Parasitol Res (2015) 114:795–799 DOI 10.1007/s00436-014-4305-y
SHORT COMMUNICATION
Factors affecting sporoplasm release in Kudoa septempunctata Sang Phil Shin & Kosuke Zenke & Hiroshi Yokoyama & Tomoyoshi Yoshinaga
Received: 18 September 2014 / Accepted: 23 December 2014 / Published online: 8 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The myxosporean parasite Kudoa septempunctata has been isolated from cultured olive flounder (Paralichthys olivaceus) and was recently identified as a cause of food poisoning in humans. Since the sporoplasm plays an important role in causing diarrhea by invading intestinal cells, the specific factors affecting the release of sporoplasm from spores should be determined. Thus, we investigated the effect of digestive and serum enzymes, fetal bovine serum (FBS), temperature, and the role of glucose in cell culture media on the release of sporoplasm. Sporoplasm release was observed in the groups treated with FBS and media containing glucose. In addition, 1,10-phenanthroline inhibited the release of sporoplasm in the FBS medium. These results indicate that K. septempunctata uses glucose for releasing its sporoplasm and that zinc or metalloprotease is related to the release mechanism. The present study provides important information for the development of agents to prevent sporoplasm release and the consequent food poisoning caused by K. septempunctata.
Keywords Kudoa septempunctata . Sporoplasm release . Food-borne illness . Glucose S. P. Shin (*) : K. Zenke : H. Yokoyama : T. Yoshinaga Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan e-mail: [email protected] S. P. Shin e-mail: [email protected] S. P. Shin Fisheries Laboratory, Kinki University, Shirahama, Wakayama 649-2211, Japan
Introduction Kudoa septempunctata (Myxosporea: Multivalvulida) has been reported from the trunk muscles of aquacultured olive flounder (Paralichthys olivaceus) in Korea and Japan (Harada et al. 2012; Song et al. 2013; Jeon et al. 2014). The spores of K. septempunctata are irregularly stellate in apical view and have six or seven unequal valves, each with a polar capsule of variable size (Matsukane et al. 2010). A lot of Kudoa species are histozoic, inducing macroscopic cysts in various organs including somatic muscles; however, some species form a pseudocyst in the myofibers and cause post-mortem myoliquefaction (Moran et al. 1999; Matsukane et al. 2010). The species K. septempunctata forms pseudocysts in the fish musculature without inducing inflammatory reactions, and the infection is not evident macroscopically. There are some human illnesses related with Kudoa sp. such as allergic symptoms (Martinez de Velasco et al. 2008) or food-borne diarrhea, especially, serial food poisonings associated with ingestion of raw olive flounder have been recently reported in Japan, and epidemiological analysis demonstrated that K. septempunctata was associated with these illnesses (Kawai et al. 2012). When ingested spores of K. septempunctata reach the human intestine, they release sporoplasms which can invade the human intestinal cell monolayer, resulting in the loss of intestinal epithelial integrity with inflammation that leads to diarrhea by an exudative mechanism. It suggests that the serial processes caused by sporoplasms are one of the primary mechanisms of the food-borne illness (Zhang et al. 2003; Ohnishi et al. 2013). Therefore, it is important to reveal the factors and conditions that are related with the release of the sporoplasm to provide fundamental information for preventing the food poisoning caused by K. septempunctata. In the present study, we investigated the effect of different factors on the discharge of sporoplasm in K. septempunctata, such as digestive and serum
796
Parasitol Res (2015) 114:795–799 Effect of different factors on the release of sporoplasm
enzymes and inhibitors, fetal bovine serum (FBS), temperature, and glucose in cell culture media.
Table 1
Materials and methods
Digestive enzymes α-amylase (1 unit/ml) in PBS Carboxypeptidase A (0.7 unit/ml) in PBS Carboxypeptidase B (0.6 unit/ml) in PBS
Parasite samples Olive flounder samples (n=20) suspected of being infected with K. septempunctata were obtained from an olive flounder farm of Kagawa Prefecture in Japan. The fish samples were examined microscopically to detect K. septempunctata spores in squash preparations, and the spores were purified by a modified Percoll (Sigma-Aldrich; St. Louis, MO, USA) density-gradient centrifugation method, as previously described (Chase et al. 2001). Briefly, the collected muscle tissue (2–3 g) was minced and sequentially sieved through 200- and 100-μm nylon mesh screens; 50-ml phosphate-buffered saline (PBS) was gradually added during sieving. The resultant slurry was collected into 50-ml polypropylene centrifuge tubes and was centrifuged at 1500 rpm for 15 min. The pellets were resuspended in 4 ml of PBS and added to 15-ml polypropylene centrifuge tubes containing discontinuous gradients of Percoll (4-ml layers of 15 and 30 % Percoll in PBS, pH 7.4). The tubes were centrifuged at 3500 rpm for 30 min, and the Percoll was removed from the resulting pellets of K. septempunctata spores by resuspending in 10 ml of PBS followed by centrifugation at 1500 rpm for 15 min. Finally, the supernatant was removed until 2-ml PBS remained. The sample was collected and preserved at 4 °C. Enzyme assay and effect of protease inhibitors K. septempunctata spores purified as described above were diluted in PBS to a final concentration of 105 spores/ml. The suspensions were incubated with several kinds of enzymes, including α-amylase (Wako; Osaka, Japan), carboxypeptidase A (Sigma-Aldrich), carboxypeptidase B (Sigma-Aldrich), enterokinase (Sigma-Aldrich), lipase (Sigma-Aldrich), leucine (Wako), trypsin (Difco; Detroit, MI, USA), plasmin (SigmaAldrich), and thrombin (Sigma-Aldrich). The final concentrations of enzymes were decided by manufacturer’s enzymatic assay except trypsin and indicated in Table 1. The sporoplasm release was observed in a 96-well plate under a bright-field inverted microscope. The effects of FBS (used 100 % of concentration) and culture temperature on the sporoplasm release were also evaluated. Ninety microliters of FBS was incubated at 25, 56, and 70 °C for 30 min, respectively, and cooled at room temperature. Ten microliters of K. septempunctata suspensions were added to the FBS and incubated at room temperature for 1.5 h. In addition, the effects of various inhibitors such as pepstatin A, phenylmethanesulfonyl fluoride, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline,
Factors
Enterokinase (0.4 unit/ml) in PBS Leucine (0.07 unit/ml) in PBS Lipase (0.2 unit/ml) in PBS Trypsin (500 μg/ml) in PBS Serum enzymes Plasmin (0.03 unit/ml) in PBS Thrombin (3 unit/ml) in PBS FBS Incubated FBS at 25 °C Incubated FBS at 55 °C Incubated FBS at 70 °C Protease inhibitors E64 (0.1 mM; cysteine proteases inhibitor) in FBS Leupeptin (0.1 mM; cysteine proteases and trypsin-like serine proteases inhibitors) in FBS PMSF (1 mM; serine proteases inhibitors) in FBS Pepstatin A (0.1 mM; asparatic proteases inhibitors) in FBS 1,10-phenanthroline (2 mM; metalloproteases inhibitors) in FBS EDTA (2 mM; metalloproteases inhibitors) in FBS Culture media DMEM L15 F-12/HAM Glucose (25 mM) added in DMEM Glucose (25 mM) added in L15
Release of sporoplasm
– – – – – – – – – + + + + + + + – + – – + + +
PBS phosphate-buffered saline, FBS fetal bovine serum, PMSF phenylmethanesulfonyl fluoride, EDTA ethylenediaminetetraacetic acid, DMEM Dulbecco’s modified Eagle’s medium, L15 Leibovitz’s medium, F-12/HAM nutrient mixtures F-12 HAM
trans-epoxysuccinyl-L -leucylamido-(4-guanidino)butane (E64), and leupeptin were evaluated by observing sporoplasm release in FBS. Ten microliters of inhibitors (used 10-fold high concentration than final concentration) were added to the 80 μl of FBS with 10 of K. septempunctata suspensions. The final concentrations of inhibitors are indicated in Table 1.
Medium assay for the release of sporoplasm The K. septempunctata suspensions were mixed with three kinds of media for cell culture: Dulbecco’s modified Eagle’s medium (DMEM; Sigma cat. No. D5030), Leibovitz’s medium (L15; Sigma cat. No. L4386), and Nutrient Mixtures F-12
Parasitol Res (2015) 114:795–799
797
HAM (F-12/HAM; Sigma cat. No. N6760) containing sodium bicarbonate at concentrations of 44, 25, and 14 mM, respectively. The new DMEM and L15 that contain 25 mM of glucose and 44 and 25 mM of sodium bicarbonate, respectively, were prepared. Ten microliters of K. septempunctata suspensions were added to the 90 μl of each of media, and the mixtures were incubated at room temperature for 1.5 h and sporoplasm release was monitored.
Results and discussion The present study was carried out to reveal the effect of intestinal enzymes, including trypsin and serum proteases, on the sporoplasm release of K. septempunctata. The mechanism of food poisoning caused by K. septempunctata is related to the invasion of sporoplasm to intestine epithelial cells. Although a previous study reported the sporoplasm release is stimulated by trypsin and the proteases of fetal calf serum (Ohnishi et al. 2013), the present results showed that the digestive enzymes plasmin and thrombin do not have an effect on the release of
sporoplasm (Table 1) even at three to ten times higher concentrations than enzymatic assay recommended by manufacturer (data not shown). Thus, we aimed to determine whether or not the enzymes are the primary stimulants for the release of sporoplasm in FBS. Since FBS and most enzymes are inactivated at 56 and 70 °C (Adams 1991; Daniel et al. 1996; Okano et al. 2006), we incubated the FBS at 25, 56, and 70 °C for 30 min. However, the results showed that sporoplasm release occurs in FBS regardless of temperature pre-treatment and the enzymes in FBS are not critical. It would need to be tested further through enzyme assays (Table 1, Fig. 1a). Interestingly, we observed inhibition of sporoplasm release in FBS samples treated with 1,10-phenanthroline, whereas no such inhibition was observed when FBS was treated with EDTA (Table 1). Although both 1,10-phenanthroline and EDTA are metalloprotease inhibitors, they differ with respect to their permeability through the cell membrane (Krishnamurti et al. 1980; Lin 1998). Further, where 1,10-phenanthroline can chelate the divalent ions in the cytoplasm, especially zinc, EDTA cannot penetrate the cell membrane. Therefore, we speculated that the inhibition of sporoplasm release was
a
b
c
d
e
f
Fig. 1 Microscopic observation of sporoplasm release in Kudoa septempunctata spores. Sporoplasms (arrows) were observed in samples treated with fetal bovine serum pre-incubated at 70 °C (a) and HAM/F12 medium (b), whereas no sporoplasms were observed in
samples treated with Dulbecco’s modified Eagle’s medium (c) and L15 (d). However, sporoplasm release did occur in DMEM and L15 to which 25 mM of glucose was added (e and f, respectively) (scale bar: 10 μm)
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caused by the effect of 1,10-phenanthroline in chelating the zinc or inactivating the metalloprotease in K. septempunctata. Several kinds of media have been used to culture parasites and cells in vitro (Casas et al. 2002; Ferruzza et al. 2013). We investigated the effect of conventional culture media, such as DMEM, L15, and F-12/HAM, on the release of sporoplasm, which was observed only in F-12/HAM medium (Table 1, Fig. 1b–d). Although all of the media consist of inorganic salts, amino acids, vitamins, and other components (such as a sugar source, phenol red, and pyruvic acid), these components are found at different concentrations and there are also some different components. In particular, the media used in the present study consisted of different sugar sources; for example, F-12/HAM includes glucose, L15 includes galactose, and DMEM did not include a sugar source. Therefore, we supplemented the DMEM and L15 medium with glucose to a final concentration of 25 mM and observed the release of sporoplasms from spores (Table 1, Fig. 1e, f). This clear effect of glucose on sporoplasm release suggests that K. septempunctata might utilize glucose as an energy source to release the sporoplasm and/or the glucose might activate cell signaling for releasing the sporoplasm. Glucose is used as a substrate for energy metabolism in many parasites, producing acetate or lactate as end products of metabolism (Tielens et al. 2010). These products result in acidification of the culture media, which can affect the physiological properties of parasites or cells, such as growth and actin polymerization (Faff and Nolte 2000; AlKurashi et al. 2011). We observed inhibition of sporoplasm release and acidification of the medium containing glucose and without sodium bicarbonate (data not shown). Based on this phenomenon, we suggest that K. septempunctata also uses glucose as an energy substrate for physiological responses related to sporoplasm release. Previous studies have reported a relationship between actin polymerization and the movement of sporoplasm in Myxozoa; for example, Ohnishi and colleagues have demonstrated inhibition of sporoplasm release by controlling actin polymerization in K. septempunctata (Alama-Bermejo et al., 2012; Ohnishi et al., 2013). Since actin polymerization is mediated through cell signaling molecules such as Rho small guanosine triphosphatases, which can be activated by the influx of glucose into the cell (Hall 1998; Kalwat and Thurmond 2013), we suggest that the glucose used in this study also induced the sporoplasm release by activating cell signaling. Most myxozoans exhibit a complex life cycle, developing alternately in vertebrates (mostly teleosts) and invertebrates (mostly annelids). The myxospores are thought to be released from the fish hosts by death of the host mainly. They infect the intestine of invertebrate hosts by ingestion. Vegetative and sporogonic stages of actinospores are located intercellularly in the intestinal epithelium or in the coelom. Actinospores released from the intermediate host infect the fish via gills or skin and initiate a development in fish (Lom and Dykova 2006). Based
Parasitol Res (2015) 114:795–799
on the present result, we suggest K. septempunctata might release sporoplasm for the development of actinospores in intermediate host because marine annelids have glucose in their body fluid (Wilber 1948). In a point of parasite view, it might be recognized that the intestine of humans is a proper environment for their development because humans also use glucose as an energy source. However, the parasite could not be developed in the intestine because the immune system of humans recognize the sporoplasm as a foreign substance; thus, the spores and sporoplasms were excreted with a symptom of food poisoning. Our results demonstrate the effects of glucose and 1,10phenanthroline on the sporoplasm release in K. septempunctata. The hope is that these findings will contribute to establish strategies to prevent food poisoning caused by this parasite. The strategies include the development of agents which prevent the parasite from taking glucose, which inhibit the metalloprotease in K. septempunctata, or recommendation to not eat glucose source such as rice with olive flounder. However, additional works will be required to elucidate the molecular mechanisms of sporoplasm release and confirm effect and toxicity of the agents in vivo. The obvious challenge is that once signs of food poisoning are observed, sporoplasm release has already occurred. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A6A3A03063215).
References Adams JB (1991) Review: enzyme inactivation during heat processing of food-stuffs. Int J Food Sci Technol 26:1–20. doi:10.1111/j.13652621.1991.tb01136.x Alama-Bermejo G, Bron JE, Raga JA, Holzer AS (2012) 3D Morphology, ultrastructure and development of Ceratomyxa puntazzi stages: first insights into the mechanisms of motility and budding in the Myxozoa. PLoS One 7:e32679. doi:10.1371/journal. pone.0032679 AlKurashi M, Eastick FA, Kuchipudi SV, Rauch C, Madouasse A, Zhu XQ, Elsheikha HM (2011) Influence of culture medium pH on internalization, growth and phenotypic plasticity of Neospora caninum. Vet Parasitol 177:267–274. doi:10.1016/j.vetpar.2010.11.053 Casas SM, La Peyre JF, Reece KS, Azevedo C, Villalba A (2002) Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus. Dis Aquat Org 52:217–231. doi:10.3354/dao052217 Chase JC, Dawson-Coates JA, Haddow JD, Stewart MH, Haines LR, Whitaker DJ, Ken ML, Olafson RW, Pearson TW (2001) Analysis of Kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies. Dis Aquat Org 45:121–129. doi:10.3354/ dao045121 Daniel RM, Dines M, Petach HH (1996) The denaturation and degradation of stable enzymes at high temperatures. Biochem J 317(Pt 1):1–11 Faff L, Nolte C (2000) Extracellular acidification decreases the basal motility of cultured mouse microglia via the rearrangement of the actin cytoskeleton. Brain Res 853:22–31
Parasitol Res (2015) 114:795–799 Ferruzza S, Rossi C, Sambuy Y, Scarino ML (2013) Serum-reduced and serum-free media for differentiation of Caco-2 cells. ALTEX-Altern Anim Exp 30:159–168 Hall A (1998) Rho GTPases Actin Cytoskeleton Sci 279:509–514. doi: 10.1126/science.279.5350.509 Harada T, Kawai T, Sato H, Yokoyama H, Kumeda Y (2012) Development of a quantitative polymerase chain reaction assay for detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus). Int J Food Microbiol 156:161–167. doi:10.1016/j. ijfoodmicro.2012.03.018 Jeon CH, Wi S, Song JY, Choi HS, Kim JH (2014) Development of loopmediated isothermal amplification method for detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in olive flounder (Paralichthys olivaceus). Parasitol Res 113:1759–1767. doi:10. 1007/s00436-014-3821-0 Kalwat MA, Thurmond DC (2013) Signaling mechanisms of glucoseinduced F-actin remodeling in pancreatic islet beta cells. Exp Mol Med 45:e37. doi:10.1038/emm.2013.73 Kawai T, Sekizuka T, Yahata Y, Kuroda M, Kumeda Y, Iijima Y, Kamata Y, Sugita-Konishi Y, Ohnishi T (2012) Identification of Kudoa septempunctata as the causative agent of novel food poisoning outbreaks in Japan by consumption of Paralichthys olivaceus in raw fish. Clin Infect Dis 54:1046–1052. doi:10.1093/cid/cir1040 Krishnamurti C, Saryan LA, Petering DH (1980) Effects of ethylenediaminetetraacetic acid and 1,10-phenanthroline on cell proliferation and DNA synthesis of Ehrlich ascites cells. Cancer Res 40:4092– 4099 Lin RS (1998) Zinc is essential for binding of p56lck to CD4 and CD8alpha. J Biol Chem 273:32878–32882. doi:10.1074/jbc.273. 49.32878 Lom J, Dykova I (2006) Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitol (Praha) 53:1–6 Martinez de Velasco G, Rodero M, Cuellar C, Chivato T, Mateos JM, Laguna R (2008) Skin prick test of Kudoa sp. antigens in patients
799 with gastrointestinal and/or allergic symptoms related to fish ingestion. Parasitol Res 103:713–715. doi:10.1007/s00436-008-1017-1 Matsukane Y, Sato H, Tanaka S, Kamata Y, Sugita-Konishi Y (2010) Kudoa septempunctata n. sp. (Myxosporea: Multivalvulida) from an aquacultured olive flounder (Paralichthys olivaceus) imported from Korea. Parasitol Res 107:865–872. doi:10.1007/s00436-0101941-8 Moran JDW, Whitaker DJ, Kent ML (1999) A review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial fisheries. Aquaculture 172:163–196. doi:10.1016/S0044-8486(98)00437-2 Ohnishi T, Kikuchi Y, Furusawa H, Kamata Y, Sugita-Konishi Y (2013) Kudoa septempunctata invasion increases the permeability of human intestinal epithelial monolayer. Foodborne Pathog Dis 10:137– 142. doi:10.1089/fpd.2012.1294 Okano S, Hurley DJ, Vandenplas ML, Moore JN (2006) Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro. Am J Vet Res 67:1020–1024. doi: 10.2460/ajvr.67.6.1020 Song J-Y, Choi J-H, Choi H-S, Jung SH, Park MA (2013) Monitoring of Kudoa septempunctata in cultured olive flounder and wild fish in Jeju Island during 2012. J Fish Pathol 26:129–137. doi:10.7847/jfp. 2013.26.3.129 Tielens AG, van Grinsven KW, Henze K, van Hellemond JJ, Martin W (2010) Acetate formation in the energy metabolism of parasitic helminths and protists. Int J Parasit 40:387–397. doi:10.1016/j.ijpara. 2009.12.006 Wilber CG (1948) Glucose content of the body fluid in marine annelids. J Biol Chem 173:141–143 Zhang S, Kingsley RA, Santos RL, Andrews-Polymenis H, Raffatellu M, Figueiredo J, Nunes J, Tsolis RM, Adams LG, Baumler AJ (2003) Molecular pathogenesis of Salmonella enterica serotype typhimurium-induced diarrhea. Infect Immun 71:1–12. doi:10. 1128/iai. 71.1.1-12.2003
SHORT COMMUNICATION
Factors affecting sporoplasm release in Kudoa septempunctata Sang Phil Shin & Kosuke Zenke & Hiroshi Yokoyama & Tomoyoshi Yoshinaga
Received: 18 September 2014 / Accepted: 23 December 2014 / Published online: 8 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The myxosporean parasite Kudoa septempunctata has been isolated from cultured olive flounder (Paralichthys olivaceus) and was recently identified as a cause of food poisoning in humans. Since the sporoplasm plays an important role in causing diarrhea by invading intestinal cells, the specific factors affecting the release of sporoplasm from spores should be determined. Thus, we investigated the effect of digestive and serum enzymes, fetal bovine serum (FBS), temperature, and the role of glucose in cell culture media on the release of sporoplasm. Sporoplasm release was observed in the groups treated with FBS and media containing glucose. In addition, 1,10-phenanthroline inhibited the release of sporoplasm in the FBS medium. These results indicate that K. septempunctata uses glucose for releasing its sporoplasm and that zinc or metalloprotease is related to the release mechanism. The present study provides important information for the development of agents to prevent sporoplasm release and the consequent food poisoning caused by K. septempunctata.
Keywords Kudoa septempunctata . Sporoplasm release . Food-borne illness . Glucose S. P. Shin (*) : K. Zenke : H. Yokoyama : T. Yoshinaga Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan e-mail: [email protected] S. P. Shin e-mail: [email protected] S. P. Shin Fisheries Laboratory, Kinki University, Shirahama, Wakayama 649-2211, Japan
Introduction Kudoa septempunctata (Myxosporea: Multivalvulida) has been reported from the trunk muscles of aquacultured olive flounder (Paralichthys olivaceus) in Korea and Japan (Harada et al. 2012; Song et al. 2013; Jeon et al. 2014). The spores of K. septempunctata are irregularly stellate in apical view and have six or seven unequal valves, each with a polar capsule of variable size (Matsukane et al. 2010). A lot of Kudoa species are histozoic, inducing macroscopic cysts in various organs including somatic muscles; however, some species form a pseudocyst in the myofibers and cause post-mortem myoliquefaction (Moran et al. 1999; Matsukane et al. 2010). The species K. septempunctata forms pseudocysts in the fish musculature without inducing inflammatory reactions, and the infection is not evident macroscopically. There are some human illnesses related with Kudoa sp. such as allergic symptoms (Martinez de Velasco et al. 2008) or food-borne diarrhea, especially, serial food poisonings associated with ingestion of raw olive flounder have been recently reported in Japan, and epidemiological analysis demonstrated that K. septempunctata was associated with these illnesses (Kawai et al. 2012). When ingested spores of K. septempunctata reach the human intestine, they release sporoplasms which can invade the human intestinal cell monolayer, resulting in the loss of intestinal epithelial integrity with inflammation that leads to diarrhea by an exudative mechanism. It suggests that the serial processes caused by sporoplasms are one of the primary mechanisms of the food-borne illness (Zhang et al. 2003; Ohnishi et al. 2013). Therefore, it is important to reveal the factors and conditions that are related with the release of the sporoplasm to provide fundamental information for preventing the food poisoning caused by K. septempunctata. In the present study, we investigated the effect of different factors on the discharge of sporoplasm in K. septempunctata, such as digestive and serum
796
Parasitol Res (2015) 114:795–799 Effect of different factors on the release of sporoplasm
enzymes and inhibitors, fetal bovine serum (FBS), temperature, and glucose in cell culture media.
Table 1
Materials and methods
Digestive enzymes α-amylase (1 unit/ml) in PBS Carboxypeptidase A (0.7 unit/ml) in PBS Carboxypeptidase B (0.6 unit/ml) in PBS
Parasite samples Olive flounder samples (n=20) suspected of being infected with K. septempunctata were obtained from an olive flounder farm of Kagawa Prefecture in Japan. The fish samples were examined microscopically to detect K. septempunctata spores in squash preparations, and the spores were purified by a modified Percoll (Sigma-Aldrich; St. Louis, MO, USA) density-gradient centrifugation method, as previously described (Chase et al. 2001). Briefly, the collected muscle tissue (2–3 g) was minced and sequentially sieved through 200- and 100-μm nylon mesh screens; 50-ml phosphate-buffered saline (PBS) was gradually added during sieving. The resultant slurry was collected into 50-ml polypropylene centrifuge tubes and was centrifuged at 1500 rpm for 15 min. The pellets were resuspended in 4 ml of PBS and added to 15-ml polypropylene centrifuge tubes containing discontinuous gradients of Percoll (4-ml layers of 15 and 30 % Percoll in PBS, pH 7.4). The tubes were centrifuged at 3500 rpm for 30 min, and the Percoll was removed from the resulting pellets of K. septempunctata spores by resuspending in 10 ml of PBS followed by centrifugation at 1500 rpm for 15 min. Finally, the supernatant was removed until 2-ml PBS remained. The sample was collected and preserved at 4 °C. Enzyme assay and effect of protease inhibitors K. septempunctata spores purified as described above were diluted in PBS to a final concentration of 105 spores/ml. The suspensions were incubated with several kinds of enzymes, including α-amylase (Wako; Osaka, Japan), carboxypeptidase A (Sigma-Aldrich), carboxypeptidase B (Sigma-Aldrich), enterokinase (Sigma-Aldrich), lipase (Sigma-Aldrich), leucine (Wako), trypsin (Difco; Detroit, MI, USA), plasmin (SigmaAldrich), and thrombin (Sigma-Aldrich). The final concentrations of enzymes were decided by manufacturer’s enzymatic assay except trypsin and indicated in Table 1. The sporoplasm release was observed in a 96-well plate under a bright-field inverted microscope. The effects of FBS (used 100 % of concentration) and culture temperature on the sporoplasm release were also evaluated. Ninety microliters of FBS was incubated at 25, 56, and 70 °C for 30 min, respectively, and cooled at room temperature. Ten microliters of K. septempunctata suspensions were added to the FBS and incubated at room temperature for 1.5 h. In addition, the effects of various inhibitors such as pepstatin A, phenylmethanesulfonyl fluoride, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline,
Factors
Enterokinase (0.4 unit/ml) in PBS Leucine (0.07 unit/ml) in PBS Lipase (0.2 unit/ml) in PBS Trypsin (500 μg/ml) in PBS Serum enzymes Plasmin (0.03 unit/ml) in PBS Thrombin (3 unit/ml) in PBS FBS Incubated FBS at 25 °C Incubated FBS at 55 °C Incubated FBS at 70 °C Protease inhibitors E64 (0.1 mM; cysteine proteases inhibitor) in FBS Leupeptin (0.1 mM; cysteine proteases and trypsin-like serine proteases inhibitors) in FBS PMSF (1 mM; serine proteases inhibitors) in FBS Pepstatin A (0.1 mM; asparatic proteases inhibitors) in FBS 1,10-phenanthroline (2 mM; metalloproteases inhibitors) in FBS EDTA (2 mM; metalloproteases inhibitors) in FBS Culture media DMEM L15 F-12/HAM Glucose (25 mM) added in DMEM Glucose (25 mM) added in L15
Release of sporoplasm
– – – – – – – – – + + + + + + + – + – – + + +
PBS phosphate-buffered saline, FBS fetal bovine serum, PMSF phenylmethanesulfonyl fluoride, EDTA ethylenediaminetetraacetic acid, DMEM Dulbecco’s modified Eagle’s medium, L15 Leibovitz’s medium, F-12/HAM nutrient mixtures F-12 HAM
trans-epoxysuccinyl-L -leucylamido-(4-guanidino)butane (E64), and leupeptin were evaluated by observing sporoplasm release in FBS. Ten microliters of inhibitors (used 10-fold high concentration than final concentration) were added to the 80 μl of FBS with 10 of K. septempunctata suspensions. The final concentrations of inhibitors are indicated in Table 1.
Medium assay for the release of sporoplasm The K. septempunctata suspensions were mixed with three kinds of media for cell culture: Dulbecco’s modified Eagle’s medium (DMEM; Sigma cat. No. D5030), Leibovitz’s medium (L15; Sigma cat. No. L4386), and Nutrient Mixtures F-12
Parasitol Res (2015) 114:795–799
797
HAM (F-12/HAM; Sigma cat. No. N6760) containing sodium bicarbonate at concentrations of 44, 25, and 14 mM, respectively. The new DMEM and L15 that contain 25 mM of glucose and 44 and 25 mM of sodium bicarbonate, respectively, were prepared. Ten microliters of K. septempunctata suspensions were added to the 90 μl of each of media, and the mixtures were incubated at room temperature for 1.5 h and sporoplasm release was monitored.
Results and discussion The present study was carried out to reveal the effect of intestinal enzymes, including trypsin and serum proteases, on the sporoplasm release of K. septempunctata. The mechanism of food poisoning caused by K. septempunctata is related to the invasion of sporoplasm to intestine epithelial cells. Although a previous study reported the sporoplasm release is stimulated by trypsin and the proteases of fetal calf serum (Ohnishi et al. 2013), the present results showed that the digestive enzymes plasmin and thrombin do not have an effect on the release of
sporoplasm (Table 1) even at three to ten times higher concentrations than enzymatic assay recommended by manufacturer (data not shown). Thus, we aimed to determine whether or not the enzymes are the primary stimulants for the release of sporoplasm in FBS. Since FBS and most enzymes are inactivated at 56 and 70 °C (Adams 1991; Daniel et al. 1996; Okano et al. 2006), we incubated the FBS at 25, 56, and 70 °C for 30 min. However, the results showed that sporoplasm release occurs in FBS regardless of temperature pre-treatment and the enzymes in FBS are not critical. It would need to be tested further through enzyme assays (Table 1, Fig. 1a). Interestingly, we observed inhibition of sporoplasm release in FBS samples treated with 1,10-phenanthroline, whereas no such inhibition was observed when FBS was treated with EDTA (Table 1). Although both 1,10-phenanthroline and EDTA are metalloprotease inhibitors, they differ with respect to their permeability through the cell membrane (Krishnamurti et al. 1980; Lin 1998). Further, where 1,10-phenanthroline can chelate the divalent ions in the cytoplasm, especially zinc, EDTA cannot penetrate the cell membrane. Therefore, we speculated that the inhibition of sporoplasm release was
a
b
c
d
e
f
Fig. 1 Microscopic observation of sporoplasm release in Kudoa septempunctata spores. Sporoplasms (arrows) were observed in samples treated with fetal bovine serum pre-incubated at 70 °C (a) and HAM/F12 medium (b), whereas no sporoplasms were observed in
samples treated with Dulbecco’s modified Eagle’s medium (c) and L15 (d). However, sporoplasm release did occur in DMEM and L15 to which 25 mM of glucose was added (e and f, respectively) (scale bar: 10 μm)
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caused by the effect of 1,10-phenanthroline in chelating the zinc or inactivating the metalloprotease in K. septempunctata. Several kinds of media have been used to culture parasites and cells in vitro (Casas et al. 2002; Ferruzza et al. 2013). We investigated the effect of conventional culture media, such as DMEM, L15, and F-12/HAM, on the release of sporoplasm, which was observed only in F-12/HAM medium (Table 1, Fig. 1b–d). Although all of the media consist of inorganic salts, amino acids, vitamins, and other components (such as a sugar source, phenol red, and pyruvic acid), these components are found at different concentrations and there are also some different components. In particular, the media used in the present study consisted of different sugar sources; for example, F-12/HAM includes glucose, L15 includes galactose, and DMEM did not include a sugar source. Therefore, we supplemented the DMEM and L15 medium with glucose to a final concentration of 25 mM and observed the release of sporoplasms from spores (Table 1, Fig. 1e, f). This clear effect of glucose on sporoplasm release suggests that K. septempunctata might utilize glucose as an energy source to release the sporoplasm and/or the glucose might activate cell signaling for releasing the sporoplasm. Glucose is used as a substrate for energy metabolism in many parasites, producing acetate or lactate as end products of metabolism (Tielens et al. 2010). These products result in acidification of the culture media, which can affect the physiological properties of parasites or cells, such as growth and actin polymerization (Faff and Nolte 2000; AlKurashi et al. 2011). We observed inhibition of sporoplasm release and acidification of the medium containing glucose and without sodium bicarbonate (data not shown). Based on this phenomenon, we suggest that K. septempunctata also uses glucose as an energy substrate for physiological responses related to sporoplasm release. Previous studies have reported a relationship between actin polymerization and the movement of sporoplasm in Myxozoa; for example, Ohnishi and colleagues have demonstrated inhibition of sporoplasm release by controlling actin polymerization in K. septempunctata (Alama-Bermejo et al., 2012; Ohnishi et al., 2013). Since actin polymerization is mediated through cell signaling molecules such as Rho small guanosine triphosphatases, which can be activated by the influx of glucose into the cell (Hall 1998; Kalwat and Thurmond 2013), we suggest that the glucose used in this study also induced the sporoplasm release by activating cell signaling. Most myxozoans exhibit a complex life cycle, developing alternately in vertebrates (mostly teleosts) and invertebrates (mostly annelids). The myxospores are thought to be released from the fish hosts by death of the host mainly. They infect the intestine of invertebrate hosts by ingestion. Vegetative and sporogonic stages of actinospores are located intercellularly in the intestinal epithelium or in the coelom. Actinospores released from the intermediate host infect the fish via gills or skin and initiate a development in fish (Lom and Dykova 2006). Based
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on the present result, we suggest K. septempunctata might release sporoplasm for the development of actinospores in intermediate host because marine annelids have glucose in their body fluid (Wilber 1948). In a point of parasite view, it might be recognized that the intestine of humans is a proper environment for their development because humans also use glucose as an energy source. However, the parasite could not be developed in the intestine because the immune system of humans recognize the sporoplasm as a foreign substance; thus, the spores and sporoplasms were excreted with a symptom of food poisoning. Our results demonstrate the effects of glucose and 1,10phenanthroline on the sporoplasm release in K. septempunctata. The hope is that these findings will contribute to establish strategies to prevent food poisoning caused by this parasite. The strategies include the development of agents which prevent the parasite from taking glucose, which inhibit the metalloprotease in K. septempunctata, or recommendation to not eat glucose source such as rice with olive flounder. However, additional works will be required to elucidate the molecular mechanisms of sporoplasm release and confirm effect and toxicity of the agents in vivo. The obvious challenge is that once signs of food poisoning are observed, sporoplasm release has already occurred. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A6A3A03063215).
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