J. Microbiol. Biotechnol. (2011), 21(11), 1193–1198
http://dx.doi.org/10.4014/jmb.1105.05040
First published online 22 August 2011
Cytotoxicity of Listeriolysin O Produced by Membrane-Encapsulated
Bacillus subtilis on Leukemia Cells
Stachowiak, R.1*, L. H. Granicka2, J. Wiósniewski1†, M. Lyz· niak3, J. Kawiak2,3, and J. Bielecki1
1
Department of Applied Microbiology, Faculty of Biology, University of Warsaw, 02-096 Warsaw, Poland
M. Na l cz Institute of Biocybernetics and Biomedical Engineering Polish Academy of Science, 02-109 Warsaw, Poland
3
Medical Center of Postgraduate Education, 01-813 Warsaw, Poland
2
Received: May 20, 2011 / Revised: July 4, 2011 / Accepted: July 13, 2011
Encapsulation of biological material in the permiselective
membrane allows to construct a system separating cells
from their products, which may find biotechnological as
well as biomedical applications in biological processes
regulation. Application of a permiselective membrane
allows avoiding an attack of the implanted microorganisms
on the host. Our aim was to evaluate the performance of
Bacillus subtilis encapsulated in an elaborate membrane
system producing listeriolysin O, a cytolysin from Listeria
monocytogenes, with chosen eukaryotic cells for future
application in anticancer treatment. The system of
encapsulating in membrane live Bacillus subtilis BR1-S
secreting listeriolysin O was proven to exert the effective
cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin
O showed selective cytotoxic activity on eukaryotic cells:
more human leukemia Jurkat T cells were killed than
human chronic lymphocytic B cells leukemia at similar
conditions in vitro. This system of encapsulated B. subtilis,
continuously releasing bacterial products, may affect
selectively different types of cells and may have future
application in local anticancer treatment.
Keywords: Hollow fiber, Bacillus subtilis, encapsulation,
listeriolysin
Encapsulation of biological material in the permiselective
membrane allows to construct a system separating cells
from their products, which may find biotechnological as
well as biomedical applications in biological processes
regulation [4, 5, 9, 11, 14, 15, 17, 19]. Application of a
permiselective membrane physically separating implanted
*Corresponding author
Phone: +48 22 5541312; Fax: +48 22 5541402;
E-mail: [email protected]
†
J. Wiósniewski is deceased
microorganisms from the host protects both the host (from
the attack of the implanted microorganisms on the host) and
the bacteria (from the host immunological response) [7].
Bacillus subtilis is a model Gram-positive bacterium.
This organism is widely used in industry, and several
enzymes produced by B. subtilis are generally regarded as
safe (GRAS). Its enormous secretion efficiency as well as
the remarkable knowledge about its growth and fermentation
conditions, combined with the possibility of gaining
GRAS status for B. subtilis-derived proteins, makes this
microorganism ideal for many biotechnological applications.
The most remarkable feature of B. subtilis is its enormous
secretion machinery. Consequently, this microorganism is
widely used both in research and industry for efficient
proteins production [10, 12, 16]. B. subtilis was the first
platform for heterologous expression of listeriolysin O
(LLO), a hemolysin from the pathogenic bacterium Listeria
monocytogenes [2]. This experiment demonstrated the thin
boundary between pathogenic and saprophytic microorganisms
and how important LLO is for intracellular parasitism. This
protein belongs to the family of cholesterol-dependent
cytolysins (CDCs), a group of pore-forming toxins produced
by related Gram-positive bacteria. The CDCs share very
similar amino acid sequences and therefore it is believed
that these toxins also share very similar planar structure.
Their biochemical properties are nearly identical, with
very few exceptions. Cholesterol dependence is not one of
them, hence the name, which refers to the strict requirement
of this lipid in the membrane for the toxin’s pore-forming
activity to take effect [1]. Although cholesterol is present
in all eukaryotic cells membranes, cell susceptibility to
cytolysis differs with respect to toxin and cell type. This
phenomenon was described very early [18], but still very
little is known about its mechanism. It is postulated that
cholesterol may affect the activity of CDC toxins directly,
or/and it may also influence the following factors apparently
1194
Stachowiak et al.
important for membrane-toxins interaction: membrane
fluidity, line tension, and the stabilization of lipid rafts in
the cellular membrane [13]. L. monocytogenes cytolysin
shows most of the typical CDC features, with one major
exception: LLO shows maximal activity in acidic pH,
whereas the remaining toxins favor neutral pH. Combined
with a short half-life and reduced expression when the
pathogen is already in the eukaryotic cell cytoplasm, this is
a major adaptation of L. monocytogenes to the intracellular
lifestyle [20].
A polypropylene surface-modified hollow fiber was
applied for encapsulation of the coated polyelectrolyte
shells with B. subtilis cells. The applied membranes
prevent bacterial escape from the lumen of the hollow
fiber. Our aim was to evaluate the performance of B.
subtilis encapsulated in membrane system in producing a
biologically active substance with chosen eukariotic cells,
for future application in anticancer treatment.
MATERIALS AND METHODS
HF Membrane Modification
Hollow fibers (HF) of polypropylene Q1 (Membrana, Germany),
inner diameter 0.6 mm, wall thickness 0.2 mm, were used. HF were
modified with the polyelectrolyte layer as follows: poly-L-lysine
hydroxybromide, mol. wt. 1,500-3,000 (PLL) layer and polyethyleneimine solution (PEI) layer (both from Sigma, USA) were
adsorbed to the hollow fiber membrane structure. The solution in
0.1 M NaCl of PLL or PEI was introduced into a membrane
structure by its exposure to it in a pressure compartment (-0.1 MPa)
at 20oC for 5 min. After the deposition time, HF membranes were
washed with PBS to remove unadsorbed polyelectrolyte. The
polyelectrolytes were introduced consecutively into a membrane
structure one by one: PLL at concentration 4 mg/ml and PEI at
concentration 4 mg/ml to obtain four layers. The modified HF
membrane surface is presented in Fig. 1. The modified HF were
filled up with bacterial suspension and closed at both ends.
Bacterial Strain
Bacillus subtilis strain BR1-S is an asporogenous derivative of strain
ZB307 [21] producing lysteriolysin (LLO). Gene hly encoding LLO
from L. monocytogenes strain 10403S was amplified using Taq
polymerase (Qiagen) with the following primers: forward 5'-GCTCTA
GAAGAGAGGGGTGGCAAACGGT-3' (XbaI site underlined) and
reverse 5'-GCGGTCGACGGTACCTTTCGTGTGTGTTAAGCGGT-3'
(sites for SalI and KpnI underlined). PCR was run in a Thermal
Cycler (MJ Research, Inc.). Ampified 1.7 kb DNA fragment containing
the hly gene was purified with a Gel-out kit (A&A Biotechnology)
and subsequently digested with XbaI and SalI (Fermentas) enzymes.
After treatment with a Purification Kit (A&A Biotechnology), the PCR
product was cloned in vector pAG58 using T4 ligase (Fermentas)
and transformed into Escherichia coli for further analysis. Recombinant
clones were selected with the restriction analysis method and were
sequenced for cloning verification. Plasmid pAG58-hly was isolated
from E. coli and introduced into B. subtilis strain ZB307, resulting in
strain BR1-S. Bacteria were grown at 37oC with 120 rpm agitation
Fig. 1. Electron microscopy image of modified polypropylene
HF with Bacillus subtilis (A) and unmodified polypropylene HF (B).
in LB medium (Sigma, USA) or BHI (Becton-Dickinson, USA)
supplemented with erythromycin (1 µg/ml) and chloramphenicol
(3 µg/ml). To induce LLO production and release by the living
bacteria, the HF-encapsulated bacteria were incubated in the
presence of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)
(Sigma, USA).
Human Peripheral Blood Mononuclear Cells Preparation
The suspension of peripheral blood mononuclear cells was obtained
from WBC of chronic lymphocytic B leukemia patients at 40,000 to
100,000 leukocytes/µl (B-CLL). The patient’s leukemia B cells
represented at least 90% of the whole leukocytes.
The blood was diluted twice with physiological saline, mixed,
and layered on Accu-Prep reagent for mononuclear cells isolation at
the volume ratio of 3:1, and centrifuged for 30 min at 2,000 rpm. The
mononuclear cells (MNC) suspension (involving mainly lymphocytes
and monocytes) was collected from interphase, and RPMI-1640 with
10% CS culture medium was added at 1:1 ratio, centrifuged for
15 min at 1,200 rpm and then suspended for culture. The MNC
were initially cultured for 7 days to eliminate broken cells as well as
trace erythrocytes and monocytes. Blood collection was approved by
patients, and the procedure was approved on 27 February 2008 by the
CYTOTOXICITY OF MEMBRANE-ENCAPSULATED BACTERIA
Local Ethics Committee of the Medical Center of Postgraduate
Education, Warsaw, Poland.
Evaluation of Encapsulated Bacteria Impact on Erythrocytes
The erythrocytes were prepared from 5-fold-diluted in PBS (v/v)
sheep blood (Biomed, Poland), washing three times with PBS, pH
7.4 (1,200 rpm, 5 min, at 4oC), and finally suspended in PBS, pH
7.4. The incubation medium consisted of 20% (vol) erythrocyte
suspension in BHI/5 with addition of erythromycin (Sigma, USA)
3 µg/ml, chloramfenicol (Sigma, USA) 1 µg/ml, and 1 mM IPTG.
BHI/5 is the BHI medium diluted with physiological saline at 1:4.
Encapsulated in membrane system bacteria BR1-S at initial concentration
of 108 cells/ml were placed and cultured in 0.5 ml of erythrocyte
suspension (5% CO2, 37oC). The erythrocyte suspension samples were
cultured with encapsulated bacteria BR1-S not induced with IPTG
addition as well. As a negative control, the erythrocyte suspensions
were cultured with empty membranes. The erythrocyte suspension
samples were collected after 24 h to evaluate erythrocyte density
spectrophotomerically at 410 nm (optical density changes of the
sample).
As a positive control, 0.01% sodium dodecyl sulfate (SDS)
(Sigma, USA) was applied to check for total hemolysis.
Evaluation of Encapsulated Bacteria Impact on T-Lymphocytes
Jurkat Cell Line
The Jurkat human leukemia T-lymphocyte cell line (American Type
Culture Collection, Rockville, MD, USA) was maintained in RPMI1640 supplemented with 10% newborn calf serum and 1% penicilin
and streptomycin. Cells were passaged every third day by diluting to
the concentration of about 0.5×106. The cells were grown at 37oC in
atmosphere of 5% CO2 in air. Bacteria BR1-S encapsulated in
membrane system at initial concentration of 108 cells/ml were cultured
(5% CO2, 37oC) in a suspension of Jurkat cells at the concentration
of 1.5×106 cells/ml in the culture medium with addition of erythromycin
(3 µg/ml) and 1 mM IPTG for 24 h. The cells suspension was also
cultured with encapsulated bacteria BR1-S without IPTG addition.
As a negative control, the cell suspension was cultured with empty
membranes. The samples were collected after 24 h, to evaluate the
cell viability in a Canto II flow cytometer (Becton Dickinson
Immunocytochemistry Systems, USA). The cytochemical reaction
with propidium iodide (PI, final concentration 5 µg/ml) was performed.
PI enters cells with damaged cell membrane and binds to nucleic
acids, thereby producing a red fluorescence of dead cells. The
results were processed by the FACS Diva software system (Becton
Dickinson, USA).
Evaluation of Encapsulated Bacteria Impact on Peripheral
Blood Mononuclear Cells Obtained from Leukemia Patients
Bacteria BR1-S encapsulated in membrane system at initial
concentration of 108 cells/ml were cultured (5% CO2, 37oC) in a
suspension of peripheral blood mononuclear cells (MNC) obtained from
B-CLL patients and suspended at a concentration of 1.5×106 cells/ml
with addition of 3 µg/ml erythromycin and 1 mM IPTG for 24 h.
The MNC suspension was cultured in RPMI-1640 supplemented
with 10% newborn calf serum (Gibco, USA) with encapsulated
bacteria BR1-S with/without IPTG addition. As a negative control,
the MNC suspension was cultured with empty membranes. The
samples were collected after 24 h to evaluate the cell viability in a
flow cytometer in a cytochemical reaction with propidium iodide.
1195
Evaluation of Bacteria Closure Tightness
After the procedures described above, the suspension of evaluated
eukaryotic cells was divided into two equal volumes and cultures
were continued: (i) after removal of membranes with encapsulated
within bacteria BR1-S as a negative control; if the bacteria formerly
escaped from the HF to the outside culture medium, they would
proliferate and produce toxin still influencing cells viability; (ii) with
the membranes containing encapsulated BR1-S cut in pieces for
liberation of bacteria to the culture medium, as a positive control.
After the next 24 h, the cell viability was assessed in a flow a
cytometer in cytochemical reaction with propidium iodide.
Statistics
Mean values and standard deviations as well as significance of
difference were calculated in the StatView 512 software. The values
of p <0.05 were assumed as significant.
RESULTS
HF Membrane Modification
In Fig. 1, a fragment of the polypropylene membrane with
bacterial cell is presented. Differences in pore size as
compared with dimensions of the bacteria may be noted.
Changes in bacteria conformation would allow their
passage through the membrane structure, even when the
tested membrane cut-off (200 kDa) is lower than the
bacteria size. However, to ensure tight closing of cells
within the membrane system, membrane modification was
applied. This prevented the leakage of bacterial cells from
the HF within the period of observation.
Evaluation of Encapsulated Bacteria Impact on
Erythrocytes in 24 h Experiment
The encapsulated B. subtilis exhibited different hemolytic
activity depending on pH. The hemolysis of 32% or 48%
with application of cysteine (reductive agent) was observed
at pH 7.4; whereas at pH 5.6 there was 35% or 64% (with
cysteine) of total hemolysis. The activity was in accordance
with literature data for listeriolysin [20]. Total hemolysis
(100%) was induced by 0.01% SDS.
Assessing the immobilized B. subtilis impact on
erythrocytes, it was observed that the optical density of
erythrocyte suspension (number of erythrocytes) declined
about twice when cultured with IPTG-induced, encapsulated
bacteria as compared with the non-induced BR1-S and
negative control. This is suggesting that encapsulated
BR1-S is able to produce and release LLO. The LLO
crossing the membrane wall exhibited activity in erythrocyte
culture outside the HF, resulting in hemolysis (Fig. 2).
There was significant statistical difference between the
optical density representing the erythrocyte number when
cultured with encapsulated BR1-S +IPTG and encapsulated
BR1-S or empty HF (p=0.007 and p=0.002, respectively).
There was no statistical difference (p=0.520) between
1196
Stachowiak et al.
Fig. 2. The erythrocytes suspension optical density values after
24 h culture in the presence of encapsulated B. subtilis BR1-S.
BR1-S+IPTG, the erythrocyte suspension cultured with IPTG-induced
encapsulated bacteria BR1-S (n=12); BR1-S, the erythrocytes suspension
cultured with encapsulated bacteria BR1-S, not induced (n=6); Negative
control, the erythrocytes suspension cultured with empty membranes
(n=6). The values are presented as mean±SD.
negative control (empty HF) and culture in the presence of
noninduced BR1-S.
Evaluation of Encapsulated Bacteria Impact on Human
Leukemia Jurkat Cells
The number of viable Jurkat cells declined when cultured
for 24 h with encapsulated B. subtilis, as compared with a
negative control (4.6±2.0% and 71.2±8.1% living cells,
respectively). The percent was calculated for Jurkat cells
gated (100%) on a FSC/SSC diagram. This suggests the
effective action of bacterial toxins produced by membraneencapsulated B. subtilis cells. The ratio of percent number
of viable cells cultured in the presence of encapsulated
BR1-S induced with IPTG (BR1-S+IPTG) or not induced
(BR1-S) to percent number of viable Jurkat cells cultured
without encapsulated B. subtilis (negative control) is
presented in Fig. 3.
There was no Jurkat cells viability decline observed
during subsequent 24 h culture in evaluated samples after
removal of HF with encapsulated B. subtilis from the
former Jurkat cell culture.
Evaluation of Encapsulated Bacteria Impact on
Peripheral Blood Mononuclear Cells Obtained from BCLL Patients
It was observed that the viability of leukemia cells declined
as compared with a negative control (75.4±6.4% and
93.1±3.0% live cells, respectively) when cultured for 24 h
with encapsulated bacteria. There was statistical significant
difference between the MNC viability cultured with
encapsulated BR1-S+IPTG and encapsulated BR1-S
without IPTG or empty HF (negative control) (p=0.014
and p=0.0007, respectively). There was no statistical significant
difference between negative control (empty HF) and culture
in the presence of BR1-S ( p=0.302).
Fig. 3. Evaluation of encapsulated bacteria impact on Jurkat
cells: the ratio of percent number of viable cells after 24 h culture
in the presence of encapsulated BR1-S induced with IPTG (BR1S+IPTG) or not induced (BR1-S) to percent number of viable
Jurkat cells cultured without encapsulated B. subtilis (negative
control).
BR1-S+IPTG, the Jurkat suspension cultured with induced encapsulated
bacteria BR1-S (n=12); BR1-S, the Jurkat suspension cultured with
encapsulated bacteria BR1-S, not induced (n=6); Negative control, the
Jurkat suspension cultured with empty membranes (n=6). Values are
presented as mean±SD.
There was no observed MNC viability decline during
subsequent 24 h culture after removal of HF-encapsulated
B. subtilis from the MNC culture (data not presented).
Therefore, we did not observe the result of free bacteria
activity outside of the membrane. This suggests the effective
action of the bacterial toxins produced by membraneencapsulated B. subtilis cells. In Fig. 4 is presented the
ratio of percent number of viable cells cultured in the
presence of encapsulated BR1-S induced with IPTG (BR1S+IPTG) or not induced (BR1-S) to percent number of
viable MNCs cultured without B. subtilis (negative control).
Comparision of B and T Leukemia Cells Susceptibility to
LLO
We compared the cytotoxic effect of LLO produced by
immobilized BR1-S cells on lymphocytes T (Jurkat cells)
and B (MNCs from B-CLL patients). The percent number
of living cells, after 24 h incubation with HF-encapsulated
B. subtilis with removed background (spontaneous
necrosis / apoptosis that could be detected in negative
control with empty HF) was 81% of LLO-treated B-CLLs,
which remained viable, and nearly 0% Jurkat cells, which
were nearly all eliminated. However, Jurkat cells were also
much more sensitive after incubation with noninduced
BR1S cells; only 27% of T cells was intact, pointing out
that the cells are overall less resistant to test conditions. To
evaluate the differences in lymphocytes susceptibility to
LLO, results should be presented as ratio of BR1S+IPTG
treated eukaryotic cells to lymphocytes incubated with
B. subtilis without IPTG (Fig. 5). In this way, not only the
background caused by spontaneous necrosis but also
CYTOTOXICITY OF MEMBRANE-ENCAPSULATED BACTERIA
Fig. 4. Evaluation of encapsulated bacteria impact on peripheral
blood mononuclear cells obtained from leukemia patients: the
ratio of percent number of viable cells after 24 h culture in the
presence of encapsulated BR1-S induced with IPTG (BR1S+IPTG) or not induced (BR1-S) to percent number of viable
MNC cultured without encapsulated B. subtilis (negative control).
BR1-S+IPTG, the MNC suspension cultured with induced encapsulated
bacteria BR1-S (n=12); BR1-S, the MNC suspension cultured with
encapsulated bacteria BR1-S, not induced (n=6); Negative control, the
MNC suspension cultured with empty membranes (n=6). Values are
presented as mean±SD.
activity caused by other naturally produced B. subtilis
proteins (i.e., proteases) can be removed, enabling the
LLO cytotoxicity to be revealed. Results processed in such
manner show that 15% of viable B-CLLs were eradicated
with LLO, whereas 22% of viable Jurkat T cells were
eliminated.
DISCUSSION
Some encapsulated microorganisms may carry a transfected
gene of another organism, and thus become a source of
Fig. 5. Evaluation of the differences in lymphocytes susceptibility
to LLO: the percent number of viable cells eradicated with B.
subtilis-produced LLO.
Results are presented as the ratio of BR1S+IPTG-treated eukaryotic cells
to lymphocytes incubated with B. subtilis without IPTG compared with the
negative control (cells suspension cultured with empty membranes).
1197
valuable regulatory factors. Such factors released in strategic
locations may direct or modify the biological processes in
the eukaryotic organism [8].
Encapsulated Bacillus subtilis demonstrated hemolytic
as well as cytolytic activity, producing and secreting the
toxin listeriolysin O (LLO), which diffuses to the culture
medium and kills the eukaryotic cells: sheep erythrocytes,
human T- lymphocyte cell line Jurkat cells, and peripheral
blood mononuclear cells from leukemia patients. LLO
production by B. subtilis BR1S strain is IPTG-dependent.
Accordingly, the encapsulated not-induced B. subtilis
exerted the toxic impact on Jurkat cells or MNC cells,
respectively, at 21% or 14% lower as compared with B.
subtilis induced with IPTG, suggesting that the induction of
LLO production enhanced the toxic impact of bacteria on
leukemia cells. There was statistical difference between
the percent number of viable cells of both types of
leukemia cultured in the presence of BR1-S or BR1-S +
IPTG.
Two types of target leukemia cells were analyzed during
the culture with encapsulated bacteria presence: Jurkat
(human T-lymphocyte cell line) and peripheral blood
mononuclear cells of patients with chronic lymphocytic B
leukemia. The main cell population in the leukemia cells
suspension were B cells, while T cells and NK lymphocytes
were only the minor fraction. It was observed, that the
viability of the Jurkat cells incubated 24 h with the HFencapsulated and IPTG-induced bacteria declined, as
compared with the negative control. A similar effect of
encapsulated B. subtilis BR1-S was observed with
leukemia mononuclear cells. This suggests an effective
activity of bacterial toxins produced by HF membraneencapsulated Bacillus subtilis BR1-S cells. Interestingly,
the killing by encapsulated B. subtilis toxins of B-leukemia
cells was less effective than in the experiment with Jurkat
T-cells (Fig. 5). There was observed difference in the
viability of cells tested in the same conditions with IPTG
as compared with a negative control (culture with empty
HF). The difference was 18% and 67% for human B
leukemia and Jurkat T leukemia cells, respectively,
suggesting the selective activity of bacteria toxins to
different leukemia cells. More research is necessary to
confirm this hypothesis, since T and B cells came from
different sources and Jurkat cells may be simply more
sensitive to the testing conditions, as indicated by the low
viability of these cells in the negative control compared
with B-CLLs (Fig. 5). Still, a more specific action of LLO
towards T cells is possible, since more viable lymphocytes
T than B were killed. Such a specifity is a very attractive
hypothesis, since lymphocytes T are the main obstacle to
succesfull intracellular proliferation of L. monocytogenes,
and more mechanisms than direct cytolysis were described
for LLO-dependent T cells elimination like apoptosis [3]
and induced T cell unresponsiveness [6].
1198
Stachowiak et al.
In conclusion, the applied membrane system allows to
avoid the release of Bacillus subtilis through the
membrane wall. The system of membrane-encapsulated
living Bacillus subtilis BR1-S secreting listeriolysin O
proves the selective cytotoxic activity on eukaryotic cells,
as more Jurkat leukemia T cells were killed than leukemia
B cells under similar conditions in vitro. The system of
encapsulated B. subtilis locally, continuously releasing
bacterial products, may affect selectively different types of
cells and may have future application in local anticancer
treatment.
8.
9.
10.
Acknowledgments
This study was partly supported by grant N N401 015936
obtained from MNiSW, Polish Ministry of Science and
Higher Education; grant CMKP 2008/2009, UW Faculty
of Biology Grant BW-179157; and grant N R13 0046 06
funded by NCBiR, National Center of Research and
Development. This work was supported by the European
Union structural funds, Innovative Economy Operational
Program POIG.01.01.02-00-109/09-00.
11.
12.
13.
14.
15.
REFERENCES
1. Alouf, J. E., S. J. Billington, and B. H. Jost. 2005. Repertoire
and general features of the family of cholesterol-dependent
cytolysins, pp. 643-658. In J. E. Alouf and M. R. Popoff (eds.).
The Comprehensive Sourcebook of Bacterial Protein Toxins, 3rd
Ed. Academic Press, London; San Diego.
2. Bielecki, J., P. Youngman, P. Connelly, and D. A. Portnoy.
1990. Bacillus subtilis expressing a haemolysin gene from
Listeria monocytogenes can grow in mammalian cells. Nature
345: 175-176.
3. Carrero, J. A., H. Vivanco-Cid, and E. R. Unanue. 2008.
Granzymes drive a rapid listeriolysin O-induced T cell apoptosis.
J. Immunol. 181: 1365-1374.
4. Chang, T. M. and S. Prakash. 2001. Procedures for microencapsulation
of enzymes, cells and genetically engineered microorganisms.
Mol. Biotechnol. 17: 249-260.
5. Charalampopoulos, D., R. Wang, S. S. Pandiella, and C. Webb.
2002. Application of cereals and cereal components in functional
foods: A review. Int. J. Food Microbiol. 79: 131-141.
6. Gekara, N. O., N. Zietara, R. Geffers, and S. Weiss. 2010.
Listeria monocytogenes induces T cell receptor unresponsiveness
through pore-forming toxin listeriolysin O. J. Infect. Dis. 202:
1698-1707.
ówiez·ewski, D.
7. Granicka, L. H., M. Wdowiak, A. Kosek, S. S
Wasilewska, E. Jankowska, A. Weryónski, and J. Kawiak. 2005.
16.
17.
18.
19.
20.
21.
Survival analysis of Escherichia coli encapsulated in a hollow
fiber membrane in vitro and in vivo: Preliminary report. Cell
Transplant. 14: 323-330.
Granicka, L. H., J. Z·olnierowicz, D. Wasilewska, A. Weryónski,
and J. Kawiak. 2010. Induced death of Escherichia coli
encapsulated in a hollow fiber membrane as observed in vitro or
after subcutaneous implantation. J. Microbiol. Biotechnol. 20:
224-228.
Hou, R. C., M. Y. Lin, M. M. Wang, and J. T. Tzen. 2003.
Increase of viability of entrapped cells of Lactobacillus
delbrueckii ssp. bulgaricus in artificial sesame oil emulsions. J.
Dairy Sci. 86: 424-428.
Ling Lin, F., X. Zi Rong, L. Wei Fen, S. Jiang Bing, L. Ping,
and H. Chun Xia. 2007. Protein secretion pathways in Bacillus
subtilis: Implication for optimization of heterologous protein
secretion. Biotechnol. Adv. 25: 1-12.
Moslemy, P., R. J. Neufeld, and S. R. Guiot. 2002. Biodegradation
of gasoline by gellan gum-encapsulated bacterial cells. Biotechnol.
Bioeng. 80: 175-184.
Nijland, R. and O. P. Kuipers. 2008. Optimization of protein
secretion by Bacillus subtilis. Recent Pat. Biotechnol. 2: 79-87.
Palmer, M. 2004. Cholesterol and the activity of bacterial
toxins. FEMS Microbiol. Lett. 238: 281-289.
Prakash, S. and T. M. Chang. 2000. Artificial cells microencapsulated
genetically engineered E. coli DH5 cells for the lowering of
plasma creatinine in-vitro and in-vivo. Artif. Cells Blood Substit.
Immobil. Biotechnol. 28: 397-408.
Prakash, S. and T. M. Chang. 2000. In vitro and in vivo uric
acid lowering by artificial cells containing microencapsulated
genetically engineered E. coli DH5 cells. Int. J. Artif. Organs.
23: 429-435.
Schallmey, M., A. Singh, and O. P. Ward. 2004. Developments
in the use of Bacillus species for industrial production. Can. J.
Microbiol. 50: 1-17.
Shah, N. P. 2000. Probiotic bacteria: Selective enumeration and
survival in dairy foods. J. Dairy Sci. 83: 894-907.
Smyth, C. J. and J. L. Duncan. 1978. Thiol-activated (oxygenlabile) cytolysins, pp. 129-183. In J. Jejaszewicz and T. Wadstrom
(eds.). Bacterial Toxins and Cell Membranes. Academic Press
Ltd, London.
Sultana, K., G. Godward, N. Reynolds, R. Arumugaswamy, P.
Peiris, and K. Kailasapathy. 2000. Encapsulation of probiotic
bacteria with alginate-starch and evaluation of survival in
simulated gastrointestinal conditions and in yoghurt. Int. J.
Food Microbiol. 62: 47-55.
Vazquez-Boland, J. A., R. Stachowiak, L. Lacharme, and M.
Scortti. 2005. Listeriolysin, pp. 700-716. In J. E. Alouf and M.
R. Popoff (eds.). The Comprehensive Sourcebook of Bacterial
Protein Toxins, 3rd Ed. Academic Press, London; San Diego.
Zuber, P. and R. Losick. 1987. Role of AbrB in Spo0A- and
Spo0B-dependent utilization of a sporulation promoter in Bacillus
subtilis. J. Bacteriol. 169: 2223-2230.