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Infection and Immunity, July 2000, p. 4363-4367, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cytocidal and Apoptotic Effects of the ClyA Protein from
Escherichia coli on Primary and Cultured Monocytes
and Macrophages
Xin-He
Lai,1,2,
Ignacio
Arencibia,3
Anders
Johansson,4
Sun Nyunt
Wai,1,5
Jan
Oscarsson,1
Sotos
Kalfas,4
Karl-Gösta
Sundqvist,3
Yoshimitsu
Mizunoe,5
Anders
Sjöstedt,2 and
Bernt Eric
Uhlin1,*
Departments of
Microbiology,1 Clinical
Bacteriology,2 Clinical
Immunology,3 and Oral
Biology,4 Umeå University, S-901 87 Umeå,
Sweden, and Department of Bacteriology, Graduate School of
Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan5
Received 4 November 1999/Returned for modification 3 February
2000/Accepted 1 April 2000
 |
ABSTRACT |
Cytolysin A (ClyA) is a newly discovered cytolytic protein of
Escherichia coli K-12 that mediates a hemolytic phenotype.
We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured
human and murine monocytes/macrophages.
 |
TEXT |
Recently it was discovered that a
chromosomal gene denoted clyA (also referred to as
sheA and hlyE) in Escherichia coli
K-12 encodes a novel hemolytic protein (5, 20, 21, 24, 25, 28; Y. Mizunoe and B. E. Uhlin, Abstr. 34th Intersci.
Conf. Antimicrob. Agents Chemother., abstr. B37, 1994). However, the gene product, the 34-kDa cytolysin A (ClyA) protein, does not seem to
be expressed under normal laboratory conditions. This normally latent
clyA gene can be activated either by mutation in the
hns locus or by overexpression of several putative
regulatory genes (5, 8, 9, 19-21, 24, 25, 28;
Mizunoe and Uhlin, 34th ICAAC). Both purified ClyA and ClyA-expressing
E. coli are able to lyse erythrocytes from several mammalian
species in both solid and liquid media, and we recently found that the
protein is cytotoxic to macrophages grown in tissue culture
(5, 8, 9, 19-21, 24, 25, 28; Mizunoe and Uhlin,
34th ICAAC). Those findings prompted us to further investigate the
interaction of ClyA-producing bacteria and purified ClyA with mammalian
cells.

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FIG. 1.
Cytotoxicity as manifested by LDH release from
murine macrophages J774. About 2 × 104 cells were
seeded each well in 96-well plates and treated with highly purified
ClyA or infected at different MOIs with ClyA-expressing or vector
control E. coli as described elsewhere (17, 18,
24). Killing was assayed at several time intervals and expressed
as percent cytotoxicity. Data are means ± standard deviations
(n = 4) from one representative experiment of three.
The E. coli strains used here do not have endogenous LDH
activity when grown aerobically. (A) Dose-dependent toxicity of
purified ClyA. Cells were pretreated with PMA as described elsewhere
(24). (B) Cytotoxicity of ClyA-expressing E. coli
MC1061/pYMZ80 compared with its plasmid vector control MC1061/pUC18 at
an MOI of 100. (C) Infection dose-dependent toxicity of ClyA-expressing
E. coli (MOI of 100 versus 10).
|
|
Cytotoxic effects of purified ClyA protein and ClyA-expressing
E. coli on fresh or cultured human and murine
monocytes/macrophages.
Cell morphology changes and detachment from
culture plates are conventional and useful indicators to monitor
bacterial cytotoxicity. We recently described that highly purified ClyA
can detach J774 macrophages from culture plates and change their cell
morphology (24). In the present study, we extended our
cytotoxicity measurements to other types of host cells by using a
neutral red uptake assay (2) and a quantitative lactate
dehydrogenase (LDH) release assay based on the fact that LDH is a
strictly cytoplasmic enzyme and its presence in the culture medium
reflects the disruption of the host cell plasma membrane
(16).
The macrophage cell lines J774 (murine) and U937 (human) were
maintained and treated as described previously (
17,
18,
24,
26,
27). Human polymorphonuclear leukocytes and monocytes
were isolated following a standardized procedure as described
previously (
11,
13). Highly purified ClyA preparations were
obtained from
E. coli K-12 cells carrying clone pYMZ80
(
24;
S. N. Wai and B. E. Uhlin,
unpublished data). Proteins were diluted
in complete medium and
sterilized by filtration through a 0.22-µm-pore-size
membrane
(Schleicher & Schuell FD 030/3). J774 cells, polymorphonuclear
leukocytes, or monocytes were treated with purified and filtrated
ClyA
in 200 µl (total volume) of cell medium. In some experiments,
cells
were pretreated with phorbol 12-myristate 13-acetate (PMA;
Sigma) as
described before (
24). For testing the effect of
cytochalasin
D (
4) on cytotoxicity, cells were pretreated
with 1 µg of cytochalasin
D (Sigma) ml
1 for 30 min
before bacterial infection, and cytochalasin D was
maintained
throughout the experiment. Treatment of the bacteria
and eukaryotic
cells with cytochalasin D at the above concentration
did not
significantly reduce cell or bacterial viability (data
not shown).
E. coli strains MC1061/pUC18 and MC1061/pYMZ80 were
used as described elsewhere (
24). J774 cells were infected
as
described elsewhere (
17,
18,
24) with bacteria at a
multiplicity
of infection (MOI) of 100, unless otherwise
indicated.
As shown in Fig.
1, LDH release was both ClyA concentration and
bacterial infection dose (i.e., MOI) dependent. ClyA at 20
µg
ml
1 caused more than 20% LDH release after 2 h of
treatment, while
no detectable LDH release came from cells treated with
one-sixth
as much ClyA (Fig.
1A). LDH release remained at baseline
level
for the vector control MC1061/pUC18, whereas the ClyA-expressing
strain MC1061/pYMZ80 was cytotoxic to J774 macrophages at each
time
interval tested (Fig.
1B and C). MC1061/pYMZ80 showed
approximately
one-third as much cytotoxicity at an MOI
of 10 compared with that
at an MOI of 100, at both 2 and 4 h
postinfection (p.i.). The
cytotoxicity was even greater at 6 h
p.i. and approached a level
similar to that at the higher MOI (Fig.
1C). The kinetics of cytotoxicity
within 6 h of treatment over
three time intervals are shown in
Fig.
1.
The effect of ClyA on viability of human primary monocytes was
monitored using a neutral red uptake assay as described before
(
2,
11,
13). The viability of freshly isolated human peripheral
monocytes showed a dose-related decline after treatment with ClyA
for
20 h (Fig.
2), and the effect was
similar to that seen with
cultured J774 macrophages. Exposure of human
peripheral blood
lymphocytes or monocytes to 10 µg of ClyA
ml
1 for 1 h caused about 20% LDH release, which is
comparable to
the cytotoxicity level of ClyA on cultured macrophages
(Fig.
1A).

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FIG. 2.
Viability results of neutral red uptake by human
peripheral monocytes after 20 h of ClyA treatment. Cells (about
2 × 105 cells per well in 96-well plates) were
prepared, treated, and assayed as described elsewhere (2, 11,
13). Viability value (neutral red uptake) of untreated cells was
taken as 100%. Note the ClyA dose-dependent tendency.
|
|
Apoptosis judged by DNA fragmentation of macrophages infected with
ClyA-expressing E. coli or treated with purified ClyA.
The biochemical hallmark of apoptosis is the cleavage of chromatin into
nucleosomal fragments, resulting in multimers of 180 to 200 bp
(15, 30). However, it has been reported that necrotic cells
may have irregular DNA fragmentation and generate
higher-molecular-weight DNA fragments (23). We used three
complementary assays to determine whether the predominant macrophage
cell death induced by these treatments was due to apoptosis.
Photometric determination of the histone-associated DNA fragments
released by the treated cells was performed with the sensitive
cell
death detection enzyme-linked immunosorbent assay (ELISA;
Boehringer
Mannheim GmbH) according to the manufacturer's instructions
and as
described elsewhere (
18). Substrate reaction time was
15 min. Both ClyA-expressing
E. coli MC1061/pYMZ80 (Fig.
3A and
C) and purified ClyA (Fig.
3B)
induced strong signals representing
small DNA fragments released to the
cell supernatant.

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FIG. 3.
Fragmentation of DNA in human and murine macrophages
(about 2 × 104 cells per well in 96-well plates) as
assayed by ELISA. Data are means ± standard deviations of
duplicate samples from one representative experiment of two. Shown are
results for infected murine macrophages J774 at 12 h p.i. at an
MOI of 100 (A), J774 cells treated with purified ClyA for 12 h
(B), and infected human macrophages U937 at 6 h p.i. at an MOI of
100 (C). Bars: 1, vector strain MC1061/pUC18; 2, ClyA-expressing strain
MC1061/pYMZ80; 3, Tris-HCl buffer (20 mM, pH 7.5); 4, purified ClyA
protein (20 µg ml 1); 5, MC1061/pUC18; 6, MC1061/pYMZ80.
|
|
Macrophage apoptosis was further quantified by TUNEL (terminal
deoxynucleotidyltransferase [TdT]-mediated dUTP nick end labeling
[7])-FACS (fluorescence-activated cell sorting) analysis of the
treated cells exactly as described before (
18). A
ClyA-expressing
strain caused about the same percentage of apoptosis in
both murine
and human macrophages (Fig.
4B and G), while the levels with the
vector control remained at baseline (Fig.
4A and F). The dose-
and
time-dependent effect of purified ClyA shown in the cytotoxicity
measurements was also evident in this TUNEL-FACS analysis. J774
cells
had an apoptosis percentage of about 15% when exposed to
10 µg of
ClyA for 36 h (Fig.
4D), and there were 96% apoptotic
cells when
10 times more ClyA was added (Fig.
4E); in contrast,
values for cells
in the control wells (containing Tris-HCl buffer
[20 mM, pH 7.5]
instead of ClyA protein) remained at the background
level (Fig.
4C).
The ClyA-treated cells did not show values above
the baseline level
during the first 12 h (data not shown). It
should be noted that
more cells were needed in the TUNEL-FACS
experiment (about 5 × 10
6 cells) than in the cytotoxicity and ELISA experiments
(about
2 × 10
4 cells) described above.

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FIG. 4.
Quantitative TUNEL-FACS measurements of apoptosis of
infected (MOI of 100) J774 (A and B) and U937 (F and G) macrophages at
12 h (data from one of two repeated tests which gave similar
results) or ClyA-treated J774 cells at 36 h (C to E), using
FACScan LYSIS II or CellQuest. The quantified percentages of apoptosis
are shown. The y axis represents the number of cells
(10,000); the x axis represents fluorescence intensity.
Shaded areas are macrophages with TdT and overlaid by cells without
TdT. The initial cell number in each well of a six-well plate was about
5 × 106. (A) Vector control strain MC1061/pUC18; (B)
ClyA-expressing strain MC1061/pYMZ80; (C) Tris-HCl buffer (20 mM, pH
7.5); (D) ClyA (10 µg); (E) ClyA (100 µg); (F) vector control
strain MC1061/pUC18; (G) ClyA-expressing strain MC1061/pYMZ80.
|
|
DNA of the infected macrophages was extracted as follows. Cells were
harvested and treated with lysis buffer (1% NP-40 in
20 mM EDTA-50 mM
Tris-HCl [pH 7.5]) (
10) at 37°C. The lysates
were
extracted once with an equal volume of phenol and once with
an equal
volume of chloroform-isoamyl alcohol (24:1, vol/vol)
before
precipitation with ethanol. The precipitates were dried
and solubilized
in 10 mM Tris (pH 8.0)-1 mM EDTA. Electrophoresis
was performed with a
1.5% agarose gel containing 0.5 µg of ethidium
bromide
ml
1 in Tris-acetate-EDTA buffer (pH 8.2). DNA was
visualized by UV
light and photographed. As evidenced by
electrophoresis of genomic
DNA, a nucleosome ladder pattern of
DNA degradation was observed
in J774 cells infected with MC1061/pYMZ80
but not in J774 cells
infected with the vector control MC1061/pUC18
(Fig.
5). It was
also evident that
cytochalasin D did not inhibit the apoptogenic
property of
MC1061/pYMZ80 on J774 cells (Fig.
5, lane 4) under
the conditions used
here.

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FIG. 5.
Fragmentation of genomic DNA from about 5 × 106 to 1 × 107 J774 macrophages infected
with MC1061/pYMZ80 at an MOI of 100 with or without cytochalasin D
(Cyt-D) at 12 h p.i. DNA purification and gel electrophoresis were
carried out as described in the text. MC1061/pYMZ80-infected cells
generated ladders of multimers of 180 bp characteristic of apoptosis.
Lanes: 1, 100-bp DNA ladder; 2, vector control strain MC1061/pUC18; 3, ClyA-expressing strain MC1061/pYMZ80; 4, ClyA-expressing strain
MC1061/pYMZ80 with 1 µg of Cyt-D ml 1.
|
|
It is roughly estimated in recent studies that pores generated by ClyA
(about 2.5 to 3 nm) appear to be somewhat larger than
those generated
by HlyA (
20,
21,
24,
29). The damaged
host cell membrane
might allow the influx and/or efflux of certain
ions which could
trigger apoptosis directly or indirectly (
1,
12,
22,
23).
Thus, the pore-forming activity of ClyA might
be responsible for
induction of apoptosis as described for other
pore-forming toxins
(
6,
14). Ca
2+ is generally regarded as a common
signal for initiation of apoptosis.
Increase in calcium concentration
has been shown to activate degradative
processes in programmed cell
death directly by stimulating endonucleases
or indirectly by promoting
activation of calcium-dependent proteases
and phosphatases (
1,
3,
12,
22,
23). One may speculate
that the pore-forming property of
ClyA could cause the modification
of the intracellular level of
Ca
2+, which may in turn trigger the apoptosis
cascade. The Ca
2+- and Mg
2+-dependent
endonuclease activated during the apoptotic process
cleaves the genomic
DNA at the internucleosomal regions, thereby
generating mono- and
oligonucleosomes.
Conclusions.
Taken together, our data demonstrate that
purified ClyA and a ClyA-expressing E. coli strain were
cytotoxic to both human and murine macrophages in a dose- and
time-dependent way and induced a massive amount of apoptosis as
determined by several assays showing host cell DNA fragmentation.
Further studies will hopefully elucidate the precise mechanisms of
ClyA-induced apoptosis of host cells. Our findings that this protein,
in addition to being merely a hemolysin, is more widely cytocidal and
has the capacity to induce macrophage apoptosis should prompt studies
of how ClyA might contribute to pathogenicity of certain
E. coli strains.
 |
ACKNOWLEDGMENTS |
We are grateful to Guangqian Zhou for supplying a sample of PMA.
This work was supported by grants from the Swedish Natural Science
Research Council, the Swedish Medical Research Council, the Swedish
Institute, the Wenner-Gren Foundations, and the Göran Gustafsson
Foundation for Research in Natural Sciences and Medicine.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Umeå University, S-901 87 Umeå, Sweden. Phone: 46 90 785 6731. Fax: 46 90 772630. E-mail:
bernt.eric.uhlin{at}micro.umu.se.
Permanent address: Priority Laboratory of Molecular Medical
Bacteriology of Ministry of Public Health, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing, China.
Editor:
J. T. Barbieri
 |
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Infection and Immunity, July 2000, p. 4363-4367, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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