Parasitol Res (2014) 113:2053–2058 DOI 10.1007/s00436-014-3853-5
ORIGINAL PAPER
Autophagy activated by Toxoplasma gondii infection in turn facilitates Toxoplasma gondii proliferation Dongmei Gao & Jing Zhang & Jun Zhao & He Wen & Jiwen Pan & Shouzhu Zhang & Yong Fang & Xiuyi Li & Yu Cai & Xuelong Wang & Shiping Wang
Received: 17 December 2013 / Accepted: 5 March 2014 / Published online: 3 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Autophagy was found to play an antimicrobial or antiparasitic role in the activation of host cells to defend against intracellular pathogens, at the same time, pathogens could compete with host cell and take advantage of autophagy to provide access for its proliferation, but there are few articles for studying the outcome of this competition between host cell and pathogens. Therefore, the aim of our study was to investigate the relationship between autophagy activated by Toxoplasma gondii (T. gondii) and proliferation of T. gondii affected by autophagy in vitro. Firstly, human embryonic fibroblasts (HEF) cells were infected with T. gondii for different times. The monodansylcadaverine (MDC) staining, acridine orange (AO) staining, punctuate GFP-LC3 distribution, and transmission electron microscopy (TEM) assays were conducted, and the results were consistent in showing that gondii infection could induce autophagy. Secondly, HEF cells were infected with T. gondii and treated with autophagy inhibitor bafilomycin A1 or inducer lithium chloride for different times. Giemsa staining was conducted, and the results exhibited that T. gondii infection-induced autophagy could in turn promote T. gondii proliferation. Simultaneously, the results of Giemsa D. Gao : H. Wen (*) : J. Pan : S. Zhang : Y. Fang : X. Li : Y. Cai Department of Clinical Laboratory, Third Affiliated Hospital of Anhui Medical University, Hefei 230032, China e-mail: [email protected] J. Zhang Department of Parasitology, Anhui Medical University, 69 Meishan Road, Hefei 230032, China J. Zhao : X. Wang (*) Department of Microbiology, Anhui Medical University, 69 Meishan Road, Hefei 230032, China e-mail: [email protected] S. Wang Department of Parasitology, Xiangya Medical School, Central South University, 110 Xiangya Road, Changsha 410078, China
staining also revealed that autophagy inhibitor could reduce the number of each cell infected with T. gondii and inhibit T. gondii proliferation. In contrast, autophagy inducer could increase the number of each cell infected with T. gondii and encourage T. gondii proliferation. Therefore, our study suggests that T. gondii infection could activate autophagy, and this autophagy could in turn facilitate T. gondii proliferation in HEF cells for limiting nutrients. Keywords Toxoplasma gondii . Autophagy . LC3 II Beclin 1 . Proliferation
Introduction Autophagy is a catabolic process in eukaryotic cells involved in cytoplasmic degradation of endogenous protein and organelles to promote cell survival and cellular homeostasis. The process of autophagy is triggered by the formation of unique double membranes which mature into autophagosomes and subsequently fuse with lysosomes, finally resulting in the degradation of captured components. Autophagy is generally induced in response to the nutrient depletion, misfolded protein aggregation, oxidative stress, and pathogen infection (Weidberg et al. 2011). Autophagy stimulated by nutrient starvation allows unneeded proteins to be degraded and the amino acids recycled for the synthesis of proteins that are essential for survivals. Autophagy for degradation of misfolded protein and damaged organelles can serve to maintain cellular homeostasis and reduce cellular stress. When pathogens including bacteria, virus, fungus, and protozoa infect host cell, autophagy can be activated to generate antimicrobial or antiparasitic function to defend against the invaders. T. gondii is an important opportunistic parasite that infects almost warm-blooded animals including humans and causes a
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chronic asymptomatic infection of the central nervous system (Saki et al. 2013). In healthy individuals, the infection is largely asymptomatic, but in immunocompromised people, the parasite can become widely disseminated, and the infection can be life threatening such as AIDS and organ transplantation to patients (Wang et al. 2014). So, many researchers devote themselves to study the pathogenic effect and the mechanisms of T. gondii infection. For example, Suzuki Y’s paper revealed that T. gondii infection could induce remarkable inflammatory in mice brain (Suzuki Y 1994) and Gatkowska l’s study showed that T. gondii cysts existing both in the hippocampus and the amygdale of the infected mice caused significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central compared to uninfected controls (Gatkowska et al. 2012). It is worth noting that T. gondii infection is actually a more significant health problem for pregnant women. On the one hand, infection in pregnant women may lead to abortion, stillbirth, or other serious consequences in newborns (Peng Zhou et al. 2011; Lin et al. 2008; Vaz et al. 2010). On the other hand, T. gondii infection causes mental retardation and loss of vision in congenitally infected children (Dubey 1996). When host cells were infected with T. gondii, on the one hand, intracellular autophagy was switched on immediately to defend against T. gondii; on the other hand, T. gondii was able to exploit host cell autophagy for its own nutrition and replicated with the parasitophorous vacuole protected from lysosomal fusion (Orlofsky 2009). In addition, autophagy activated in response to amino acid limitation with rapid kinetics led to selective mitophagy and death, which was an activatable and irreversible cell death pathway (Ghosh et al. 2012). Therefore, the outcome of competition between T. gondii and host cell for limiting nutrients was well worth discussing. In this study, we investigated whether autophagy activated by T. gondii infection could in turn promote T. gondii proliferation and the effect of autophagy inhibitor and activator on T. gondii proliferation in vitro.
Parasitol Res (2014) 113:2053–2058
Fig 1 T. gondii infection increased autophagy in HEF cells as measured by fluorescence microscopy and TEM. a AO staining of HEF cells infected with or without T. gondii for 48 h (×400). The images show that HEF cells infected with T. gondii for 48 h promoted autophagic vacuole formation compared with uninfected cells. b MDC staining of HEF cells infected with or without T. gondii for 48 h (×1,000). Increase in fluorescence intensity and numbers of autophagic vacuoles show that HEF cells with infection by T. gondii for 48 h had more autophagic vacuole compared with uninfected cells. c Ultrastructural observation (×15,000) of HEF cell infected with or without T. gondii for 48 h by TEM. Many autophagic vacuoles were observed in cells infected with T. gondii for 48 h, but no autophagic vacuoles were discovered in uninfected cells. The illustrations are shown with a 0.5-μm bar for scale
Results Infection by T. gondii induces HEF cells autophagy To confirm autophagy stimulated by T. gondii, the human foreskin fibroblast (HEF) cells were infected with T. gondii at a parasite-cell ratio of 2 for 0, 8, 24, 48, 72 h. Acridine orange (AO, Sigma, USA) staining of HEF cells was performed to evaluate autophagosome formation. Images of cells of 48 h postinfection acquired by invert microscope showed that infection by T. gondii could apparently induce autophagic vacuoles formation compared with uninfected cells (Fig. 1a) and flow cytometry analysis showed that T. gondii could
modulate autophagic vacuoles formation in a timedependent manner (Table 1). To further confirm autophagy stimulated by T. gondii, monodansyl cadaverine (MDC, Sigma, USA) staining and transmission electron microscopy (TEM) observation were exploited to detect numerous autophagic vacuoles at 48 h postinfection. In MDC staining, distinct fluorescent dot-like structures corresponding to numerous autophagic vacuoles were observed in periplasmic space and cytoplasm of the infected cells, while no fluorescent dot-like structures were discovered in the uninfected cells (Fig. 1b). In TEM experiments, images acquired by TEM showed that many
Parasitol Res (2014) 113:2053–2058
2055
Table 1 T. gondii increased autophagic vacuoles formation in a timedependent manner. HEF cells were routinely infected with T. gondii at a parasite-cell ratio of 2 for 0, 8, 24, 48, and 72 h. Then, cells were stained with AO, and autophagic vacuoles were quantified by flow cytometry Time (h)
Acridine orange cells (%)
0 8 24 48 72
7.3±2.5 18.2±4.6* 24.0±3.8* 36.3±4.2** 44.2±7.2**
*P
ORIGINAL PAPER
Autophagy activated by Toxoplasma gondii infection in turn facilitates Toxoplasma gondii proliferation Dongmei Gao & Jing Zhang & Jun Zhao & He Wen & Jiwen Pan & Shouzhu Zhang & Yong Fang & Xiuyi Li & Yu Cai & Xuelong Wang & Shiping Wang
Received: 17 December 2013 / Accepted: 5 March 2014 / Published online: 3 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Autophagy was found to play an antimicrobial or antiparasitic role in the activation of host cells to defend against intracellular pathogens, at the same time, pathogens could compete with host cell and take advantage of autophagy to provide access for its proliferation, but there are few articles for studying the outcome of this competition between host cell and pathogens. Therefore, the aim of our study was to investigate the relationship between autophagy activated by Toxoplasma gondii (T. gondii) and proliferation of T. gondii affected by autophagy in vitro. Firstly, human embryonic fibroblasts (HEF) cells were infected with T. gondii for different times. The monodansylcadaverine (MDC) staining, acridine orange (AO) staining, punctuate GFP-LC3 distribution, and transmission electron microscopy (TEM) assays were conducted, and the results were consistent in showing that gondii infection could induce autophagy. Secondly, HEF cells were infected with T. gondii and treated with autophagy inhibitor bafilomycin A1 or inducer lithium chloride for different times. Giemsa staining was conducted, and the results exhibited that T. gondii infection-induced autophagy could in turn promote T. gondii proliferation. Simultaneously, the results of Giemsa D. Gao : H. Wen (*) : J. Pan : S. Zhang : Y. Fang : X. Li : Y. Cai Department of Clinical Laboratory, Third Affiliated Hospital of Anhui Medical University, Hefei 230032, China e-mail: [email protected] J. Zhang Department of Parasitology, Anhui Medical University, 69 Meishan Road, Hefei 230032, China J. Zhao : X. Wang (*) Department of Microbiology, Anhui Medical University, 69 Meishan Road, Hefei 230032, China e-mail: [email protected] S. Wang Department of Parasitology, Xiangya Medical School, Central South University, 110 Xiangya Road, Changsha 410078, China
staining also revealed that autophagy inhibitor could reduce the number of each cell infected with T. gondii and inhibit T. gondii proliferation. In contrast, autophagy inducer could increase the number of each cell infected with T. gondii and encourage T. gondii proliferation. Therefore, our study suggests that T. gondii infection could activate autophagy, and this autophagy could in turn facilitate T. gondii proliferation in HEF cells for limiting nutrients. Keywords Toxoplasma gondii . Autophagy . LC3 II Beclin 1 . Proliferation
Introduction Autophagy is a catabolic process in eukaryotic cells involved in cytoplasmic degradation of endogenous protein and organelles to promote cell survival and cellular homeostasis. The process of autophagy is triggered by the formation of unique double membranes which mature into autophagosomes and subsequently fuse with lysosomes, finally resulting in the degradation of captured components. Autophagy is generally induced in response to the nutrient depletion, misfolded protein aggregation, oxidative stress, and pathogen infection (Weidberg et al. 2011). Autophagy stimulated by nutrient starvation allows unneeded proteins to be degraded and the amino acids recycled for the synthesis of proteins that are essential for survivals. Autophagy for degradation of misfolded protein and damaged organelles can serve to maintain cellular homeostasis and reduce cellular stress. When pathogens including bacteria, virus, fungus, and protozoa infect host cell, autophagy can be activated to generate antimicrobial or antiparasitic function to defend against the invaders. T. gondii is an important opportunistic parasite that infects almost warm-blooded animals including humans and causes a
2054
chronic asymptomatic infection of the central nervous system (Saki et al. 2013). In healthy individuals, the infection is largely asymptomatic, but in immunocompromised people, the parasite can become widely disseminated, and the infection can be life threatening such as AIDS and organ transplantation to patients (Wang et al. 2014). So, many researchers devote themselves to study the pathogenic effect and the mechanisms of T. gondii infection. For example, Suzuki Y’s paper revealed that T. gondii infection could induce remarkable inflammatory in mice brain (Suzuki Y 1994) and Gatkowska l’s study showed that T. gondii cysts existing both in the hippocampus and the amygdale of the infected mice caused significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central compared to uninfected controls (Gatkowska et al. 2012). It is worth noting that T. gondii infection is actually a more significant health problem for pregnant women. On the one hand, infection in pregnant women may lead to abortion, stillbirth, or other serious consequences in newborns (Peng Zhou et al. 2011; Lin et al. 2008; Vaz et al. 2010). On the other hand, T. gondii infection causes mental retardation and loss of vision in congenitally infected children (Dubey 1996). When host cells were infected with T. gondii, on the one hand, intracellular autophagy was switched on immediately to defend against T. gondii; on the other hand, T. gondii was able to exploit host cell autophagy for its own nutrition and replicated with the parasitophorous vacuole protected from lysosomal fusion (Orlofsky 2009). In addition, autophagy activated in response to amino acid limitation with rapid kinetics led to selective mitophagy and death, which was an activatable and irreversible cell death pathway (Ghosh et al. 2012). Therefore, the outcome of competition between T. gondii and host cell for limiting nutrients was well worth discussing. In this study, we investigated whether autophagy activated by T. gondii infection could in turn promote T. gondii proliferation and the effect of autophagy inhibitor and activator on T. gondii proliferation in vitro.
Parasitol Res (2014) 113:2053–2058
Fig 1 T. gondii infection increased autophagy in HEF cells as measured by fluorescence microscopy and TEM. a AO staining of HEF cells infected with or without T. gondii for 48 h (×400). The images show that HEF cells infected with T. gondii for 48 h promoted autophagic vacuole formation compared with uninfected cells. b MDC staining of HEF cells infected with or without T. gondii for 48 h (×1,000). Increase in fluorescence intensity and numbers of autophagic vacuoles show that HEF cells with infection by T. gondii for 48 h had more autophagic vacuole compared with uninfected cells. c Ultrastructural observation (×15,000) of HEF cell infected with or without T. gondii for 48 h by TEM. Many autophagic vacuoles were observed in cells infected with T. gondii for 48 h, but no autophagic vacuoles were discovered in uninfected cells. The illustrations are shown with a 0.5-μm bar for scale
Results Infection by T. gondii induces HEF cells autophagy To confirm autophagy stimulated by T. gondii, the human foreskin fibroblast (HEF) cells were infected with T. gondii at a parasite-cell ratio of 2 for 0, 8, 24, 48, 72 h. Acridine orange (AO, Sigma, USA) staining of HEF cells was performed to evaluate autophagosome formation. Images of cells of 48 h postinfection acquired by invert microscope showed that infection by T. gondii could apparently induce autophagic vacuoles formation compared with uninfected cells (Fig. 1a) and flow cytometry analysis showed that T. gondii could
modulate autophagic vacuoles formation in a timedependent manner (Table 1). To further confirm autophagy stimulated by T. gondii, monodansyl cadaverine (MDC, Sigma, USA) staining and transmission electron microscopy (TEM) observation were exploited to detect numerous autophagic vacuoles at 48 h postinfection. In MDC staining, distinct fluorescent dot-like structures corresponding to numerous autophagic vacuoles were observed in periplasmic space and cytoplasm of the infected cells, while no fluorescent dot-like structures were discovered in the uninfected cells (Fig. 1b). In TEM experiments, images acquired by TEM showed that many
Parasitol Res (2014) 113:2053–2058
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Table 1 T. gondii increased autophagic vacuoles formation in a timedependent manner. HEF cells were routinely infected with T. gondii at a parasite-cell ratio of 2 for 0, 8, 24, 48, and 72 h. Then, cells were stained with AO, and autophagic vacuoles were quantified by flow cytometry Time (h)
Acridine orange cells (%)
0 8 24 48 72
7.3±2.5 18.2±4.6* 24.0±3.8* 36.3±4.2** 44.2±7.2**
*P
