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Protistology 6 (1), 45—54 (2009)
Protistology
Exocytotic and phagocytotic activities of Tetrahymena pyriformis are not influenced by Clostridium botulinum neurotoxins
N. M. Staender, W. Schroedl and M. Krueger
Institut fur Bakteriologie und Mykologie, Veterindrmedizinische Fakultat der Universitat Leipzig
Summary:
The ciliate Tetrahymena pyriformis GL was tested for its applicability to the detection of botulinum neurotoxins. Botulinum neurotoxins attack different proteins of the SNARE-complex, which is involved in fusion processes of the cellular membrane traffic. The exocytosis of enzymes and the phagocytosis of germs include several presumptive SNARE-dependent pathways within T. pyriformis. Acid phosphatase was chosen as indicator enzyme for the quantification of the exocytotic activity. It was determined by photometric measurement after the addition of the substrate p-nitrophenylphosphate. The phagocytotic activity was quantified with Escherichia coli as prey germ. Botulinum neurotoxins were produced by cultivating reference strains of the seven Clostridium botulinum toxovars A to G in TPGY broth with and without the addition of trypsin. The success of the neurotoxin production was tested by the mouse bioassay, which is the standard test for botulinum neurotoxins. The neurotoxins were added to the assays used for the determination of the exocytotic and the phagocytotic activity of T. pyriformis in final concentrations of1.5-102 mouse lethal doses-ml-1, except E: 1.5 mouse lethal doses -ml-1. No significant influence of the toxins could be detected. Hence, T. pyriformis cannot be used as a test-organism for the detection of botulinum neurotoxins.
Key words: BoNTs, botulinum neurotoxins, exocytosis, indicator organism, phagocytosis, Tetrahymena pyriformis GL
Abbreviations: BoNT(s) = botulinum neurotoxin(s), cfu = colony-forming units, DIN = Deutsche Industrie Norm, MLD = mouse lethal dose, NSF = N-Ethyl-maleimid sensitive fusion protein, SD = standard deviation, SNARE = Soluble NSF acceptor protein receptor
Introduction
Tetrahymena pyriformis is a very popular test and indicator organism for various substances (Dayeh et al., 2004; Muller et al., 2006; Leitgib et al., 2007),
including bacterial toxins (Schlimme et al., 1999). This is due to minor demands on cultivation conditions and a responsiveness comparable to that of higher eukaryotic cells.
© 2009 by Russia, Protistology
The botulinum neurotoxins A to G, according to the nomenclature of the seven toxovars, are produced by the anaerobic, spore-forming bacterium Clostridium botulinum. They act as metalloprotein-ases and are considered to be the most poisonous poisons (Lamanna, 1959). Furthermore, these toxins are shown to be the most selective proteases according to their substrate specificity (Arndt et al., 2006). They interact with different proteins of the SNARE-complex (Singh, 2000). These proteins mediate homo- and hetero-typic vacuolar fusion processes within eukaryotic cells (Nichols et al., 1997) and are highly conserved (Ferro-Novick and Jahn, 1994). The points of attack differ depending on the type of toxin. As a result of the interaction, SNARE-dependent membrane fusion processes, such as the exocytosis of neurotransmitter, are stopped (Rossetto et al., 2001), which is the reason for the clinical symptoms of the widespread disease “botulism” (Bohnel and Gessler, 2005).
Besides their pivotal role in the exocytosis, SNARE-proteins were proven to be essential for the phagocytosis: it was inhibited in murine macrophages by the application of BoNT B (Hackam et al., 1998). Their relevance for the fusion of en-dosomes and lysosomes was also demonstrated in rabbit alveolar macrophages (Ward et al., 2000).
To date, there is no satisfying assay allowing one to detect BoNTs in biological matrices because of the minimal amount of an effective dose. The mouse bioassay is the only test accredited by law to prove BoNTs in suspicious samples in Germany. However, it involves different problems, namely, ethical aspects (the mice die slowly with preserved sensorium), high limits of detection (cattle is about 13 times more sensitive; Moeller et al., 2003) and the impossibility to standardize this test. Alternative detection tests are urgently needed, also in order to meet new diagnostic challenges, such as the “visceral botulism” (Bohnel and Gessler, 2005).
Some authors (Graf, 1985; De Waart et al., 1972) have already discussed the applicability of T. pyriformis to the detection of BoNTs. Their approaches were unsuccessful, since they decided to choose the growth pattern of a protozoan culture as a presumably sensitive parameter. However, there is hitherto no evidence of an involvement of the SNARE-proteins in growth pattern and cell division.
Encouraged by the detection of SNARE-dependent steps during the membrane traffic in protozoa as different as Giardia intestinalis (Dacks and Doolittle, 2002), Paramecium tetraurelia (Kissmehl et al., 2002; Froissard et al., 2002) and
Dictyostelium discoideum (Bogdanovic et al., 2002), we studied again the applicability of T. pyriformis to indication of BoNTs, but focused on exocytosis and phagocytosis as presumably SNARE-dependent and sensitive parameters.
The exocytosis ofthe acid phosphatase is mainly constitutive and quite insensitive to alterations in the medium compared to the other enzymes released by T. pyriformis (Banno et al., 1987). The process of release seems to be analogous to the release of neurotransmitter in metazoans (Hutton, 1997).
The phagocytotic activitiy in T. pyriformis can be determined in different ways, e.g. with Indian ink (Kovacs et al., 198б), latex particles (Batz and Wunderlich, 197б) or spores of Glugea spp. (Wei-dner and Sibley, 1985). Escherichia coli was also used (Watson et al., 1981; Burkharin and Nemtseva, 2001).
Material and Methods
Tetrahymena pyriformis
T. pyriformis strain GL was used. The cells were counted in a Neubauer chamber. They were cultivated in 25 cml-cell culture-flasks (TPP AG Tras-adingen, Switzerland) filled with 10 ml of PPYG (20 g proteose-peptone, 1 g yeast-extract, 10 g glucose ad 1 l aqua dest.).
Escherichia coli
The apathogenic strain K12 of E. coli was chosen as prey germ and stored at -80°C. Before using them, the bacteria were incubated overnight on a blood agar plate at 37°C. Prior to the experiment they were washed down from the agar with VOLVIC®-water. They were quantified photometrically as suspension in VOLVIC®-water as described by Schroedl et al., 2003.
Botulinum neurotoxins
The BoNTs were produced according to the DIN 10102. Briefly, two media were used: TPGY (50 g casein-peptone, 5 g mixed peptone, 4 g glucose, 1 g sodium thioglycolate, 20 g yeast extract ad 1 l aqua dest.) without and with (1:15) trypsin solution (porcine trypsin, activity 1:250, 0.15 g ad 10 ml aqua dest.).
Reference strains of the Cl. botulinum toxovars A to G and of Cl. sporogeneswere incubated anaerobically on blood agar plates for three days at 37°C. Afterwards, the cells were washed down and one half of this suspension was inoculated into 100 ml of TPGY with trypsin solution and the other one into 100 ml of TPGY without it. These cultures were
incubated for seven days at 25°C under anaerobic conditions. Afterwards, the suspensions were centrifuged (1500 g for 15 min, 8°C) and the supernatants were sterile-filtrated. The concentrations of neurotoxins in these supernatants were determined in the mouse bioassay. Blank media without inocula were treated in the same manner in order to serve as controls (control 1). The amount of protein was measured photometrically.
PRELIMINARY INVESTIGATIONS
The viability of the protozoa during both of our assays was assessed by light optical microscopy at 0 h and after 1h, 2h, 4h and 5h.
The culture supernatants and blank media controls were incubated with an E. coliK12-suspension in VOLVIC®-water (7.00 log10cfu-mH) at 28°C for 4h. The effective concentrations ofneurotoxins were the same as in the assay to determine the phago-cytotic activities. Before and after the incubation period, samples were taken to determine the cfu-mH via microdilution method (plate count).
The activity of acid phosphatase was measured as follows: a culture of T. pyriformis in PPYG (б.00 log10cells-mH) was centrifuged (1500 g for 5 min) and sterile-filtrated. The supernatants and blank media controls were added to the sterile filtrate using the same effective concentrations as in the assay to determine the exocytotic activities. Samples of 50 ^l were taken after 5 h and diluted 1:4.3 with 100 mM sodium acetate (pH 4.0). To 200 ^l of this solution, 5 ^l of 300 mM p-nitrophenylphosphate were added. After 15 min of shaking (400 U/min at room temperature), the reaction was stopped by 50 ^l of 1 N NaOH. The extinction was measured at 405 nm (б20 nm as reference). PPYG without protozoa was treated in the same way in order to serve as control for unspecific background signals, which were then subtracted.
EXOCYTOTIC ACTIVITY
Cells in the early static growth phase from cultures with PPYG were centrifuged (1500 g for 5 min). The pellet was resuspended in fresh PPYG and the cells were adjusted to a concentration of 5.35 log10cells-mH. Seventy ^l of this suspension were incubated at 28°C with 35 ^l of the supernatants and the blank media. Samples with pure PPYG instead of PPYG with protozoa served as controls for unspecific background signals again. After an incubation period of 5 h, 100 mM sodium acetate (pH 4.0) were added (1:4.3), which immobilized but did not destroy the cells. After centrifugation (1500 g for 5 min), 5 ^l of300 mM p-nitrophenylphosphate
were added to 200 Щ of the supernatants. The activity of the acid phosphatase released into the medium during the incubation period was measured as described above.
The supernatant of toxovar E was detoxified by heating (100°C, 15 min).
PHAGOCYTOTIC ACTIVITY
The possible influence of protozoal secretion products on the bacteria was examined first. An overnight culture of T. pyriformis in VOLVIC®-water in the beginning of the exponential growth phase was centrifuged (1500 g for 5 min) and the pellet was resuspended in VOLVIC®-water. This culture was adjusted to 4.б0 log10cells-mH according to the assay-setting for the determination of the phagocytotic activity. The protozoa were removed by sterile filtration. The filtrate was incubated with bacteria in VOLVIC®-water (7.00 log10cfu-mH). A sample with pure VOLVIC®-water instead of the sterile filtrate served as control. After an incubation period of 4 h at 28°C, cfu-mH of E. coli were determined as described above. The results obtained with the sterile filtrate were compared to the results of the control.
The supernatants and blank media controls were tested afterwards. They were incubated with protozoa (prepared as above) for 1 h at 28°C before the bacteria were added. This mixture containing 4.б0 log10cells-mH of T. pyriformis and 7.00 log10cfu-mH of E. coli was incubated further at 28°C for another 4 h. Afterwards, the remaining cfu-mH of the bacteria in the suspensions were determined as described above. Since the concentration of the bacteria during the incubation with the supernatants and blank media controls did not vary and since protozoal secretion products had no effect, either, a decrease of the cfu-mH had to be due to the phagocytosis of T. pyriformis solely. Thus, the phagocytotic activity could be calculated by a comparison of the cfu-mH added at the beginning of the incubation period and the cfu-mH in the suspension after incubation with protozoa. The possible effect of the neurotoxins on the phagocytosis was determined by comparing the results of the approaches with neurotoxins to the results of the approaches with the blank media controls (control 1) and the supernatant of Cl. spo-rogenes (control 2).
The supernatants of toxovar E were detoxified as described above.
STATISTICAL ANALYSIS
The results ofthe assays aimed at the determination of an influence ofthe BoNTs on the exocytotic
and phagocytotic activity were analysed statistically. The Mann-Whitney Rank Sum Test was used to show significant differences between the approaches with culture supernatants and those with blank media controls.
Results
Botulinum neurotoxins
The reference strains of the toxovars A — D, F and G produced BoNTs in concentrations of 5-102 MLDml-1 in Trypticase-peptone-glucose-yeast-extract broth with addition of trypsin solution (TPGYT), as well as in the broth without it (TPGY). Strain E produced a concentration of only 5 MLD-ml-1 in the trypsinized variant (TPGYT) and no detectable BoNT in the non-trypsinized one (TPGY).
The culture supernatants of Cl. sporogenes (TPGY and TPGYT), which should serve as another non-neurotoxin-containing control (= control 2), did not show any mouse toxicity.
T. pyriformis and BoNTs
Preliminary investigations
The microscopical examination demonstrated that there was no negative influence of the BoNTs either on the viability or on the quantity of the protozoa, when these approaches were compared to the controls (controls 1 and 2).
The growth of the prey germ E. coli K12 in suspensions with the culture supernatants of the Cl. botulinum toxovars and Cl. sporogenes did not differ from the growth in suspensions with blank media (TPGY and TPGYT, control 1 without neurotoxins) after an incubation period of 4h (Table 1). Furthermore, there was no difference in the measurable enzymatic activity of acid phosphatase between the approaches incubated with the blank media controls for 5 h and the ones incubated with culture supernatants of the Cl. botulinum toxovars and Cl. sporogenes (Table 2).
Exocytotic activity
The results of the photometrical measurements of the amounts of acid phosphatase released into the medium were initially adjusted by subtracting the unspecific background-signals. Afterwards, the results of the assays with the blank media controls were defined as 100% exocytosis-activity and the results of the approaches with the neurotoxin-containing supernatants of the Cl. botulinum toxovars and the supernatants of Cl. sporogenes were adjusted accordingly.
The results of the approaches with the supernatants of the Cl. sporogenes cultures did not differ from the results with the blank media controls.
The number of cells of T. pyriformis remained constant during the assay determining the exocytotic activity (Table 3).
In the assays with the different neurotoxins, the trypsin-added culture supernatant of toxovar E was
Table 1. Log10cfu-ml-1 of E. coli K12 after incubation in a suspension with blank media (control) and with culture supernatants of Cl. botulinum and Cl. sporogenes for 4h.
Cultivation medium Control Mean (n=3), log10cfu-ml_1 SD Supernatant of Mean (n=3), log10cfu-ml_1 SD
Without trypsin Blank medium б.90 0.14 Toxovar A 7.17 0.27
(TPGY) Blank medium 7.50 0.27 Toxovar B 7.50 0.27
Blank medium 7.03 0.11 Toxovar C 7.0б 0.11
Blank medium 7.81 0.1б Toxovar D 7.58 0.31
Blank medium 8.1б 0.12 Toxovar E 7.89 0.24
Blank medium б.90 0.14 Toxovar F 7.24 0.21
Blank medium 7.81 0.1б Toxovar G 7.75 0.28
Blank medium 7.42 0.09 Cl. sporogenes 7.3б 0.08
With trypsin Blank medium 7.78 0.10 Toxovar A 7.89 0.11
(TPGYT) Blank medium 7.78 0.10 Toxovar B 7.59 0.19
Blank medium 7.92 0.43 Toxovar C 7.90 0.17
Blank medium 7.92 0.43 Toxovar D 7.81 0.12
Blank medium 8.24 0.08 Toxovar E 7.93 0.30
Blank medium 7.78 0.10 Toxovar F 7.91 0.12
Blank medium 7.92 0.43 Toxovar G 7.93 0.08
Blank medium 7.37 0.04 Cl. sporogenes 7.23 0.10
Table 2. Milli-optical density of the approaches to determine the enzymatic activity of the acid phosphatase after the addition of the substrate in sterile filtrates of a T. pyriformis culture after incubation with blank media (control) and with culture supernatants of Cl. botulinum and Cl. sporogenes for 5h, unspecific background signals subtracted.
Cultivation medium control Mean (n=3), mOD* SD Supernatant of Mean (n=3), mOD* SD
Without trypsin Blank medium 1535 31 Toxovar A 1515 бб
(TPGY) Blank medium 1535 31 Toxovar B 1495 38
Blank medium 1459 31 Toxovar C 14б4 1б
Blank medium 1459 31 Toxovar D 14б3 43
Blank medium 1б0б 5б Toxovar E 1598 57
Blank medium 10б9 33 Toxovar F 10б2 3
Blank medium 1459 31 Toxovar G 1459 10б
Blank medium 1380 40 Cl. sporogenes 13б2 29
With trypsin Blank medium 1059 23 Toxovar A 1031 58
(TPGYT) Blank medium 1059 23 Toxovar B 1040 18
Blank medium 1531 47 Toxovar C 1508 37
Blank medium 1531 47 Toxovar D 1530 б9
Blank medium 181б 105 Toxovar E 1859 102
Blank medium 2027 100 Toxovar F 2072 154
Blank medium 1891 85 Toxovar G 2039 225
Blank medium 181б 105 Cl. sporogenes 1805 94
* mOD = milli-optical density
the only one to cause a significantly lower exocyto-sis-activity (Table 4). After detoxification of the supernatant, a significantly lower exocytosis-activity was still observed. The measurement of the protein amounts in the supernatant and in the blank medium control (TPGYT) revealed differences (145 mg-mH and 4б mg-ml-1, resp.). They were adjusted by dilution. Afterwards, the exocytotic activities did not differ significantly anymore (Table 5).
PHAGOCYTOTIC ACTIVITY
Sterile filtrates of protozoan cultures did not show any detectable influence on the concentration of the bacteria (Table б), compared with the control (VOLVIC®-water instead of sterile filtrate).
Table 3. Number of cells of T. pyriformis at the beginning and the end of the assays to determine the exocytotic and phagocytotic activity.
Group Time in h Mean (n=3) log10cfu-ml-1 SD
Exocytosis assay 0 5.22 0.02
5 5.24 0.03
Phagocytosis assay 0 4.53 0.08
4 4.45 0.13
Therefore, the decrease of the cfu of E. coli K12 in a suspension with T. pyriformis was only due to the
Table 4. Exocytotic activity of T. pyriformis after incubation with culture supernatants of Cl. botulinum and Cl. sporogenes, resp., for 5 h, unspecific background signals subtracted, propotional (exocytotic activity in the blank media controls = 100%).
Cultivation medium Supernatant of Mean (n=9), % SD
Without trypsin Toxovar A 84 б
(TPGY) Toxovar B 88 3
Toxovar C 109 8
Toxovar D 11б 7
Toxovar E 88 17
Toxovar F 91 18
Toxovar G 103 12
Cl. sporogenes 94 9
With trypsin Toxovar A 89 14
(TPGYT) Toxovar B 92 2
Toxovar C 100 12
Toxovar D 102 8
Toxovar E бб* 2
Toxovar F 99 14
Toxovar G 9б 22
Cl. sporogenes 87 8
* p < 0,01
Table S. Exocytotic activity of T. pyriformis after incubation with culture supernatants of Cl. botulinum for 5 h, unspecific background signals subtracted, propotional (exovytotic activity in the blank media controls = 100%).
Cultivation medium Supernatant of Mean (n=9), % SD
With trypsin Toxovar E бб* 1
(TPGYT) Toxovar E, detoxified б5* 10
Toxovar E, protein amount adjusted 98 24
* p < 0,01
phagocytotic activity of the protozoa. The number of cells remained constant during the assay determining the phagocytotic activity (Table 3).
The culture supernatants of Cl. sporogenes did not affect the phagocytotic activity of the protozoa compared with the blank media controls. The neurotoxin-containing supernatants of the different Cl. botulinum toxovars did not significantly affect the phagocytotic activity of T. pyriformis, either, neither in the trypsinized nor in the non-trypsinized variant,
Table б. Log10cfu-mH of E. coli K12 after incubation with a sterile filtrate of a T. pyriformis culture and VOLVIC®-water, resp., for 4 h.
Group Mean (n=3), log10cfu^ml-1 SD
Sterile filtrate VOLVIC®-water control б.58 б.79 0.22 0.20
except the supernatants of toxovar E (Table 7). The detoxified variants still impaired the phagocytotic activities in the same way. The amount of protein in the non-trypsinized supernatant of toxovar E differed from the blank medium control (TPGY) as well (100 mg-ml-1 and 43 mg-ml-1, resp.). The protein concentrations in the culture supernatants were adjusted by dilution. No further effect on the phagocytotic activities was observed (Table 8).
Discussion
The addition of trypsin to the culture medium of the Cl. botulinum strains is shown to enhance the toxicity of the neurotoxins A (Krysinski and Sugi-
Table 7. Log10cfu-mH of E. coli K12 after incubation with T. pyriformis and blank media (control) and culture supernatants of Cl. botulinum and Cl. sporogenes, resp., for 4 h.
Cultivation medium control Mean (n=9), log10cfu^ml-1 SD Supernatant of Mean (n=9), log10cfu^ml-1 SD
Without trypsin Blank medium 4.39 0.22 Toxovar A 4.37 0.18
(TPGY) Blank medium 4.33 0.19 Toxovar B 4.47 0.14
Blank medium 4.73 0.27 Toxovar C 4.7б 0.24
Blank medium 4.б1 0.40 Toxovar D 4.б2 0.32
Blank medium 5.23 0.3б Toxovar E 7.51* 0.28
Blank medium 4.4б 0.1б Toxovar F 4.5б 0.14
Blank medium 4.б1 0.40 Toxovar G 5.05 0.5б
Blank medium 4.71 0.28 Cl. sporogenes 5.13 0.25
With trypsin Blank medium 4.48 0.17 Toxovar A 4.7б 0.42
(TPGYT) Blank medium 4.48 0.17 Toxovar B 4.59 0.19
Blank medium 4.б1 0.22 Toxovar C 4.б7 0.25
Blank medium 4.б1 0.22 Toxovar D 4.б4 0.40
Blank medium 4.40 0.47 Toxovar E 7.03* 0.19
Blank medium 4.48 0.17 Toxovar F 4.55 0.14
Blank medium 4.б1 0.22 Toxovar G 4.50 0.37
Blank medium 4.99 0.43 Cl. sporogenes 4.б9 0.29
* p < 0,001
Table S. Log10cfu-ml-1 of E. coli K12 after incubation with T. pyriformis and blank media (control) and culture supernatants of Cl. botulinum toxovar E, resp., for 4 h.
Cultivation medium control Mean (n=9), log10cfu^ml-1 SD Supernatant of Mean (n=9), log10cfu^ml-1 SD
Without trypsin Blank medium 5.23 0.3б Toxovar E 7.51* 0.28
(TPGY) Blank medium 5.09 0.53 Toxovar E, detoxified 7.4б* 0.25
Blank medium 4.79 0.70 Toxovar E, protein amount adjusted 4.97 0.42
With trypsin Blank medium 4.45 0.38 Toxovar E 7.03* 0.19
(TPGYT) Blank medium 4.34 0.14 Toxovar E, detoxified 7.00* 0.50
Blank medium 4.б8 0.48 Toxovar E, protein amount adjusted 4.57 0.33
yama, 1981); B (Hallis et al., 1996); D (Miyazaki et al., 1977); E (Duff et al., 1957); F (Holdeman and Smith, 1965) and G (Gimenez and Ciccarelli, 1970). There are controversial discussions about toxovar C enhancement (Baumgart, 1970), no effect (Eklund and Poyski, 1972), nevertheless, it was treated like the others.
Differences in the mouse bioassay due to the addition of trypsin were only seen in toxovar E, but BoNT concentrations were not specified further than the decimal power for ethical reasons, and so it is possible that the differences caused by the addition of trypsin were not revealed. The different toxovars produced their toxins in the same amounts, except toxovar E. Since no other strain of toxovar E was available, we had to work with a lower toxin concentration.
Cl. sporogenes served as additional unspecific control because of its similarity to Cl. botulinum. There is a strong DNA-homology and it is not possible to distinguish between those two species just by metabolic or biochemical parameters (Cato et al., 1986). Therefore, we concluded that Cl. sporogenes releases into the medium the same or very similar products as Cl. botulinum, except the neurotoxins.
Due to the assay design, the effective doses of the neurotoxins were not as high as those detected in the mouse bioassay because they were automatically diluted by the usage in both of our assays. Thus, the effective doses were 1.5-102 MLD-ml-1 (toxovars A-D, F and G) and 1.5 MLD-ml-1 (toxovar E).
All available BoNT-containing supernatants were tested in both of our assays for the exocy-totic and phagocytotic activities. Though the non-
trypsinized toxovar E did not show any toxicity in the mouse bioassay, it cannot be excluded that our assays could have been more sensitive.
The microscopical examination of protozoan cultures that were exposed to the different neurotoxins revealed no differences compared to the controls without BoNTs in contrast to the results published by Graf (1985) and De Waart et al. (1972). Although our observation period was shorter than in their assays (4 and 5 h, resp. compared to 1—5 days), there is no hint in the literature that SNARE-proteins play any role in cell division.
The acid phosphatase is transported to the exocytotic sites via lysosomes (Tiedtke et al., 1993). The amount of enzyme released into the medium can be determined by its activity (Rasmussen et al., 1992) and is influenced by the age of a Tetrahymena culture. Cells in a static growth phase release more acid phosphatase than those in an exponentially growing phase. Therefore, we used older cultures in the exo-cytosis assay than in the phagocytosis assay.
Since the detoxified supernatant of toxovar E impaired the release of acid phosphatase in a similar degree as the one with active neurotoxin, the lower performance had to be attributed to other reasons. The considerably higher amount of protein in the supernatant compared to the blank medium control could be proven to cause the differing exocytotic activities.
The usage of a vital prey germ offered several advantages. The phagocytotic activity is more than the simple incorporation of particles. A complex interaction of different membrane fusion processes is required. All of them are presumably mediated by
proteins of the SNARE-complex: the transport of membrane vesicles from the cytoplasm to the cyto-stome to form the future food or digestive vacuole (Hausmann and Radek, 1996), the lysosomal traffic from the derivation in the endoplasmatic reticulum via processing in the Golgi apparatus to the fusion with the digestive vacuole and the process of egestion (Nilsson, 1987).
Rasmussen et al. (1992) assumed a contribution of secretion products to the extracellular digestion. Therefore, their influence on the bacteria was tested. The amount of enzymes released by the number of cells which we used was not sufficient to degrade vital germs of E. coli K12.
It was shown that the phagocytotic activity of T. pyriformis was not affected by BoNTs but was affected by the amount of protein introduced into the assay. The additional protein entry seemed to be more critical for the phagocytosis than for the exocytotic activity, since the non-trypsinized supernatant of toxovar E impaired the phagocytosis but not the release of acid phosphatase.
There might be several reasons why no effect of the neurotoxins either on the exocytotic or on the phagocytotic activity of T. pyriformis could be measured.
Firstly, the concentration of the BoNTs might have been insufficient. As they could be easily detected in the mouse bioassay, it did not seem necessary to use higher concentrations when looking for an alternative assay.
It is also not clear whether the BoNTs could reach their substrates in an appropriate way. But even if Tetrahymena does not possess receptors to take up the BoNTs via endocytosis as other eukaryotic cells, the neurotoxins might have gained access to their substrates at least via phagocytosis of medium or during the engulfment of the bacteria. Since the contents of the digestive vacuoles also undergo acidification, the conditions in them are probably similar to those in the vesicles ofthe motor nerve cells (Singh, 2000). Nevertheless, this remains speculative, for we did not examine the way of the BoNTs into or through the protozoa.
There is also a probability that SNARE-proteins are not involved in the exocytosis and phagocytosis of T. pyriformis. However, since such an involvement is described in many eukaryotic cells (Brumell et al., 1995; Desjardins et al., 1997; Hackam et al., 1998; Bogdanovic et al., 2002; Kissmehl et al., 2002; Froissard et al., 2002), it is more likely that the effect was not visible due to mutations in the binding or cleaving sites of the presumable SNARE-isoforms in T. pyriformis.
Thus, T. pyriformis cannot provide an alternative for the detection of BoNTs.
Acknowlegdements
The authors are grateful to Arne Rodloff and Joerg Beer (Faculty for Medicine, University of Leipzig) for providing bacteria strains, Helge Boehnel and Frank Gessler (Faculty ofAgricultural science, Georg-August-University, Goettingen) for providing the missing toxovars and for testing the supernatants in the mouse bioassay, Wilfried Pauli (Department of Biology, Chemistry and Pharmacy, Freie University, Berlin) for providing T. pyriformis and Ilse Staender and Anja Mueller for the revision of the English version.
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Address for correspondence: Norman Martin Staender. Institut fur Bakteriologie und Mykologie, Veterinar-medizinische Fakultat der Universitat Leipzig, An den Tierkliniken 29, 04103 Leipzig, e-mail: staender@ vetmed.uni-leipzig.de
