similar manner, LD formation may play important roles in the growth and/or survival of O. tsutsugamushi in host cells. Future studies should address molecular mechanisms of alteration in lipid metabolism, and critical LD roles in O. tsutsugamushi infection. Conflict of interest There is No conflict. Acknowledgments This study was supported by a grant from the Ministry of Health, Labour and Welfare, Japan (H23-Shinkou-Ippan-007). References Fig. 3. Cellular lipid content of infected and uninfected cells with O. tsutsugamushi. Cellular contents in (A) TG, (B) cholesterol and (C) total phospholipid were extracted from infected and uninfected cells. Each assay was performed on cells cultured in medium with (lipid-rich condition) or without FCS (non-lipid condition). Statistical significance was evaluated by Welch ttest (n ¼ 3; P < 0.01). ns: no statistical difference, OTS: O. tsutsugamushi.
Time-course analysis demonstrated that the accumulation of LDs occurred in parallel with the multiplication of O. tsutsugamushi in L-929 cells. Finally, lipid composition analysis indicated that the pathogen selectively induced accumulation of TG content (not phospholipids or cholesterol contents) both in the presence and absence of fatty acids. The most remarkable finding is that the increase of TG content was not substantiated only by the addition of a source of fatty acids in the culture medium, however it was extremely substantiated by combination of O. tsutsugamushi infection and the fatty acids source. It is widely believed that mammalian cells store excess fatty acids as neutral lipids (i.e., TGs) within LDs to prevent cytotoxicity of free fatty acids, as in obese animals [7]. The present study suggests that, regardless of the presence or absence of externally supplied fatty acids, O. tsutsugamushi altered the cellular metabolism of fatty acids to increase TG content and finally induced the accumulation of LDs in host mammalian cells (mouse fibroblast L-929 cells). Nevertheless, the extracellular supply of fatty acids dramatically upregulated the TG accumulation and LD formation in response to O. tsutsugamushi infection. We interpreted these observations to suggest the existence of, at least, two pathways that additively or synergetically contribute to the LD-relevant response in host cells: one is dependent on extracellular fatty acids, whereas another is independent of them. Recent reports revealed interesting functions for LDs, including cellular signaling and membrane trafficking [4,16].
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