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Applied and Environmental Microbiology, July 2000, p. 3031-3036, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Penetration of the Coral-Bleaching Bacterium
Vibrio shiloi into Oculina
patagonica
E.
Banin,1
T.
Israely,1
A.
Kushmaro,2
Y.
Loya,2
E.
Orr,3 and
E.
Rosenberg1,*
Department of Molecular Microbiology and
Biotechnology1 and Department of
Zoology,2 The George S. Wise Faculty of
Life Sciences, Tel Aviv University, Ramat Aviv, Israel, and
Department of Genetics, University of Leicester, Leicester
LE1 7RH, United Kingdom3
Received 18 January 2000/Accepted 11 April 2000
 |
ABSTRACT |
Inoculation of the coral-bleaching bacterium Vibrio
shiloi into seawater containing its host Oculina
patagonica led to adhesion of the bacteria to the coral surface
via a 
-D-galactose receptor, followed by penetration of
the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of
the coral by electron microscopy and fluorescence microscopy using
specific anti-V. shiloi antibodies to stain the
intracellular bacteria. At 29°C, 80% of the bacteria bound to
the coral within 8 h. Penetration, measured by the viable count
(gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and
21.7% of the initial inoculum at 8, 12, and 24 h, respectively.
The viable count in the coral tissue decreased to 5.3% at 48 h,
and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloi
antibodies) in the coral tissue showed results similar to viable counts
for the first 12 h of infection. After 12 h, however, the
total count more than doubled from 12 to 24 h and continued to
rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were
transformed into a form that could multiply inside the coral tissue but
did not form colonies on agar medium. Internalization of the bacteria
was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and
multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.
| |
INTRODUCTION |
During the past two decades there
have been an increasing number of reports of a disease of corals
referred to as coral bleaching (2, 8, 10). Coral bleaching
is the disruption of the symbiotic association between the coral hosts
and their photosynthetic microalgal endosymbionts (zooxanthellae).
Bleaching or paling of corals results from a reduction in the density
of zooxanthellae in the coral's gastrodermal tissues and/or from
decreased concentrations of photosynthetic pigments in the algal cells
(6). The loss of zooxanthellae greatly affects the coral
host, because these photosynthetic symbionts supply as much as 63% of
the coral's nutrients (5). Algae remaining in bleached
corals suffer severe damage to photosystem II (24).
Coral bleaching is a widespread disease that occurs in the world's
three major oceans and involves more than 50 countries (25).
It has been suggested that coral bleaching is triggered by
environmental factors that impose stress on the coral. The most
frequently reported stress condition is increased seawater temperature
(2, 5, 9, 12, 15). Thus, it is possible that global warming
could result in alterations to or destruction of coral reef systems,
the consequences of which could be devastating
to tourist and fishing
industries, to islands that are protected by coral reefs, and, most
importantly, to the health of the sea. Consequently, it is essential to
understand the mechanism(s) of coral bleaching.
Recently, it was reported that bleaching of the coral Oculina
patagonica from the Mediterranean Sea is the result of a bacterial infection (13-15, 20). The causative agent, Vibrio
shiloi (1, 20), was obtained in pure culture and shown
to cause bleaching in controlled aquarium experiments. Furthermore, it
was shown that bacterium-induced bleaching by V. shiloi
could be inhibited by antibiotics. The infection and resulting coral
bleaching were temperature dependent, occurring only at elevated
seawater temperatures (15).
By using the V. shiloi-O. patagonica model system
to study coral bleaching, it was demonstrated that the first step in
the infectious process is the adhesion of V. shiloi to a
-galactoside-containing receptor on the coral surface
(23). The temperature of bacterial growth is critical for
the adhesion of V. shiloi to the coral. When the
bacteria were grown at a low temperature, there was no adhesion to the
coral, regardless of the temperature at which the coral had been
maintained. However, bacteria grown at the elevated seawater
temperature adhered avidly to corals maintained at either low or high
temperatures. The important ecological aspect of these findings was
that the environmental stress condition was causing the coral-bleaching
pathogen to express its virulence determinants.
In an attempt to understand how V. shiloi causes the
destruction or loss of the algae, it was discovered that V. shiloi cells produce both a heat-stable extracellular toxin that
inhibits the photosynthesis of zooxanthellae and also heat-sensitive
toxins that bleach and lyse algal cells isolated from corals (1,
20). In this report, we demonstrate that V. shiloi
penetrates into the coral epidermis following adhesion to the coral
surface. Shortly after penetrating the epidermis, the bacteria multiply
in the tissue and enter a state in which they fail to form
colonies on media that normally support the growth of V. shiloi.
| |
MATERIALS AND METHODS |
Microorganisms and corals.
V. shiloi AK-1 was
isolated from a bleached coral as described previously (13,
14). The strain was maintained on MB agar (1.8% marine broth
plus 0.9% NaCl solidified with 1.8% agar [both products of Difco
Laboratories, Detroit, Mich.]). After being streaked onto MB agar, the
cultures were incubated at 30°C for 2 days and then allowed to stand
at room temperature for 1 week. The coral Oculina patagonica
was collected and maintained as described previously (13, 14,
23).
Adhesion and penetration of V. shiloi onto and into
O. patagonica.
An overnight culture of V. shiloi, grown at 30°C in MB broth with aeration, was centrifuged
at 5,000 × g for 10 min, and the cell pellet was
washed twice and then resuspended to ca. 109 cells per ml
in sterile seawater. The bacteria were inoculated into a 125-ml flask
containing fragments of O. patagonica (~1 cm3)
in 25 ml of sterile seawater to the desired initial bacterial concentration. The flasks were incubated at 29 ± 1°C with
gentle shaking on a "Belly Dancer" (Stovall Life Sciences Inc.,
Greensboro, N.C.). During the incubation period, the coral fragments
were illuminated with a fluorescent lamp on cycles alternating 12 h of light and 12 h of darkness. Adhesion was determined by
removing water samples at timed intervals and plating on MB agar and/or thiosulfate-citrate-bile-sucrose (TCSB) agar (Difco) as described previously (23). In each experiment, a no-coral control was included and the percent bacteria adhering to these flasks was subtracted from the values obtained with the coral experiments to
obtain net adhesion. All values reported refer to net adhesion.
Penetration of V. shiloi into the coral tissue was
determined by modifications of the gentamicin invasion assay
(11). The infected coral was removed from the flask at the
appropriate time, rinsed with sterile seawater, and then transferred to
a 50-ml tube with 5 ml of sterile seawater containing 200 µg of
gentamicin per ml and 0.01% methyl--D-galactopyranoside
in order to desorb and kill noninternalized bacteria. After incubation
for 3 h at 29°C, the coral was removed, rinsed in sterile
seawater, then crushed in 5 ml of seawater with a mortar and pestle,
and finally vortexed in a tube for 1 min. The first method (viable
count) involved estimating the number of internal bacteria by plating appropriate dilutions on MB agar and TCSB agar. V. shiloi
has a characteristic colony morphology on TCSB agar. Confirmation that
the CFU were due to V. shiloi was obtained by checking the cells with anti-V. shiloi antibodies. The second method
(total count) involved determining the number of V. shiloi
cells in the tissue microscopically after staining with a specific
polyclonal anti-V. shiloi antiserum as described below.
Microscopy.
Crushed coral samples (0.5 ml) were fixed with
freshly prepared 4% paraformaldehyde in seawater for 1 to 3 h.
The fixed samples were then washed three times in Tris-buffered saline
(TBS) (10 mM Tris-HCl [pH 7.5]-150 mM NaCl) and attached to
microscope slides covered with poly-L-lysine (50 µg/ml).
After incubation for 1 h, the slides were washed once in TBS and
incubated for 12 h at 4°C with polyclonal antibodies to V. shiloi (1:500 dilution in TBS). The antibodies were affinity
purified by using fixed Escherichia coli and Vibrio
mediterranei cells. The slides were then washed three times in TBS
and incubated with 5 µg of Amca-conjugated anti-rabbit immunoglobulin
G (IgG) (Jackson ImmunoResearch, West Grove, Pa.)/ml. After the
incubation, the slides were washed three times in TBS and mounted with
a solution of 90% glycerol containing 1 mg of
p-phenylenediamine (Sigma, St. Louis, Mo.)/ml. Coverslips were sealed, and the sample was stored at 
20°C until examination. Examination was carried out using a Leica fluorescence microscope (model DMR) with filter A (UV) for Amca.
The viability of intracellular
V. shiloi was examined with
the Live/Dead Baclight Bacterial Viability Kit (Molecular Probes,
Eugene, Oreg.). The bacteria were stained according to the
manufacturer's
protocol and then examined by fluorescence microscopy
with a Leica
B/G/R filter. Live bacteria fluoresce green, and dead
bacteria
fluoresce
red.
For examining coral sections, samples at different stages of infection
were fixed in 4% formaldehyde in seawater for 24 h,
rinsed in
fresh water, and transferred to 70% ethanol for preservation.
Decalcification was carried out using a solution of formic acid
(25%)
and sodium citrate (10%) for 15 to 25 h (
19). After
decalcification,
the tissue was rinsed in fresh water and transferred
into 70%
ethanol. The tissue was embedded in paraffin by use of a
Citadel
Embedding apparatus. Sections (4 to 6 µm thick) of polyps
were
attached to microscope slides covered with
poly-
L-lysine. The
paraffin was removed by successive
washing with
O-xylene and decreasing
concentrations of
ethanol. The slides were then washed in TBS
and stained with the
antibodies as described
above.
For electron microscopy, coral fragments were fixed in 2.5%
glutaraldehyde in filtered (pore size, 0.2 mm) seawater and decalcified
in a mixture of equal volumes of formic acid (50%) and sodium
citrate
(15%) for 15 h. They were then dehydrated in graded series
of
ethyl alcohol and embedded in Epon. Sections stained with uranyl
acetate and lead citrate were viewed with a JEOL 1200 EX electron
microscope.
Measurement of V. shiloi toxin P activity.
A
portable underwater Mini Pulse-Amplitude-Modulation (PAM) fluorometer
(Walz GmbH, Effeltrich, Germany) was used to measure the quantum yield
of zooxanthellae. This instrument allows the direct, noninvasive
measurement of the effective quantum yield (Y) of
photosystem II under ambient light. Good correlations between measurements of quantum yield and photosynthetic rates (determined by
O2 evolution and CO2 uptake) have been reported
for plants (7) and cyanobacterial symbionts of lichens
(22).
In the experimental procedure used here, the quantum yields of 0.05-ml
samples containing zooxanthellae in seawater (5 × 10
6
algae ml
1) were measured in enzyme-linked immunosorbent
assay (ELISA) plates
at room temperature with the Mini-PAM
(
Y0). Then 0.05 ml of the
experimental sample
was added to the algae, and the quantum yield
was measured after 5 min
(
Yt). The percent quantum yield at each
time was
Yt/
Y0 × 100. One
unit of toxin P activity is defined
as the amount that causes a 10%
net inhibition of the quantum
yield in the presence of 10 mM
NH
4Cl. NH
4Cl is required for toxin
P activity
(
1). Toxin P was obtained from the infected corals
by
extraction with ethyl acetate, removal of the solvent with
a stream of
nitrogen gas, and dissolving of the dried material
in sterile
seawater.
| |
RESULTS |
Qualitative microscopic observations of corals infected with
V. shiloi.
Thin sections of O. patagonica that
were infected with V. shiloi showed numerous bacteria in the
coral tissue visualized in the electron microscope (Fig.
1). The intracellular bacteria were short
rods, approximately 2.5 by 0.8 µm. The fact that these intracellular bacteria were V. shiloi was demonstrated using specific
anti-V. shiloi antibodies and fluorescence microscopy (Fig.
2). Control experiments demonstrated that
the antibodies did not react with other bacteria, including the closest
known relative of V. shiloi, V. mediterranei
(14). Eight hours after inoculation of V. shiloi into seawater containing O. patagonica, most of the
bacteria were seen adhering to the coral surface. After 24 h, many
V. shiloi bacteria had penetrated into the coral tissue.
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FIG. 2.
Section of O. patagonica 24 h after
infection with V. shiloi stained with anti-V.
shiloi antibodies.
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Adhesion and penetration of V. shiloi onto and into
O. patagonica as determined by viable counts.
As
summarized in Table 1, approximately 80%
of the bacteria inoculated into seawater adhered to the coral within
8 h. At that time only 6% of the input bacteria were recovered as
CFU in the crushed tissue after desorbing and killing of external bacteria with methyl--D-galactopyranoside and
gentamicin, respectively. The number of internalized CFU increased to
21% of the input bacteria after 12 h, remained constant
until 24 h, and then decreased to 5% at 48 h. No CFU could
be recovered inside the tissue after 72 h. Uninoculated controls
showed no detectable internalized CFU throughout the experiment.
When different inoculum sizes of
V. shiloi were compared, it
appeared that both adhesion and penetration onto and into
O. patagonica with the lower inoculum concentration (1.6 × 10
5 per ml) and the higher inoculum concentration (4 × 10
6 per ml) were similar with regard to both extent and
kinetics.
With both inocula, the number of internalized CFU decreased
dramatically
after 24
h.
Penetration and multiplication of V. shiloi in coral
tissue.
As shown in Fig. 2, antibodies specific for V. shiloi can be used to visualize the bacteria inside coral tissue.
This technique was applied to quantitate the number of V. shiloi bacteria inside the coral as a function of time after
infection, and these data were compared to the data obtained from
viable counts (Fig. 3). During the first
12 h after infection, the number of V. shiloi bacteria
that penetrated into the tissue was the same regardless of whether the
bacterial concentration was determined by viable counts or total
counts. From 24 h onward, however, the data obtained by the two
methods were very different. Whereas the CFU count of V. shiloi inside the tissue decreased rapidly after 24 h, the total counts more than trebled from 24 to 36 h and continued to rise until 48 h, reaching a value of six times the number of
bacteria that were introduced in the inoculum. Since the total number
of V. shiloi bacteria initially inoculated into the 25 ml of
seawater was 4 × 106, the number of V. shiloi cells in the 1-cm3 piece of coral at 72 h
was 2.4 × 107.
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FIG. 3.
Multiplication of V. shiloi bacteria in coral
tissue. Healthy pieces of coral were inoculated with 1.6 × 105 bacteria per ml and incubated with shaking as described
in Table 1, footnote a. At timed intervals, bacterial
adhesion ( ) to the coral, culturable internal bacteria ( ), and
total internal bacteria ( ) were measured. Total internal V. shiloi bacteria were quantitated by using specific anti-V.
shiloi antibodies. The ordinate indicates the number of cells
relative to the inoculum. Error bars, standard errors.
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Further evidence that intracellular V. shiloi is viable
but not culturable.
One possible explanation for the failure of
intracellular V. shiloi to form colonies was that the
treatment of the corals with relatively high concentrations of
gentamicin and methyl--D-galactopyranoside, prior to
crushing, actually killed the intracellular bacteria. To test
this possibility, a control experiment was performed (Table 2). Extracellular V. shiloi bacteria, obtained from the mucus on the outside of the
coral, were efficiently killed when treated with a mixture of
gentamicin and methyl--D-galactoside. However, when the
coral fragments were treated with the antibiotic, essentially the same
CFU count of intracellular bacteria was obtained after crushing the
coral as with no antibiotic treatment. Thus, the fact that the CFU
count of intracellular bacteria was more than a thousand times less
than the microscopic count of specifically stained V. shiloi
bacteria was not the result of the treatment killing the intracellular
bacteria.
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TABLE 2.
Sensitivity of free and intracellular V. shiloi to treatment with gentamicin
and methyl--D-galactopyranosidea
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Another possible explanation for the unculturability of the
intracellular
V. shiloi is that the coral host responds to
the
infection by producing a compound that depresses plate counts.
This hypothesis was eliminated by showing that adding
V. shiloi to crushed coral did not change the CFU
count.
Intracellular bacteria that bound the anti-
V. shiloi
antibodies were also scored as viable (green) by the Live/Dead BacLight
Viability Kit (Fig.
4). Although it was
not quantitated, it appeared
that a high percentage of the
intracellular
V. shiloi bacteria
were in close contact with
the zooxanthellae in the crushed infected
coral.
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FIG. 4.
Viability of V. shiloi in coral tissue. The
experiment was performed as described in the legend to Fig. 3. After
incubation with the bacteria for 24 h, the washed coral was
crushed and examined by fluorescence microscopy for V. shiloi by staining with anti-V. shiloi antibodies
(blue) (a) and viable cells (green) by staining with the Live/Dead
BacLight Bacterial Viability Kit (b).
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Production and degradation of toxin P activity in coral tissue
following infection with V. shiloi.
Flask cultures of
V. shiloi produce a heat-stable extracellular photosynthesis
inhibitor (toxin P) during stationary phase (1, 20). If
toxin P plays a role in coral bleaching, then it must reach the
intracellular zooxanthellae in the coral tissue. Because the bacteria
penetrate into the coral during the first 24 h of infection, it
was interesting to determine the amount of toxin P activity inside the
coral at different times after infection (Fig.
5). The rise and fall of toxin P activity
in the coral tissue followed kinetics similar to those of the
intracellular CFU. The toxin P activity in the coral tissue 24 h
after infection was more than 10 times higher than the level that was
produced in flask cultures.
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FIG. 5.
Production and degradation of V. shiloi
photosynthesis inhibitor toxin (toxin P) in coral tissue ( ) compared
to internal viable counts of the bacterium (). The experiment was
performed as described in Table 1, footnote a. Levels of
toxin P activity were determined on extracts of coral tissue.
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| |
DISCUSSION |
The data presented here demonstrate that the coral pathogen
V. shiloi penetrates into the epidermis of its coral host
following adhesion to the coral surface. Then, once inside the coral,
the intracellular V. shiloi bacteria multiply and are
transformed into a state that does not form colonies on media that
normally support the growth of extracellular V. shiloi.
These results have significant implications with regard to both the
mechanism of coral bleaching and the difficulties that may be
encountered in isolating other potential coral-bleaching pathogens from
different species of corals.
With regard to the mechanisms of pathogenesis, we have previously
demonstrated that V. shiloi produces extracellular
heat-resistant and heat-sensitive toxins that cause inhibition of
photosynthesis and that bleach as well as lyse zooxanthellae
isolated from the coral host (1, 20). The fact that
V. shiloi penetrates into the coral tissue would facilitate
contact between these toxins and their target algae. The high tissue
activity of toxin P following infection, shown in this
article, supports this hypothesis. Since toxin P activity
requires the presence of ammonia to inhibit zooxanthella photosynthesis, it is unlikely that the toxin would function when V. shiloi is outside of its host because the
concentration of ammonia in seawater is extremely low (21).
Entry of bacteria into a state described as viable but not culturable
(VBNC) has been reported repeatedly with a large number of bacterial
species, including several Vibrio species, Vibrio vulnificus (18), Vibrio parahaemolyticus
(3), Vibrio cholerae (4), and
Vibrio fischeri (16). A bacterium in the VBNC
state has been defined "as a cell which can be demonstrated to be
metabolically active, while being incapable of undergoing the sustained
cellular division required for growth in or on a medium normally
supporting growth of that cell" (17). Intracellular
V. shiloi cells fit that definition precisely, but unlike
most cases of VBNC that have been studied, this is not brought about by
starvation or low temperatures. Rather, the entry of V. shiloi into the VBNC state occurs inside the coral epidermis,
where nutrients are abundant and, in fact, V. shiloi
multiplies. It is possible that the intracellular differentiated
V. shiloi becomes dependent on one or more nutrients present
in the coral cell. This hypothesis could be tested by attempting to
plate intracellular V. shiloi on media containing coral
tissue homogenate.
The gentamicin invasion assay (11) was developed for
measuring the penetration of Yersinia pseudotuberculosis
into human epithelial cells and has been found to be generally useful
for determining the invasion of animal cells by different bacteria. Since V. shiloi was efficiently killed only by relatively
high concentrations of gentamicin (200 µg/ml), it was necessary to demonstrate that the antibiotic did not kill V. shiloi
present in coral cells. Whereas the gentamicin treatment killed
bacteria in the coral mucus with more than 99% efficiency, there was
no killing of intracellular bacteria. In both cases, the samples were
treated with 0.01% methyl--galactopyranoside as well as the
antibiotic to desorb V. shiloi (23) that had not
been internalized.
The VBNC state of intracellular bacteria in corals could make it
difficult to demonstrate that they are the causative agents of coral
bleaching. In the case of V. shiloi, it was possible to
isolate the bacterium from the coral mucus and then apply Koch's postulates to prove that it is the causative agent of bleaching of the
coral O. patagonica. However, culturing potential pathogens from other corals may present a problem if they exist in the VBNC state. It would therefore be desirable to develop a general method for
culturing intracellular coral bacteria. Further studies on the
VBNC state of intracellular V. shiloi may be
useful in this regard.
| |
ACKNOWLEDGMENTS |
This work was supported by United States-Israel Binational
Science Foundation grant 95-00177, the Porter Super-Center for Ecological and Environmental Studies, the Pasha Gol Chair for Applied
Microbiology, and the Center for the Study of Emerging Diseases.
We thank Ralph Martinez of UCLA for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology & Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel. Phone: 972 3 640 9838. Fax: 972 3 642 9377. E-mail:
eueqene{at}ccsg.tau.ac.il.
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Abstract
Introduction
