Nitric oxide stimulates early egress of Toxoplasma gondii tachyzoites from Human foreskin fibroblast cells

Yan et al. Parasites & Vectors (2015) 8:420 DOI 10.1186/s13071-015-1037-5 RESEARCH Open Access Nitric oxide stimulates early egress of Toxoplasma gondii tachyzoites from human foreskin fibroblast cells Xinlei Yan1,2, Yongsheng Ji1,2, Xianyong Liu1,2,3 and Xun Suo1,2,3* Abstract Background: Egress is a vital step in the life cycle of Toxoplasma gondii which attracts attentions of many groups. Previous studies have shown that exogenous nitric oxide (NO) stimulates the early egress of T. gondii from infected peritoneal macrophages, a kind of immune cells. However, because Toxoplasma forms cysts in brain and muscle tissues, the development of autonomous immunity in non-immune cells is vital for limiting parasite burden and cyst formation. Therefore, we attempted to investigate whether exogenous NO could induce the early egress of T. gondii from infected non-immune cells. Methods: T. gondii tachyzoites were cultured in human foreskin fibroblast (HFF) cells and were then treated with NO released by sodium nitroferricyanide (III) dihydrate (SNP). The egressed parasites were analysed by flow cytometry. Results: The results showed that NO induced the early egress of parasites from HFF cells before completing their intracellular life cycles. We also found that the occurrence of egress was dependent on intracellular calcium (Ca2+) levels and the mobility of the parasite. Compared with freshly isolated tachyzoites, the developmental ability and virulence of egressed tachyzoites presented no difference. Conclusions: Taken together, our findings demonstrate a novel assay for the analysis of egress signalling mechanisms and an avenue of parasite clearance by hosts of T. gondii. Keywords: Toxoplasma gondii, Nitric oxide, Non-immune cells Background Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects a wide range of vertebrate hosts including humans [1]. One third of the world’s population has been reported to be chronically infected by Toxoplasma [2]. Immuno-compromised individuals, such as those with acquired immunodeficiency syndrome (AIDS), and transplant patients with acute or reactivated infections can develop severe infections, which may even lead to death [3]. A few of the devastating consequences caused by the parasite are due to lysis of the host cell during egress [4]. Egress of T. gondii was initially studied by inducing elevated levels of intracellular * Correspondence: 1 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China 2 National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Full list of author information is available at the end of the article calcium (Ca2+) by ionophore A23187 [5]. Ethanol was also used to produce the secretion of microneme proteins, which results in the early egress of the parasite [6, 7]. Another chemical, dithiothreitol (DTT), causes an acute egress of tachyzoites within 60 sec by activating isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) [8]. In addition, a type of potassium ionophore triggers egress by causing an increase in the cytoplasmic Ca2+ levels within the parasite through the inositol-1,4,5-triphosphate (IP3) pathway [9]. One of the characterised mechanisms of resistance to T. gondii in human non-immune cells involves a disruption of the intracellular life cycle of the parasite. Recently, many studies have focused on early egress from non-immune cells induced by immune molecules. Death receptor ligation in T. gondii infected cells results in the early egress of infectious parasites via an active process mediated by the release of intracellular Ca2+ [10]. In addition, © 2015 Yan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yan et al. Parasites & Vectors (2015) 8:420 interferon-γ (IFN-γ)-induced cell death leads to early egress of Toxoplasma, which may promote the clearance of the parasite by immune cells [11]. Nitric oxide (NO) is produced by a number of different cells in response to cytokine stimulation and has been found to play roles in immunologically mediated protection against a growing list of protozoans, including T. gondii [12]. A previous study indicated that NO production during an acute infection with T. gondii can kill intracellular parasites [13], and the opposing effects of NO on the parasite contributed towards the establishment of a chronic state of host parasite equilibrium [14]. Moreover, when the parasites replicate in microlia, their multiplication could be prevented by activating the cells with IFN-γ or lipopolysaccharide (LPS), which is a treatment that upregulates upregulate NO synthase activity [15]. Our recent study uncovered another effect of NO against T. gondii: exogenous NO induced the the early exit of tachyzoites from infected macrophages [16]. This finding introduced another type of immunemediated egress for Toxoplasma, which may become a new means for the study of parasite clearance by the immune system of host cells. However, the previous study did not determine whether NO could induce egress of tachyzoites from non-immune cells and exposed little information on the mechanism of this immune-mediated egress. In this study, we attempted to determine whether NO could induce the egress of T. gondii tachyzoites from non-immune cells and investigated the mechanism of NO-induced egress. Our results showed that NO could trigger the early egress of T. gondii tachyzoites from infected human foreskin fibroblast (HFF) cells by elevating the concentration of the cytoplasmic Ca2+ of the parasites and that the occurrence of egress required the parasite motility. Moreover, virulence of the egressed tachyzoites was not decreased. Taken together, our discovery presents a novel assay for the analysis of the signalling mechanisms of egress and the study of parasite clearance by hosts of T. gondii. Page 2 of 6 Animals and ethical approval C57BL/6 mice (6–8-weeks old) were maintained in a pathogen-free facility. All animal research was approved by the Beijing Association for Science and Technology (approval ID SYXK (Beijing) 2007–0023) and complied with the guidelines of the Beijing Laboratory Animal Welfare and Ethics of the Beijing Administration Committee of Laboratory Animals. SNP-induced egress assay RH-YFP parasites (2 × 105) were allowed to infect HFF cells for 2 h. Free parasites were washed w (...truncated)