Production of p57 by Renibacterium salmoninarum
Within Rainbow Trout Macrophages
Archana Dhawan
Hopkins Marine Station
Stanford University
Spring Quarter 1995
Abstract
Renibacterium salmoninarum is an intracellular pathogen that survives
within the macrophages of salmonids. The ability of R.salmoninarum to
produce p57, a major surface antigen of the bacterium, while inside
rainbow trout macrophages was determined. R. salmoninarum was
incubated with rainbow trout macrophages isolated from the head kidney
and spleen, and phagocytosis occurred as demonstrated by
immunofluorescence. Macrophages were then lysed and a Western blot
was run on the lysate, to determine the presence of p57. Macrophages
incubated with only L-15 media showed no p57 production nor did
macrophages incubated with killed R. salmoninarum. Macrophages
incubated with R. salmoninarum showed the presence of p57, thus
indicating that p57 was produced by R. salmoninarum while inside the
trout macrophages. These results suggest that p57 may play a role in the
intracellular survival of R. salmoninarum..
Introduction
Bacterial kidney disease (BKD) is a serious infection of salmonids that
progresses slowly and is often fatal (Evelyn 1987). The disease has been
shown to be both vertically and horizontally transmitted. Horizontal
transmission occurs in the wild with several species of trout while vertical
transmission has recently been shown to occur though the egg (Evelyn et
al. 1984). Renibacterium salmoninarum, the bacteria that causes the
disease is able to infect the egg through the micropyle without producing
toxins that would damage the egg (Evelyn 1987). The disease does not
appear until the fish are 6-12 months of age by which time the industry
has spent a considerable amount of effort and money in rearing the fish.
R. salmoninarum is a gram positive, non-motile diplobacillus. The
bacterium is an intracellular pathogen that enters the macrophage, the
antigen presenting cell of the immune system, and is able to survive there
despite the toxic conditions of the cell (Evenden 1993). The mechanisms of
R. salmoninarum survival are unknown, however several recent studies on
the relationship between macrophages and R. salmoninarum have been
made (Bandin et al. 1993). They have shown that upon infection with R.
salmoninarum, the macrophage is activated and responds by an increase in
superoxide anion production, indicating respiratory burst. In addition, this
research demonstrated that macrophages incubated with R. salmoninarum
and subsequently incubated with Aeromonas salmonicida, a second fish
pathogen, were able to kill A. salmonicida but unable to kill R.
salmoninarum. This suggests that R. salmoninarum does not completely
inhibit the macrophage bactericidal activity but instead is able to resist
killing.
The major surface antigen of R. salmoninarum is p57. This protein has
been cloned and sequenced and has been shown to be produced in large
quantities by R. salmoninarum, in culture (Evenden 1993). Antibodies to
the protein have been detected in the sera of trout with BKD. In addition,
several monoclonal antibodies specific to p57 epitopes have been produced
to further characterize p57 (Wiens and Kataari 1991). This major protein
of R. salmoninarum has been described as an agglutinin (Daly and
Stevenson 1990) and has been shown to have hemolysin-like qualities
(Turaga et al. 1987).
It is theorized that p57 might play a role in the intracellular survival
of R. salmoninarum. Our purpose was to determine whether p57 was
produced by R. salmoninarum, inside the macrophage. This information
would illustrate the viability of R. salmoninarum while inside the
macrophage as well as indicate whether p57 was in a location to play a
role in the intracellular survival of the bacteria.
Materials and Methods
Bacteria. R. salmoninarum were obtained from Oregon.
Serum. Blood from the caudal vein of the trout was collected in a glass
tube and allowed to clot at 4°C for 4-6 hours. The serum was extracted
and then spun at 18,000 g for 10 minutes. The supernatant was removed
and stored at -70°C.
Isolation of Macrophages. Rainbow trout were maintained at 12°C in a
freshwater circulating tank. Macrophages were isolated using the method
of Rose and Levine (1992). The fish were killed and blood for serum was
collected from the caudal vein. The head kidney and spleen of each fish
were removed aseptically and placed in 2 ml of L-15 media containing 2%
heat inactivated fetal calf serum, 100 iu/ml of penicillin-streptomycin, and
10 iu/ml of heparin. A cell suspension was created by passing the head
kidney and spleen tissue through a sieve, using a sterile syringe plunger
Percoll gradients had been previously made of 34% Percoll (3.4 ml of
Percoll, 1 ml of Hank's Balanced Salt Solution, and 5.6 ml of distilled H2O)
layered on top of 51% Percoll (5.1 mls of Percoll, Iml of Hank's Balanced
Salt Solution, and 3.9 ml of distilled H2O) in 15 ml sterile tubes. The cell
suspension was carefully layered onto the Percoll gradients using a sterile
Pasteur pipet. The gradients were then spun for 40 minutes at 400 g. The
cells were removed from the 34% and 51% Percoll interface. Cells were
counted and the cell density was adjusted to 1.4-2.5 x 10 7 cells/ml with
L-15 containing .1% Fetal Calf Serum and 100 iu/ml penicillin-
streptomycin. A final concentration of 4-7.5 x10 7 cells per well was
made. The cell cultures were incubated at 17°C for 2 h, following which
the media was replaced with 3 ml/well of L-15 containing 5% Fetal Calf
Serum and 100 iu/ml penicillin-streptomycin. The cells were observed
under an inverted microscope and appeared adherent and confluent. The
macrophages were used within 24 hours of isolation.
Test Trout for R. salmoninarum
ELISA (DiagXotics) was performed on kidney tissue from each fish,
according to the protocol provided and all samples were negative.
Polymerase Chain Reaction was used to determine whether the gene for
p57 existed in sample tissue. Following the protocol by Brown et al.(1993).
all fish proved to be negative for R. salmoninarum.
Incubation of R. salmoninarum with trout macrophages.
R. salmoninarum cells were suspended in eppendorf tubes, containing 1
ml each of phosphate buffered saline (PBS). Eppendorf tubes of cells were
washed five times with 100 ul distilled H2O, to remove p57 from the cell
surfaces. Five tubes of live R. salmoninarum cells were pooled and
resuspended in 1 ml of distilled H2O, after which an O.D. 540 reading was
taken. The cells were washed once more and resuspended in 500 ul of
PBS. They were then incubated with active trout serum for 1 h at 25°C, to
increase opsonization. The cells were resuspended to give a concentration
of 1.3 x10 9 cells/ml and 50 ul were added to each well of macrophage
culture. An aliquot of R. salmoninarum cells was incubated with 0.3%
formalin for 30 min at room temperature. These killed R. salmoninarum
cells were added to control wells of macrophage cultures. R. salmoninarum
was incubated with the macrophages for 22-24 hours.
Phagocytosis Assay. The procedure of Rose and Levine (1992) was
followed. Briefly, macrophages were adhered to slides after isolation from
rainbow trout as described above. These slides were then incubated with
R. salmoninarum cells for two hours, during which time phagocytosis
occurred. The slides were then fixed with 2% w/v formaldehyde for 15
min at 25°C and washed in PBS. In order to block excess free aldehyde
groups, the slides were incubated for 10 min at 25°C in L-15 media. The
slides were incubated in a polyclonal, goat anti-R. salmoninarum antibody
(Kirkegaard and Perry Laboratories) for one hour, washed and then
incubated in the dark with rabbit anti-goat antibody conjugated to FITC
(Sigma), diluted 1:100 in L-15. To stain the macrophages slides were
washed with PBS and incubated with 0.001% w/v Hoescht 3328 stain for 2
min at 25°C. Mounting media (.5M Na2CO3 and glycerol in a 1:l ratio) was
placed on each slide. A coverslip was added and the samples were viewed
at 380 nm for the Hoescht stain and 490 nm for the FITC.
Lysing of the macrophages. Using a sterile Pasteur pipet, the media from
each of the wells of cultured macrophages was removed. One ml of PBS
was added to wash each well and then removed thoroughly. One hundred
ul of distilled H2O was added to each well to lyse the macrophages, and the
well was scraped vigorously. The lysate of each well was collected and
pooled for each treatment group. The protein in the lysate samples was
concentrated by using centricon tubes and centrifuging at 4000 g.
Sodium dodecylsulfate polyacrilamide gel electrophoresis. Twenty one
ul of each sample were added to 9 ul of sample buffer (120 mM tris base,
4% w/v SDS, 10% v/v 2-mercaptoethanol, 20% v/v glycerol, and 3mM
bromophenol blue), then vortexed and placed in a boiling water bath for 3
min. Samples were run on 12 % acrylamide gel and 4.5 % acrylamide
stacking gel, for 1.5 h at approximately 20mAmps.
Western Blotting. Protein was transferred onto nitrocellulose by
electrophoresis at 20 mA for 30min. and then 200 mA for 2 hrs. at 4°C in a
wet transblot apparatus. The nitrocellulose membranes were blocked in
tris buffered saline (TBS)/0.1% Tween 20/5 % nonfat milk for 2 hours at
37°C. The blots were then washed with IX TBS, TBS/0.5% Tween and again
with IX TBS. The nitrocellulose membranes were incubated overnight at
4°C with different monoclonal antibodies: 4D3, and 3HI(provided by
Wiens, G. et al.), at a dilution of 1:25 and 1:50 respectively, in
TBS/Tween/milk. The Blots were washed and incubated in sheep anti-
mouse horseradish-peroxidase conjugated (Sigma) secondary antibody at a
dilution of 1:1000 in IX TBS for one hour, at 25°C. After washing in IX
TBS, the blots were placed in a Chemiluminescent Reagent (Renaissance)
for 1 min. and then were exposed to film.
Results
Evidence of Phagocytosis by Immunofluorescence
In order to determine whether phagocytosis had occurred, trout
macrophages and R. salmoninarum were stained with fluorescent dyes.
Fluorescence emitted by R. salmoninarum in the same location as the
fluorescence of the macrophage indicated that R. salmoninarum was inside
the macrophage.
Diagnosis of Fish for R. salmoninarum
The results of the ELISA and PCR showed that the trout were negative
for R. salmoninarum (data not shown).
Quantitative Analysis of p57 in the Washes of R. salmoninarum
In order to ensure the complete removal of p57 from the surface of R.
salmoninarum, bacteria were washed five times with distilled water. The
last wash shows no p57, indicating that p57 had been removed from the
cell surface.
Production of p57 by R. salmoninarum
Western blot analysis was performed on the lysate samples of the
experimentally infected macrophages, the non-infected macrophages, and
R. salmoninarum cells, in order to determine the presence of p57. Trout
macrophages incubated with L-15 media showed no p57, (Fig. 3), nor did
the trout macrophages incubated with killed R. salmoninarum.
Macrophages incubated with R. salmoninarum showed p57 production,
although less than that in R. salmoninarum alone. The supernatant of the
fifth wash unexpectedly showed a large amount of p57.
The same samples were probed with a different monoclonal antibody to
verify previous results and to avoid false positives for p57 due to cross¬
reactions. Trout macrophages incubated with only L-15 media showed no
band at the 57 kD region, however a band did appear at about 75-80 kD.
R. salmoninarum incubated with macrophages showed both a band at 57
kD (indicating p57) as well as a band at 75-80 KD. Whereas, R.
salmoninarum incubated with only L-15 media showed a band only at 57
KD which was p57. The bands of lower molecular weight in the p57
antigen (DiagXotics) positive control are the result of proteolysis.
Discussion
The Western blot of experimental and control groups of macrophages and
R. salmoninarum, indicates that R. salmoninarum when phagocytosed by
macrophages, does produce p57. Since macrophages incubated with killed
R. salmoninarum showed no presence of p57, the possibility that the p57
detected in the lysate sample of macrophages incubated with R.
salmoninarum was due to p57 released from the bacteria’s surface was
eliminated. Furthermore, since the macrophages incubated with R.
saloninarum were washed of bacteria and extracellular protein several
times before lysis, the presence of p57 must be due to its production by
the R. salmoninarum inside the macrophages.
The production of p57 by R. salmoninarum within trout macrophages
has important implications. It suggests that R. salmoninarum remains
viable while inside the macrophage, at least in terms of p57 protein
production. Furthermore p57 might have a role in the intracellular
survival of R. salmoninarum. It is possible that p57 may act on the
macrophage to alter its intracellular environment and make it more
hospitable. Or p57 might serve to protect R. salmoninarum from the
cytotoxicity of the macrophage (Bandin et al. 1993). Since R.
salmoninarum has been characterized to have hemolysin-like activity, it is
also possible that p57 acts as the hemolysin (Turaga et al. 1987).
This information, that p57 is produced intracellularly, suggests that an
antigen does exist however the macrophages may be unable to process it
and present it to T cells to elicit a normal immune response to eliminate
the bacteria. This may be the result of the biochemical properties of p57.
tolerance or the protein's possible affect on the macrophage's antigen
processing ability. In either case, if the antigen is not presented, then T
cells cannot respond by lysing the macrophage or by stimulating cytokine
production.
It is also important to examine that monoclonal antibodies specific to
p57 seem to cross react with proteins in the 70-80 kD range, such as in the
sample of macrophages incubated in L-15 media. This has two
implications. One is that the trout macrophages produce a protein with a
similar epitope as p57. Another is that there may be other intracellular
pathogens such as Pseudamonas maltophila and Carnobacteruim piscicola
that can produce a protein with common epitopes to p57 (Brown et al.
1995). In experiments we performed, trout that were negative for the p57
gene by PCR cross-reacted with polyclonal antibodies specific to R.
salmoninarum in an indirect immunofluorescence antigen test (IFAT) as
well as with monoclonal antibodies specific to p57. The presence of many
gram positive bacteria with common epitopes to p57 may indicate that p57
does play an important role in the pathogenesis of the disease.
Acknowledgements
I would like to thank Laura Brown for introducing me to the world
of BKD and for being such a supportive supervisor. I would also like
to thank Paul Levine for being a truly inspirational advisor and I
would like to thank Marta Gomez for making it all come together at
the end.
Works Cited
Bandin, I., Ellis, A.E., Barja, J.L., and Secombes, C.J. 1993. Interaction
between rainbow trout macrophages and Renibacterium
salmoninarum in vitro. Fish & Shellfish Immunology 3, 25-33.
Brown, L.L., Albright, L.J., Evelyn, T.P.T. 1990. Control of vertical
transmission of Renibacterium salmoninarum by injection of
antibiotics into maturing female coho salmon Oncorhynchus
kisutch. Diseases of Aquatic Organisms 9, 127-131.
Brown, L.L., Evelyn, T.P.T., Iwama, G.K., Nelson, W.S., Levine, R. P. 1995.
Bacterial species other than Renibacterium salmoninarum cross¬
react with antisera against R. salmoninarum but are negative for
the p57 gene of R. salmoninarum as detected by the polymerase
chain reaction (PCR). Diseases of Aquatic Organisms 21, 227-231.
Daly, James G., Stevenson, M. W. 1990. Characterization of the
Renibacterium salmoninarum haemagglutinin. Journal of General
Microbiology 136, 949-953.
Evenden, A. J., Grayson, T. H., Gilpin, M. L., Munn, C. B. 1993. Renibacterium
salmoninarum and bacterial kidney disease- the unfinished
jigsaw. Annual Review of Fish Diseases, 87-104.
Evelyn, T.P.T. 1987. Bacterial kidney disease in British Columbia, Canada:
Comments on its epizootiology and on methods for its control on
fish farms. Proc. of Trondheim International Conference ,51-57.
Evelyn, T. P. T., Prosperi-porta, L., J.E. Ketcheson. 1984. The salmonid egg as
a vector of the kidney disease bacterium Renibacterium
salmoninarum. Fish Diseases,11-117.
Rose, A. S., Levine, R. P. 1992. Complement-mediated opsonisation and
phagocytosis of Renibacterium salmoninarum. Fish and Shellfish
Immunology 2, 223-240.
Turaga, P., Wiens, G. Kaatari, S. 1987. Bacterial kidney disease : the
potential role of soluble protein antigen(s). Journal of Fish Biology
31, 191-194.
Wiens, Gregory D., Kaatari, Stephen L. 1989. Monoclonal Antibody Analysis
of Common Surface Proteins of Renibacterium salmoninarum. Fish
pathology 24,1-7.
Wiens, Gregory D., Kaatari, Stephen L.. 1991. Monoclonal antibody
characterization of a leukoagglutinin produced by Renibacterium
salmoninarum. Infection and Immunity Feb, 631-637.
Fig. la-b Photographs of immunofluorescence demonstrating phagocytosis by rainbow trout
macrophages of R. salmoninarum. Fig. la Hoescht 3328 counterstain showing macrophage
nuclei, viewed at wavelegnth of 380 nm. Fig. Ib FITC conjugated secondary antibody specific for
goat anti-R. salmoninarum (polyclonal) showing bacteria, viewed at wavelegnth 490 nm.
Fig. 2 Western blot analysis of the supernatants collected from each of five washes of R.
salmoninarum using a mixture of monoclonal antibodies (DiagXotics). Lanes 1-5 represent
supernatant of washes 1-5, respectively
Fig. 3 Western Blot of various treatment groups and controls of rainbow trout macrophages and
R. salmoninarum, using the monoclonal antibody 4D3. Lanes are as follows: 1. Bio-Rad
prestained molecular weight standard, 2. trout macrophages incubated with L-15 media, 3. trout
macrophages incubated with R. salmoninarum, 4. trout macrophages incubated with killed R.
salmoninarum, 5. R. salmoninarum incubated in L-15 and washed 2X with Phosphate Buffered
Saline, 6. R. salmoninarum incubated with distilled H2O, 7. supernatant of the fifth wash of R.
salmoninarum, 8. p57 antigen (DiagXotics), 9.Bio-Rad prestained molecular weight standards.
The weights of Bio-Rad prestained molecular markers are listed on the left.
Fig. 4 Western blot analysis of the treatment group and controls of rainbow trout macrophages
and R. salmoninarum, using the monoclonal antibody 3HI. Lanes are as follows: 1. p57 antigen
(DiagXotics), 2. R. salmoninarum incubated in L-15 media, 3. trout macrophages incubated with
R. salmoninarum and L-15, 4. trout macrophages incubated with L-15, 5. Bio-Rad prestained
molecular weight standards.
Figure 1
1.

Figure 2
Figure 3
-





10





14 —
3 —



3







3


















—
—.— —













(






44.


43
1.
—
—
—
+
—
—
Figure 4