DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 78: 115–127, 2007
doi: 10.3354/dao01859
Published December 13
INTRODUCTION
Adherence to host tissues is an essential requirement
for many bacterial pathogens to establish infections
in specific hosts and cause disease. Gram-positive
bacteria achieve this in part by employing an array
of surface-displayed protein virulence factors called
MSCRAMs (microbial surface components recogniz-
ing adhesive matrix molecules) (Patti et al. 1994).
These surface-displayed proteins mediate initiation
and propagation of infection through adherence to
host endothelial tissue and immune system evasion
(Schneewind et al. 1995) often determining both bacte-
rial host range and site of infection (Foster & McDevitt
© Inter-Research 2007 · www.int-res.com*Corresponding author. Email: mark.strom@noaa.gov
Sortase inhibitor phenyl vinyl sulfone inhibits
Renibacterium salmoninarum adherence and
invasion of host cells
Ponnerassery S. Sudheesh1, 2, Samuel Crane1, 3, Kenneth D. Cain2, Mark S. Strom1,*
1Northwest Fisheries Science Center, NOAA Fisheries Service, 2725 Montlake Boulevard East, Seattle,
Washington 98112, USA
2Department of Fish and Wildlife Resources and The Aquaculture Research Institute, University of Idaho,
Moscow, Idaho 83844, USA
3Present address: Graduate Center, The City University of New York and the Sackler Institute for Comparative Genomics,
American Museum of Natural History, Central Park West at 79th Street, New York, New York 10024, USA
ABSTRACT: Renibacterium salmoninarum, the causative agent of bacterial kidney disease in
salmonid fishes, is a Gram-positive diplococcobacillus belonging to the family Micrococcaceae.
Analysis of the genome sequence of the bacterium demonstrated the presence of a sortase homolog
(srtD), a gene specifying an enzyme found in Gram-positive bacteria and required for covalent
anchoring of cell surface proteins. Interference of sortase activity is being examined as a target for
therapeutic prevention of infection by several pathogenic Gram-positive bacterial species. In silico
analysis identified 8 open reading frames containing sortase recognition motifs, suggesting these
proteins are translocated to the bacterial cell wall. The sortase and potential sortase substrate genes
are transcribed in R. salmoninarum, suggesting they encode functional proteins. Treatment of R.
salmoninarum with phenyl vinyl sulfone (PVS) significantly reduced bacterial adherence to Chinook
salmon fibronectin. In addition, the ability of the PVS-treated bacteria to adhere to Chinook salmon
embryo cells (CHSE-214) in vitro was dramatically reduced compared to that of untreated bacteria.
More importantly, PVS-treated bacteria were unable to invade and replicate within CHSE-214 cells
(demonstrated by an intracellular growth assay and by light microscopy). When treated with PVS, R.
salmoninarum was not cytopathic to CHSE-214 cells, whereas untreated bacteria produced
cytopathology within a few days. These findings clearly show that PVS, a small molecule drug and a
known sortase inhibitor, can interfere with the ability of R. salmoninarum to adhere and colonize fish
cells, with a corresponding decrease in virulence.
KEY WORDS: Renibacterium salmoninarum · Bacterial kidney disease · Anti-virulence chemotherapy ·
Adherence · Invasion · Host cell sortase inhibitor · Phenyl vinyl sulfone
Resale or republication not permitted without written consent of the publisher

Dis Aquat Org 78: 115–127, 2007
1994, Kehoe 1994). MSCRAMs have specific C-termi-
nal anchoring signals or cell wall sorting (CWS)-motifs
that are recognized by a membrane transpeptidase
called sortase. Sortase catalyzes covalent anchoring of
these proteins to the peptidoglycan surface during cell
wall synthesis (reviewed in Navarre & Schneewind
1999, Ton-That et al. 2004, Marraffini et al. 2006).
Elimination of sortase activity and the consequent dis-
ruption of surface protein anchoring correlates with a
dramatic decrease in pathogenicity of Gram-positive
bacteria in animal infections (Mazmanian et al. 2000,
Bolken et al. 2001, Bierne et al. 2002, Garandeau et al.
2002, Jonsson et al. 2002, Weiss et al. 2004). Sortase is
also involved in the assembly of cell surface pili on
Gram-positive bacteria, which often aid attachment to
host cells (Ton-That & Schneewind 2003). Collectively,
these and other findings suggest that sortases are
promising targets for new antibacterial drugs (Schnee-
wind et al. 1993, Frankel et al. 2004, Ton-That et al.
2004, Weiss et al. 2004, Zink & Burns 2005). However,
there have been few studies focused on the develop-
ment of specific inhibitors of sortases for the treatment
of Gram-positive bacterial infections.
Vinyl sulfones are a group of small molecule elec-
trophilic inactivators of cysteine proteinases (Hanzlik
& Thompson 1984) that inhibit sortase enzyme activity.
Significant attenuation of virulence with vinyl sulfones
has been demonstrated for Gram-positive bacteria
(Frankel et al. 2004) with no effects on viability, thus
avoiding selective pressure to develop resistance
(Aberg & Almqvist 2007). Since there is development
of resistance against almost any antibiotic available for
treatment today, such an anti-infective strategy may
prove valuable in the treatment of bacterial infections.
Moreover, because vinyl sulfones are small molecule
drugs, they may have the added advantage of easy
absorption into host tissues.
Renibacterium salmoninarum is a Gram-positive,
slow growing bacterium and the causative agent of
bacterial kidney disease (BKD), a chronic granuloma-
tous infection in wild and cultured salmonid fish (Fryer
& Sanders 1981). A common practice for managing
the disease in fish supplementation and conservation
hatcheries is prophylactic use of a macrolide drug, ery-
thromycin under a FDA-investigational new animal
drug (INAD) approval system (FDA = US Food and
Drug Adminstration). However, erythromycin treat-
ment does not completely eliminate the pathogen, and
in fact the bacterium and clinical signs of BKD
reemerge soon after treatment is stopped (Wolf & Dun-
bar 1959). Persistence of the bacterium in the host has
been attributed to its ability to survive intraovum
(Bruno & Munro 1986, Evelyn et al. 1984) or intracellu-
larly (Bandín et al. 1993, Gutenberger et al. 1997,
McIntosh et al. 1997, Young & Chapman 1978). Intra-
cellular survival allows the bacteria to escape both
humoral and cellular immune responses as well as the
action of antibiotics that normally act in the extracellu-
lar milieu (Fryer & Lannan 1993). In order to improve
treatment efficacy, azithromycin (another macrolide
drug with better tissue permeability and persistence) is
being used to treat BKD in valuable endangered
salmon stocks reared in captivity (Fairgrieve et al.
2005, 2006). However, development of resistance to
antibiotics is an increasingly important concern, not
only for human health implications, but also in aquatic
organisms (WHO 1997, Cabello 2006). Recent evi-
dence also indicates that long-term prophylactic use
of erythromycin can produce toxicity and cause a
reduction in reproductive success in Chinook salmon
(Fairgrieve et al. 2006). Clearly there is a strong
need for alternative, non-antibiotic based drugs for
treating BKD.
Genome sequencing of Renibacterium salmoni-
narum was recently completed and offers tremendous
opportunities for identifying novel vaccine and drug
targets. Until annotation of the genome, the presence
of sortase and sortase-mediated translocation of pro-
teins to the cell surface were unknown in this organ-
ism. We present a characterization of the R. salmoni-
narum sortase and sortase substrate genes, and the
inhibitory effect of a known sortase inhibitor, phenyl
vinyl sulfone (PVS), on the adherence, invasion and
replication of the bacteria in a Chinook salmon embryo
cell line. In addition, we investigated the effect of PVS
on the expression and localization of cell-wall bound
proteins in R. salmoninarum.
MATERIALS AND METHODS
Bacteria and culture conditions. Renibacterium
salmoninarum ATCC 33209 (Rs-33209, American Type
Culture Collection), which was originally isolated from
a Chinook salmon in Oregon (Sanders & Fryer 1980)
was used in this study. Bacteria were grown at 15°C
with constant stirring in selective kidney disease
medium (SKDM-2) broth (Evelyn 1977) containing
0.05% (w/v) cysteine-HCl and 10% (v/v) fetal bovine
serum. Glycerol stocks of the bacteria were prepared
in SKDM-2 medium containing 15% glycerol, and
were kept at –80°C for long-term storage.
Bioinformatic analysis and database searches. In sil-
ico discovery of putative sortase enzymes was con-
ducted by scanning all open reading frames (ORFs) in
the Rs-33209 genome with a published sortase profile
hidden Markov model (HMM) of 6 sortase subfamilies
(Comfort & Clubb 2004) using the program HMMER,
version 2.3 (Eddy 1998). To further verify subfamilial
assignment, potential sortase proteins were aligned to
116

Sudheesh et al.: Inhibition of Renibacterium salmoninarum adherence
a published set of sortase enzymes (Dramsi et al. 2005).
A neighbor-joining tree was built from 54 aligned sor-
tase amino acid sequences (bootstrapped 100 times)
using the program MAFFT (Katoh et al. 2005).
After sortase gene discovery, sortase substrate pro-
teins were identified using a modified version of the
substrate search of Boekhorst et al. (2005). Amino acid
sequences were scanned for one tripartite pattern and
one cleavage motif using ScanProsite (de Castro et al.
2006). The cleavage motif used L-[PA]-X-[TA]-[GNSD]
is the most relaxed query (more matches at the cost of
more false positives) and is based on the subfamily 5
sorting signal. The tripartite pattern L-A-X-[TA]-
[GNSD]-X(1,11)-[VIFAGTSMLWCNRK](14,20)-X(6)-
[RK](1,5) incorporates the cleavage motif, the trans-
membrane region and the anchor site typical of sortase
substrates. Matches to either pattern were retained
and HMMER was used to build a HMM. The entire
Rs-33209 genome was scanned for matches using
this HMM.
Sequences containing the query cell wall sorting sig-
nal (cleavage or tripartite) were considered true puta-
tive sortase substrates if they had 3 properties: (1) they
contained a transmembrane region in the C-terminus,
(2) they had no more than 3 transmembrane helices
and (3) they showed no homology to known intracellu-
lar proteins (Boekhorst et al. 2005). Transmembrane
regions were predicted by the program TMHMM, ver-
sion 2.0 (Krogh et al. 2001, Sonnhammer et al. 1998).
The genome was also analyzed using the MEME/
MAST software, as outlined by Boekhorst et al. (2005)
PCR and RT-PCR analysis. The gene sequences
specifying sortase and sortase substrate proteins were
PCR amplified from genomic DNA of Rs-33209. Tran-
scription of sortase and sortase substrates was assessed
by reverse transcription (RT)-PCR. The primer pair
used for the sortase gene were 5’-AGCAATTCTA-
GCGGCAAAAC-3’ and 5’-GTTCGGAAGATCCAAC-
CGTA-3’ and the primer pairs used for sortase sub-
strates are listed in Table 1. Total DNA and RNA were
extracted from Rs-33209 cells by DNeasy genomic
DNA extraction kit (Qiagen) and Ribopure bacterial
RNA extraction kit (Ambion), respectively, according
to the manufacturers’ instructions. Purified RNA was
treated with DNase I (0.16 U µl–1) at 37°C for 30 min to
remove any residual DNA. First-strand cDNA synthe-
sis was done using the SuperscriptTM III first-strand
synthesis system for RT-PCR (Invitrogen). After the
first strand synthesis, the cDNA was treated with
RNase H (0.2 U µl–1) at 37°C for 20 min to remove the
RNA template from the cDNA:RNA hybrid. PCR and
the second step of RT-PCR reactions were carried out
in 50 µl reaction volumes containing 50 ng genomic
DNA for PCR or 0.3 µg of cDNA as template DNA for
RT-PCR, 200 µM concentration of each dNTP, 30 pM of
each primer, 1.5 mM MgCl2 and 2.5 U of Taq DNA
polymerase (Promega) in 1× Taq reaction buffer. PCR
cycling conditions consisted of an initial denaturation
at 95°C for 1 min followed by 30 cycles of denaturation
at 95°C for 30 s, primer annealing at 48°C for 20 s, and
primer extension at 72°C for 1 min. This was followed
by a final extension at 72°C for 2 min. The size and
quality of PCR products were determined by elec-
trophoresis on 1.5% agarose gel at a constant current
of 100 V and visualized under UV transillumination.
One kb and 100 bp DNA ladder (New England Biolab)
were used as the standard for size comparison.
Growth of bacteria in SKDM-2 medium containing
vinyl sulfones. Six vinyl sulfone drugs, viz. phenyl
vinyl sulfone (PVS), methyl vinyl sulfone, ethyl vinyl
117
ORF ID Primer pairs used in PCR and RT-PCR Function/Annotation CWS- motif
(Accession no.)
RRSA00417 5’-CCTTGTCGGCCTTGTCTTAG-3’ Possible proteinase
(EF426712) 5’-GACGACGTCGAACGGTCAG-3’ (Rhodococcus sp. RHA1) LAnTG
RRSA00636 5’-AAGCGCCAGTCAAGAACG-3’ Putative glycosyl hydrolases family 31
(EF426714) 5’-ACGACGAAGCAGCAGTAAGC-3’ (Arthrobacter aurescens TC1) LAaTG
RRSA01045 5’-GCCAAGATGGCAGCGTTT-3’ Hypothetical membrane protein
(EF426715) 5’-CGAGCGACCACCGTCATAC-3’ (Bifidobacterium longum NCC2705) LAaTG
RRSA01248 5’-GATATGAAATCCCTCTTTTACCG-3’ Putative partial cell-surface adhesin, Cna B-type
(EF426716) 5’-CCGAGCGAAGAAAACTGATG-3’ (Chloroflexus aggregans DSM 9485) LAnTG
RRSA01787 5’-ACCAATTCGTGCAGAAAACC-3’ Acid phosphatase (EC 3.1.3.2) LAaTG
(EF426717) 5’-TAGTTGAAGCGTTTGCGTTG-3’ (Corynebacterium jeikeium K411)
RRSA02499 5’-TTGGTGGACATCGCGAAC-3’ Hypothetical protein AAur_1465 LAeTG
(EF426718) 5’-GAGTGACGGTGCCTTCGTA-3’ (Arthrobacter aurescens TC1)
RRSA00949 5’-CGGGGACGACCAAAACTT-3’ Hypothetical protein Arth_2238 LPvAG
(EF426719) 5’-TAAATTTACCGGGTAGCGATAAG-3’ (Arthrobacter sp. FB24)
RRSA02971 5’-GAAAATCGCTGGAACTGTTG-3’ Putative integral membrane protein LAiTG
(EF426720) 5’-AGAGCCGCCCTGGCTTAC-3’ (Arthrobacter aurescens TC1)
Table 1. Renibacterium salmoninarum-33209: probable SrtD substrates

Dis Aquat Org 78: 115–127, 2007
sulfone, divinyl sulfone, phenylsulfonyl ethylene and
phenyl trans-styryl sulfone (all from Sigma-Aldrich)
were screened for their effect on cell growth and via-
bility. One molar stock solutions of all drugs were pre-
pared in 100% sterile dimethyl sulfoxide (DMSO)
(Research Organics). Bacteria were grown for 14 d at
15°C with constant stirring in SKDM-2 broth contain-
ing different concentrations (0.625 to 10 mM) of the
vinyl sulfone drugs. As a control, sterile DMSO alone
was added to bacterial cultures not treated with drugs.
In order to assess the effect of PVS on bacterial growth,
14 d old 10 ml broth cultures were used to inoculate
100 ml SKDM-2 medium with and without PVS.
Aliquots of cultures were drawn on alternate days for
14 d and optical density (OD525) was measured to
assess the growth.
Purification of Chinook salmon fibronectin. Fibro-
nectin protein from the muscle tissue of healthy Chin-
ook salmon was purified by affinity chromatography.
Muscle tissue was aseptically removed from Chinook
salmon and homogenized in binding buffer (PBS, pH
7.2) using a sterile tissue homogenizer. One milliliter of
the macerated tissue extract (~5 mg) was applied to a
10 ml gelatin sepharose™ 4B column (Amersham
Pharmacia Biotech) that had been previously equili-
brated with binding buffer. Following 2 washes with
binding buffer, the bound fibronectin was eluted with
binding buffer containing 8 M urea. Eluted proteins
were dialyzed for 24 h against PBS (pH 7.2), concen-
trated, and stored at –20°C until used. The purity of the
protein was ascertained by SDS-polyacrylamide gel
electrophoresis. The protein concentration was deter-
mined using a NanoDrop ND-1000 spectrophotometer
(NanoDrop Technologies).
Chinook salmon fibronectin binding assays. The
ability of bacteria to bind Chinook salmon fibronectin
was assayed using a fibronectin-enzyme linked im-
munosorbent assay (ELISA). Bacteria grown in SKDM-
2 medium in the presence of each of 6 different vinyl
sulfone drugs were screened for their ability to bind
Chinook salmon fibronectin and compared to the
fibronectin binding ability of normal untreated bacte-
ria. Wells of 96 well flat-bottom microtitre ELISA plates
were coated with 100 µl of Chinook salmon fibronectin
(10 µg ml–1) in 0.1 mM carbonate buffer (pH 9.6). The
plates were covered and incubated overnight at 4°C.
After rinsing 4 times with PBS containing 0.75%
Tween-20 (PBS-T), the wells were blocked with 100 µl
of 3% BSA (bovine serum albumin) in PBS for 1 to 2 h
at 37°C and then rinsed once with PBS-T.
Bacteria were grown at 15°C in SKDM-2 broth
medium containing different concentrations of vinyl
sulfone drug ranging from 0.625 to 10 mM or without
the drug for 14 d. Bacteria were then harvested by cen-
trifugation at 5000 × g for 10 min, washed once with
PBS and resuspended in PBS-T containing 1% BSA.
The bacterial samples were adjusted to an OD525 of 0.5,
and 100 µl of each suspension was added to a
fibronectin-coated well and incubated at 37°C for 2 h.
After washing the wells 4 times with wash buffer
(Kirkegaard & Perry Laboratories) 200 µl of peroxidase
conjugated goat anti-Renibacterium salmoninarum
polyclonal antibody (Kirkegaard & Perry Laboratories)
diluted 1:4000 in 1× milk was added and incubated at
room temperature for 2 h. After 4 washes with wash
buffer, 200 µl of 2,2’-azino-bis (3-ethyl-benzthiazoline-
6-sulphonic acid) (ABTS) peroxidase substrate (Kirke-
gaard & Perry Laboratories) were added and incu-
bated at 37°C for 30 min. The OD405 of each reaction
was measured using an ultra microplate reader
(ELX808IU, BioTek Instruments). Triplicate samples
were used for each treatment and the assays were
repeated at least 3 times.
Cell adherence assays. The ability of Renibacterium
salmoninarum to adhere to host cells was measured in
2 different ways. Initially, adherence of Rs-33209 to
CHSE-214 cells was visualized by a fluorescent anti-
body staining procedure. Bacteria were grown in
SKDM-2 medium with and without 10 mM PVS, as
described previously. The bacteria were harvested by
centrifugation at 5000 × g for 10 min, washed once with
HBSS, and resuspended in MEM complete medium,
and the optical density (OD525) was adjusted to 0.5.
CHSE-214 cells were cultured on cell culture-treated
Thermanox sterile coverslips (Nalge Nunc) kept in
24-well culture plates and incubated at 15°C for 24 to
48 h. The cell monolayers on the coverslips were
washed once with HBSS, infected with 100 µl of the
bacterial suspension per well and incubated at 15°C
for 2 h. The monolayer was washed 4 times in PBS and
once in PBS containing 0.1% triton X-100. The washed
monolayer was then covered with 30 µl of fluorescein-
conjugated affinity purified anti-R. salmoninarum goat
antibody (50 µg ml–1 in PBS, pH 7.2) and incubated for
1 h at room temperature. After reacting with antibody
the monolayers were washed once with PBS and coun-
terstained with 0.02% Evan’s blue stain for 5 min. The
stained monolayers were rinsed with PBS and air-
dried. Epifluorescence microscopy was performed on a
light microscope (Carl Zeiss, Axioscope) using the
100× objective and appropriate filters, and imaged
using a Qimaging Micropublisher 3.3 RTV camera and
software system.
For a quantitative measure of Renibacterium sal-
moninarum adherence, CHSE-214 cells were grown in
MEM-complete medium containing 10% fetal bovine
serum at 15°C. The cells were counted using a
Neubauer hemocytometer, seeded at a density of 0.5 ×
105 cells well–1 in 24-well tissue culture plates, and
incubated at 15°C for 24 to 48 h to confluent monolay-
118

Sudheesh et al.: Inhibition of Renibacterium salmoninarum adherence
ers. Bacteria were grown in SKDM-2 tubes for 14 d at
15°C, washed once with PBS and resuspended in
MEM-complete medium. For the cell adherence assay,
CHSE-214 cells were infected with bacteria at a multi-
plicity of infection (m.o.i.) of 10 bacteria per fish cell for
2 h at 15°C. The monolayer was then washed 5 times
with Hank’s balanced salt solution (HBSS). The
washed cells were disrupted by the addition of 0.5 ml
sterile deionized ice-cold water and repeated pipet-
ting. Serial dilutions of the lysate were plated onto
SKDM-2 agar for counts of viable bacteria. The percent
adherence was calculated as follows:
(CFU on plate/CFU in original inoculum) × 100
where CFU = colony forming units. Assays were per-
formed in duplicate and repeated at least 3 times.
Intracellular growth determination. For intracellu-
lar growth assay, CHSE-214 cells were infected with
bacteria at an m.o.i. of 10 bacteria per fish cell and
incubated for 8 h at 15°C. Monolayers were washed 5
times with HBSS, followed by the addition of MEM
complete medium containing 10 µg ml–1 gentamycin
and incubation at 15°C for an additional 30 min. Mono-
layers were then washed 3 times with HBSS, fresh
MEM complete medium without gentamycin was
added and incubated at 15°C. After incubating the
plates for different time points, the monolayer was
washed again with HBSS and lysed with sterile deion-
ized ice-cold water and repeated pipetting. The cell
monolayers were observed microscopically to assure
complete lysis. Serial dilutions of the lysate were
plated onto SKDM-2 agar for counts of viable bacteria.
The time immediately following the removal of the
gentamycin was taken as the zero time point. Assays
were performed in triplicate and repeated at least 3
times.
For Giemsa staining, the monolayers were grown on
cell culture-treated Thermanox sterile coverslips
(Nalge Nunc) kept in 24-well culture plates, and infec-
tion was performed as described in the intracellular
growth assay. The coverslips were removed from the
wells at different time points after the gentamycin
treatment and incubation, and stained with 0.01%
Giemsa stain for 45 min. The stained monolayers on
the coverslips were observed under a light microscope
(Carl Zeiss, Axioscope) using a 100× objective, and
representative cells from at least 3 repeated experi-
ments were photographed using a Qimaging Micro-
publisher 3.3 RTV camera and software system.
Cytopathic effect (CPE) of Renibacterium salmoni-
narum. The CPE assay was performed by preparing
CHSE-214 cell monolayers and bacteria as described
above. CHSE-214 cells were infected at an m.o.i. of 10
bacteria per fish cell and observed for CPE for up to
2 wk. Typical CPE of R. salmoninarum on CHSE-214
cells included morphological changes such as forma-
tion of rounded cells and cell detachment as the infec-
tion progressed. Representative monolayers from at
least 3 repeated experiments were photographed
using a Nikon digital camera mounted on an inverted
microscope (Carl Zeiss, Axiovert 135). Toxicity of PVS
to CHSE-214 cells was assayed by growing the cell
monolayers in 24-well tissue culture plates for 24 to
48 h and then exposing them to different doses of PVS
ranging from 0.001 mM to 100 mM. The monolayers
were incubated at 15°C and observed daily for visible
CPE and morphological changes for 14 d.
Isolation of cell wall-bound proteins. Bacteria were
grown at 15°C for 14 d in SKDM-2 broth medium with
or without 10 mM PVS. Bacteria were harvested by
centrifugation at 5000 × g for 10 min. Cell wall-bound
proteins of Renibacterium salmoninarum were pre-
pared using modified methodology of Stalhammar-
Carlemalm et al. (1993). Briefly, the bacteria were
washed twice with Tris-HCl buffer (50 mM, pH 7.3)
and resuspended in osmotic digestion buffer (20%
sucrose-2.5 µM phenylmethylsulfonyl fluoride in
50 mM Tris-HCl, pH 7.3). Cell wall-bound proteins
were enzymatically released from the bacterial cells
by adding mutanolysin (Sigma-Aldrich) in potassium
phosphate buffer (10 mM, pH 6.2) to a final concentra-
tion of 350 U ml–1. The digestion reaction was incu-
bated for 18 h at 37°C with gentle shaking followed by
2 centrifugations at 20 000 × g for 15 min (4°C) in a
tabletop centrifuge (Eppendorf, 5417R) in order to
remove cell debris and remaining protoplasts. The
supernatant containing the proteins released from the
cell wall was frozen at –20°C for subsequent 2-dimen-
sional electrophoresis (2-DE). The protein concentra-
tion was determined using a NanoDrop ND-1000 spec-
trophotometer (NanoDrop Technologies).
Two dimensional polyacrylamide gel electrophore-
sis analysis. The cell wall-bound protein samples were
extracted in a sample extraction buffer containing
5 M urea, 2 M thiourea, 2 mM tributyl phosphene
(TBP), 2% CHAPS, 2% sulfobetaine 3-10, 0.5% biolyte
ampholyte, 10 mM Tris and 0.001% Orange G dye.
Extracted protein samples were applied to 7 cm IPG
strips (pH 4–7) (Bio-Rad) (25 µg total protein strip–1)
and rehydrated overnight in a humidified chamber at
room temperature. First dimension isoelectric focusing
was performed on IPG strips using a Mini Protean IEF
cell (Bio-Rad). The strips were focused initially for
15 min at 250 V, 2 h at 4000 V and then for 20 000 volt
hours at 4000 V. Focused strips were equilibrated in 5×
Tris-HCl glycine gel buffer containing 6 M urea, 2%
SDS, 20% glycerol, 5mM TBP and 2.5% acrylamide
monomer. Second dimension separation was carried
out using precast 10–20% polyacrylamide gradient
gels with a 2-D miniprep well (Bio-Rad) on a Mini-
119

Dis Aquat Org 78: 115–127, 2007
PROTEAN 3 electrophoresis cell (Bio-Rad). Electro-
phoresis was performed using the standard Laemmli
buffer system (Laemmli 1970) at a constant current of
5 mA for 30 min and then at a constant current of
12 mA for 1.5 h. Prestained BlueRanger® molecular
weight marker mix (Pierce) was used to compare the
molecular weight of protein spots. Multiple gels were
prepared for the same protein samples from both treat-
ments to ensure consistency of protein profiles on 2-D
gels, and isoelectric focusing was carried out simulta-
neously to minimize experimental variability. The gels
were silver stained and scanned.
Nucleotide sequence accession numbers. The DNA
sequences of the Renibacterium salmoninarum sor-
tase and sortase substrate ORFs RRSA01227 (Acc.
No. EF426 713), RRSA00417 (Acc. No. EF426712),
RRSA00636 (Acc. No. EF426714), RRSA01045 (Acc.
No. EF426715), RRSA01248 (Acc. No. EF426716),
RRSA01787 (Acc. No. EF426717), RRSA 02499 (Acc.
No. EF426718), RRSA00949 (Acc. No. EF426719) and
RRSA02971 (Acc. No. EF426720) have been deposited
in the GenBank database.
RESULTS
Characterization of Renibacterium salmoninarum
sortase and sortase substrates
The Rs-33209 genome sequence allowed identification
of an ORF (RRSA01227, Genbank Acc. No. EF426713)
specifying a sortase enzyme. The deduced protein
sequence has a calculated molecular weight of
30682.58 Da and a pI of 7.87. As calculated from
ClustalW pairwise alignments, the amino acid sequence
of the sortase had significant sequence identity to sortase
or sortase-like proteins of Arthrobacter sp. FB24 (61%),
Brevibacterium linens (39%), Kineococcus radiotoler-
ans (36%), Acidothermus cellulolyticus (32%), Bifido-
bacterium longum (32%) and Frankia alni (32%) (Fig. 1).
All putative genes in Rs-33209 (3515 total open reading
frames) were compared to the 6 sortase subfamily
models of Comfort & Clubb (2004). The only significant
result was ORF RRSA01227 matching to subfamily 5
(score of 220.2 and e-value of 3.1e-66). The amino acid
sequence of this sortase protein clustered in a neighbor-
joining phylogenetic tree with other Class D sortase
enzymes (result not shown). The sortase subfamily 5
described in Comfort & Clubb (2004) is equivalent to the
Class D sortase group of Dramsi et al. (2005). Therefore,
the Renibacterium salmoninarum sortase gene is de-
signated srtD based on the latest classification of sortase
enzymes (Dramsi et al. 2005).
All amino acid sequences were scanned for the tri-
partite pattern and cleavage motif of the subfamily 5
sorting signal resulting in 698 matches to the cleavage
motif (LAxTG) and 19 matches to the tripartite pattern
(results not shown). According to the criteria used, 8
of the putative sortase substrates were true positives
with both features (Table 1). Searching the Rs-33209
genome with an HMM built from these putative sor-
tase substrates or by using the MEME/MAST software
as outlined by Boekhorst et al. (2005) failed to yield any
additional novel matches (results not shown).
Specific primers were designed to PCR-amplify
these gene sequences from the bacteria. All the genes
were transcribed in the bacteria as observed by RT-
PCR (data not shown).
Use of vinyl sulfone sortase inhibitors to inhibit the
ability of bacteria to bind Chinook salmon fibronectin
Many of the sortase substrates studied to date
are MSCRAMs. This group of bacterial proteins often
includes proteins involved in adherence to eukary-
otic matrix proteins such as fibronectin. To inves-
tigate the ability of Renibacterium salmoninarum
to bind fibronectin (a common ligand of bacterial
MSCRAMs), we optimized and used a salmon fibro-
nectin ELISA. Fibronectin protein was purified from
Chinook salmon muscle tissue by gelatin sepharose
affinity chromatography; the purity of the protein
was determined by observing a single high molecu-
lar weight protein band on SDS-PAGE. Initial opti-
mization of the fibronectin ELISA was performed
using PVS (Frankel et al. 2004). By titrating different
concentrations of PVS and fibronectin, it was found
that maximum inhibition of fibronectin binding was
achieved at doses of 10 mM PVS and 10 µg ml–1
fibronectin (Fig. 2A). Fibronectin binding ability was
significantly reduced (75.8%) in bacteria treated with
PVS (Fig. 2B) in comparison with controls. The
fibronectin ELISA was used to screen 5 other vinyl
sulfone drugs at 10 mM doses for their ability to
inhibit fibronectin binding and, thus, to indirectly
assess their ability to inhibit sortase activity. The
reduction in fibronectin binding abilities of methyl
vinyl sulfone (17.53%), ethyl vinyl sulfone (12.6%),
divinyl sulfone (8.14%), phenylsulfonyl ethylene
(3.47%) and phenyl trans-styryl sulfone (1.32%) were
significantly lower than that of PVS. Therefore PVS
was selected for all other experiments.
Growth of bacteria in SKDM-2 medium containing
vinyl sulfone drugs
Growth of Rs-33209 in SKDM-2 broth medium was
screened by measuring the OD525 of the broth culture
120

Sudheesh et al.: Inhibition of Renibacterium salmoninarum adherence
in order to determine whether PVS treatment may
affect growth and viability of the bacteria. The growth
of Rs-33209 in SKDM-2 medium containing 10 mM
concentration of each of 6 vinyl sulfone drugs was not
significantly different from that of normal untreated
bacteria, indicating that they do not affect the growth
of Rs-33209 (data not shown).
Inhibition of cell adherence, cell invasion and CPE
The ability of Renibacterium salmoninarum treated with
PVS to adhere to a Chinook salmon embryo cell line
(CHSE-214) was determined by fluorescence microscopy
using a fluorescein-conjugated R. salmoninarum specific
antibody. Normal Rs-33209 cells adhered strongly to
121

R. salmoninarum --------------------------------------------------
Arthrobacter sp.FB24 --------------------------------------------------
Brevibacterium linens MSTAPELGGGQNSGAGSHPGTGSQAGTDQPLPSRRELRRREAEAAAANGA 50
Kineococcus radiotolerans --------------------------------------------------
Acidothermus cellulolyticus ------------------------------------MTRTLQKSPVTTIR 14
Bifidobacterium longum --------------------------------------------------
Frankia alni ----------------------------MPATNRRHLADSLTDLPVQPGA 22

R. salmoninarum ----------------MELGTSRGSQEANIGVDELVSGALK--------R 26
Arthrobacter sp.FB24 -------------------------------------MVLL--------E 5
Brevibacterium linens PTAVYSDAPPAYQDSTQVQQPVSSETNPNLGAHAGAGAAAAGYSAPGEGS 100
Kineococcus radiotolerans --------------------------------------------------
Acidothermus cellulolyticus G-------------------------SAP--------------------- 18
Bifidobacterium longum ------------------------MAADPANASR---------------A 11
Frankia alni GRPPAPASPTA--DPHLGEPAHRGARAAPGGPGRSRGARRPGLVRHTPWP 70

R. salmoninarum DRSDRGRPRRSRPRKGVFR---------TIVQVFGELLITLGVILMLFVG 67
Arthrobacter sp.FB24 KETTAAVPARG---VGVLR---------IVVQIVGELLITVGVILLLFVA 43
Brevibacterium linens RRTNRRRQKPEREPLGPVR---------GTIRTFGELCITAGMVLILFVV 141
Kineococcus radiotolerans ----------------MIR---------RATSVLGELLVTAGVLTLLFVV 25
Acidothermus cellulolyticus RRVLG------------VA---------------GEILITLGVVVLLFVG 41
Bifidobacterium longum RSRRAAVRGASSPQRSPVW---------QALGICAELLITAAVICALYIV 52
Frankia alni RRVQPDRVDGRRRRRNPVAGLADRPVGGRVSRGLGEVMITAGLVVVLFLA 120

R. salmoninarum WELWWTNIQSDQTQQQAVQQFAQNFKGPLTPQAS----------APTNYG 107
Arthrobacter sp.FB24 WQLWWTNVESDAKQSETIKNFAQELGGSAAPAASDAPDASTPAPTPTDYG 93
Brevibacterium linens WQLWWTDIEANRDNEQLADKLAQDWQNQDPNEL------------PDDPD 179
Kineococcus radiotolerans WQLHWTDLTSGRAQAATVTSLQQQWDAAPAPTATAG---AAATAAPTAPA 72
Acidothermus cellulolyticus YDLWFTGLYTASAQRELKHELAITWQTATANPAPPP-------APSAVPS 84
Bifidobacterium longum WQMWWTGVEAERAQNETTQSVDWSDPSNNGGTVT---------IAKAQEG 93
Frankia alni YQLWITDIFAARTQDRLRNDLTTAWSRQPHPRAP---------AEAAKPR 161

R. salmoninarum DPVVTKAPDAAGETFGLAYIPRFGADYKPRPLVQGTAQR-ELDTLGLGHH 156
Arthrobacter sp.FB24 PPRVAEAP-GHGQTIGIMYIPRFGADYT-RPIVQGTSTD-VLDTLGLG-H 139
Brevibacterium linens EP-VVADPVEKNSAFGIFYIPRFGDDYY-RTVAEGVDLEPVLNRMGVG-R 226
Kineococcus radiotolerans TARAVDETPPTGDAFAILHVPRFGEDYA-VPVVEGTGTE--ELKEGIG-H 118
Acidothermus cellulolyticus PDGVLPDDVVPGNALALIRIPRLGRHYV-YAIVEGVSTA--DLKKGPG-H 130
Bifidobacterium longum DAPVQPKDAKYGDLIAQIYIPRFGSQWH-RNIVEGTTLE-QLNRHGLG-H 140
Frankia alni P--VVPP-VELGEGVAVLRVPRFGADYA-PVVVEGVSVA--ALRRGPG-H 204

R. salmoninarum YTSTAMPGAVGNFAVAGHRQTHGAVLDAIHALVPGDKIYVQTQDGYYTYV 206
Arthrobacter sp.FB24 YSDTAMPGATGNFAVAGHRQTHGAVLDNIHTLVPGDKIYVQTRDGFYVYV 189
Brevibacterium linens YPNSAMPGEVGNFSIAGHRVTYGKPLNQIAQLRPGDEIIVQTKDGFYTYT 276
Kineococcus radiotolerans YADAALPGEVGNFAIAGHRVTYGKPFHLIADLQEGDAVVVATATQWFTYR 168
Acidothermus cellulolyticus YPGTAMPGQVGNFVVSGHRTTYLAPFNGLDKLRLGDPIVIETATMWYVYR 180
Bifidobacterium longum YDTTQMPGQVGNFAVAGHRNGYGQPLGDVDKLQEGDPIIVRTKDYWYVYH 190
Frankia alni FPGTAMPGDVGNFVVSGHRTTYGKPFSRLDELRVGDPLVVEVADRYFTYR 254

R. salmoninarum FRNSEIVLPTQTSVLAPVPTQSSAQPTDRYLTLTSCNPRFGV-AERFIAY 255
Arthrobacter sp.FB24 FRNNQIVLPSATDVLLPVPTQPAARPTEAYLTMTSCNPRFGS-QERIIAY 238
Brevibacterium linens FRNFDIILPDAVEVLAPVPNEPKFKGKDRILTMTACNPMFSA-RERYVAY 325
Kineococcus radiotolerans VRSHEVVSPKQVSVIAPVPGRPGETPTEAWLTMTACHPMHSA-RQRYVVH 217
Acidothermus cellulolyticus VTQMETVLPTDVAVILPVPDHPGERPTEALITLTTCTPKYSA-SHRLVVH 229
Bifidobacterium longum YTRYEIVLPTDVHVIAPNPEDSTANPTKRMITLTTCEPKYSTPTHRWISY 240
Frankia alni VTGSEVVDPHRLDVTYPVPGHAGVAPTRALMTLTTCHPRFSA-RSRLIVF 303

m
m

R. salmoninarum AVLESWQPASAGPPAEIAQ-----QVQAAAGQG----------------- 283
Arthrobacter sp.FB24 SLLDHWQPASAGPPAEIAA-----QVAKALGKG----------------- 266
Brevibacterium linens AELTDWTPAGDGAPDNIKDSKAYDKVSKNGGA------------------ 357
Kineococcus radiotolerans AQLESVQDRSAGPPASLTA----------AG------------------- 238
Acidothermus cellulolyticus GRLETAQPKSAGIPAVLREG------------------------------ 249
Bifidobacterium longum GELAYWAKVSDGVPKELATTDSSGAVMFSTTETPSIASRIGSLDKVVFGA 290
Frankia alni ANLDETTDKSDGPPRALADE------------------------------ 323
Fig. 1. CLUSTAL W alignment of sortase-like protein sequences from different Gram-positive bacteria. Amino acid residues iden-
tical in all 7 sequences are shown in white font on a black background. Residues present in at least 5 of the 7 sequences are
shown in black font on a grey background. Black arrows indicate the positions of the probable active site cysteine and the
conserved histidine residue. R. = Renibacterium

Sudheesh et al.: Inhibition of Renibacterium salmoninarum adherence
PVS-treated bacteria (Fig. 6B) as compared to un-
treated bacteria (Fig. 6A). Repeated extractions from
several groups of PVS-treated and untreated bacteria
gave identical results, and the consistency of the 2-DE
protein profiles on gels confirmed the differences ob-
served between PVS-treated and untreated bacteria.
Toxicity of PVS to CHSE-214 cells
PVS at concentrations up to 50 mM was non-toxic to
CHSE-214 cells. The cell monolayers grew normally
and absence of any CPE, such as visible morphological
changes or rounded and detached cells, when ob-
served for up to 2 wk in culture at this concentration of
PVS. At higher doses, precipitation of PVS in the cul-
ture medium was observed, making assessment of
CPE and morphological observation difficult.
DISCUSSION
As antibiotics are becoming increasingly inefficient
in treating bacterial infections because of the emer-
gence of antibiotic resistant bacteria, alternative thera-
peutic strategies aimed at the disruption of bacterial
virulence are gaining importance in current antibacte-
rial drug development programs. Our finding that
PVS, a known sortase inhibitor, dramatically reduces
the ability of Renibacterium salmoninarum to adhere,
123
Fig. 4. Intracellular growth of Renibacterium salmoninarum strain Rs-33209 in CHSE-214 cells. (A) CHSE-214 cells were infected
and intracellular growth was measured using the gentamycin protection assay as described in ‘Materials and methods’. Mean log
CFU ± SD estimates from viable plate counts of triplicate samples (y-axis) are plotted for different growth times. (B) Light microscopy
of intracellular survival and multiplication of Rs-33209 in CHSE-214 cells over time. CHSE cells were infected by Rs-33209 and
extracellular bacteria were killed by using gentamycin (see ‘Materials and methods’). Panels show representative Giemsa-stained
CHSE-214 cells with bacteria growing inside at (i) 1 d, (ii) 2 d, (iii) 4 d and (iv) 8 d after gentamycin treatment. Scale bars = 10 µm
Fig. 5. Cytopathic effect of PVS-treated and untreated Rs-33209 to CHSE-214 cells. (A) Uninfected normal CHSE-214 cells.
(B) CHSE-214 cells infected with washed bacteria previously grown in SKDM-2 medium containing 10 mM PVS. (C) CHSE-214
cells infected with washed normal bacteria previously grown in SKDM-2 medium. Scale bars = 50 µm

Dis Aquat Org 78: 115–127, 2007
invade, and grow intracellularly in fish cells opens up
the possibility of using vinyl sulfone drugs as a poten-
tial anti-virulence treatment option for managing BKD
in salmonid fish.
The presence of a sortase in Renibacterium salmoni-
narum was unknown until the genome was sequenced.
The Rs-33209 sortase most closely resembles the sub-
family-5 or class D of bacterial sortases based on recent
classification schemes (Comfort & Clubb 2004, Dramsi et
al. 2005). Subfamily-5 sortases recognize a LAxTG motif.
In contrast, most Gram-positive bacteria possess SrtA or
subfamily-3 sortases that recognize proteins with CWS-
motifs LPxTG[DE] and LPxTGG, respectively. SrtB sor-
tases recognize an altogether different CWS-motif in
Staphylococcus aureus (NPQTN), Listeria (NPKSS) and
Bacillus (NPKTG, NPKTD and NPQTG), while subfam-
ily-4 sortases process proteins containing LPxTA[ST]
motifs. It is interesting that R. salmoninarum has a high
G+C content (56.3%), and that high G+C Gram-positive
bacteria belonging to Actinobacteria (G+C > 55%) pos-
sess subfamily-5 sortase. Presumably these bacteria
have replaced SrtA enzymes with subfamily-5 homologs
(Comfort & Clubb 2004).
Bioinformatics analyses identified the presence of
8 ORFs in the Rs-33209 genome specifying potential
sortase substrates with some predicted to be cell sur-
face proteinases or adhesins. These proteins may be
MSCRAMs or surface virulence determinants, and
may play an important role in adhesion and coloniza-
tion of the host during Renibacterium salmoninarum
infection. Out of the 8 sortase substrate ORFs identi-
fied, 7 possess the LAxTG sorting signal recognized by
subfamily-5 sortases. Interestingly, ORF RRSA00949
has a sorting motif (LPvAG) that does not fall into any
of the proposed 5 subfamilies (Comfort & Clubb 2004).
The presence of only one complete sortase gene (srtD)
in the Rs-33209 genome and the variation in amino
acids at specific conserved positions in the sorting
motifs found in RRSA00949 point to the possible flexi-
bility of R. salmoninarum SrtD in recognizing proteins
with different sorting signals. Alternatively, this pro-
tein may not be a legitimate R. salmoninarum SrtD
substrate.
Fibronectin is abundant in the eukaryotic extra-
cellular matrix and plasma, and is the ligand for
many bacterial surface adhesins (Joh et al. 1999). Ear-
lier findings have shown that the fibronectin binding
ability of srtA− mutants of Staphylococcus aureus
(Mazmanian et al. 2000) and S. agalactiae (Lalioui et
al. 2005) was severely hampered compared to wild
type bacteria. We screened 6 vinyl sulfone drugs (by a
fibronectin-binding ELISA) for their ability to inhibit
the binding of Renibacterium salmoninarum to Chi-
nook salmon fibronectin. While all 6 vinyl sulfones
inhibited adherence to some extent, PVS was the most
efficient. Similarly, Frankel et al. (2004) have shown
that PVS inhibited fibronectin binding of S. aureus to
an extent comparable to that of an untreated srtA−
mutant strain. Although the treatment of Rs-33209
with PVS prevented its adherence to fibronectin, the
treatment did not have any significant effect on bacte-
rial cell viability and growth. Vinyl sulfones are known
to irreversibly inactivate sortase enzymes by capturing
cysteine nucleophiles in the active site of the enzyme
and forming a stable non-functional thioether adduct
(Frankel et al. 2004). Based on the fibronectin binding
results, we hypothesize that one or more proteins that
are involved in adherence of the bacterium to host tis-
sues are substrates for SrtD and are not displayed on
the cell surface of Rs-33209 after PVS treatment. The
ability of PVS to inhibit R. salmoninarum adherence to
CHSE-214 cells was further analyzed. When PVS-
treated bacteria were used to infect CHSE-214 cells in
a cell adherence assay, their binding ability was signif-
icantly reduced compared to untreated bacteria. R.
salmoninarum has a significant ability to adhere to
CHSE-214 cells and produces CPE within 4 to 5 d of
infection, whereas most of the PVS-treated bacteria
could not adhere to CHSE-214 cells. PVS-treated bac-
teria were found floating in the culture medium and
were easily removed during the washing steps. Fur-
ther evidence for the difference in adherence of PVS-
124
Fig. 6. Two-dimensional gel electrophoretic analysis of cell wall-bound proteins of Rs-33209. (A) Cell wall-bound proteins of
untreated bacteria grown in SKDM-2 medium. (B) Cell wall-bound proteins of bacteria grown in SKDM-2 medium in the
presence of 10 mM PVS. Circled and boxed protein spots are present in the normal untreated bacteria but are absent in the
PVS-treated bacteria

Sudheesh et al.: Inhibition of Renibacterium salmoninarum adherence
treated and untreated bacteria came from immunoflu-
orescence microscopy of infected monolayers. The flu-
orescein-conjugated anti-R.salmoninarum antibody
detected very few bacteria adhering to CHSE-214
monolayers infected with Rs-33209 previously grown
in the presence of PVS. Taken together, these data
strongly suggests that PVS is inhibitory to the Rs-33209
SrtD sortase, which results in the disruption of translo-
cation and surface display of many MSCRAMs or other
sortase substrates on the bacterial surface. Unequivo-
cal proof of this hypothesis will require construction
and characterization of R. salmoninarum mutants lack-
ing srtD.
Mere physical contact between the bacteria and
CHSE-214 cells did not result in CPE, indicating the
need for specific adherence of the bacteria to CHSE-
214 cells to produce CPE. The observation that the
PVS-treated bacteria did not produce any CPE even
after 1 mo of infection suggests that important sor-
tase-mediated surface proteins involved in the
virulence of the bacteria were disrupted by the PVS
treatment. This finding also supports an earlier
study demonstrating that extracellular products (ECP)
of Renibacterium salmoninarum did not possess
hemolytic or cytotoxic activity, and the injection of
ECP did not result in toxicity to fish (Bandín et al.
1991). Conversely these enzyme activities, possibly
located on the bacterial cell surface (Bandín et al.
1991, Grayson et al. 1995), are expressed only during
intracellular growth (McIntosh et al. 1997). The com-
bination of these prior studies and our current find-
ings strongly suggests a specific requirement of R.
salmoninarum to adhere to fish cells as a prerequisite
for establishing infection. Yet another possibility is the
presence of toxin secretion mechanisms similar to the
contact-dependent type III secretion systems (TTSS)
found in Gram-negative bacteria. In fact, genome
sequencing has identified a TTSS in at least one
Gram-positive bacterium (Symbiobacterium ther-
mophilum, Ueda et al. 2004). However, a search of the
Rs-33209 draft genome did not identify any gene
sequences involved in TTSS. The fact that the sortase
substrates identified by the bioinformatics approach
in this study include 2 adhesin homologs, a surface
anchor peptidase, a hydrolase, a sialidase and an acid
phosphatase suggests that some of these proteins are
involved in the adherence of R. salmoninarum to
CHSE-214 cells.
It is not surprising that impairment of Renibac-
terium salmoninarum adherence results in a failure to
invade and multiply inside CHSE-214 cells. Although
the ability of R. salmoninarum to invade phagocytic as
well as epithelial and fibroblast cells of fish has been
documented (Bandín et al. 1993, Gutenberger et al.
1997, McIntosh et al. 1997), this is the first conclusive
demonstration that R. salmoninarum replicates intra-
cellularly after invasion. Intracellular survival is an
efficient strategy exploited by many pathogenic bac-
teria to evade host immune responses and/or to allow
the bacteria to spread to adjacent tissues from a pri-
mary site of colonization. The chronic nature of BKD
(Fryer & Lannan 1993), along with the ability of R.
salmoninarum to survive intracellularly points to a
similar evasion strategy. Intracellular survival likely
protects R. salmoninarum from exposure to antibiotics
such as erythromycin, a probable contributing factor
in the reemergence of R. salmoninarum after cessa-
tion of treatment. Microscopic observation of intracel-
lular bacteria shows that the multiplication of the bac-
teria inside CHSE-214 cells over time results in lysis
of host cells and ultimate release of the bacteria into
the extracellular medium (Fig. 4Biv), leading to inva-
sion of adjacent cells and further spread of the infec-
tion. Hence, it is possible that bacteria freshly
released from fish cells several days after ery-
thromycin treatment are exposed to sub-clinical con-
centrations of the antibiotic leading to induced
macrolide drug resistance. Therefore, intracellular
survival combined with the exposure to sub-clinical
doses of antibiotic and the consequent induced drug
resistance results in macrolide resistant R. salmoni-
narum, partially explaining the chronic nature of BKD
in treated salmon populations.
Results from PCR and RT-PCR analyses confirmed
that the srtD and genes specifying 8 probable sortase
substrates are transcribed in Rs-33209. This prompted
further investigation of the cell surface protein translo-
cation in Renibacterium salmoninarum. A comparative
proteomic analysis using 2-DE of cell wall-bound
proteins from PVS-treated and untreated bacteria
revealed the absence of at least 10 protein spots in the
PVS-treated bacteria. This result is additional strong
evidence that PVS treatment inhibits R. salmoninarum
SrtD activity, preventing the translocation and anchor-
ing of these proteins to the cell wall. However, con-
firmation that these proteins are the same sortase
substrates identified by bioinformatics analysis will
require characterization using mass spectrometric
analysis. Research is underway to characterize each of
these proteins and determine their roles in host-
pathogen interactions.
Our observation that PVS-treated Rs-33209 are
incapable of invading and multiplying inside CHSE-
214 cells points to the potential use of the drug to
specifically prevent the invasion and intracellular
growth of Renibacterium salmoninarum in vivo. PVS
appears to only affect bacterial surface protein
expression and presumably virulence, and therefore
is a good example of an anti-infective class of antimi-
crobial agents (Aberg & Almqvist 2007, Lee et al.
125

Dis Aquat Org 78: 115–127, 2007
2003). Since inhibition of growth or death of the
pathogen is not required for drug effectiveness, it is
unlikely that PVS-treated R. salmoninarum will
develop resistance to the drug. This study also
showed PVS is non-toxic to CHSE-214 cells at con-
centrations 5 times that needed to show almost com-
plete inhibition of adherence of the bacteria to fish
cells. Investigations are underway to determine the
toxicity and pharmacokinetics of PVS and its ability to
reduce or eliminate R. salmoninarum infection and
clinical BKD in experimentally-infected salmonid fish.
We anticipate that reduction of virulence of R.
salmoninarum using small molecule drugs like vinyl
sulfones will form an efficient antivirulence strategy
and an alternative to antibiotics in controlling BKD.
Further experiments are being conducted to generate
srtD deletion mutants to confirm the involvement of
SrtD in R. salmoninarum virulence.
Acknowledgements. We thank S. Miller, Department of
Microbiology, University of Washington for allowing use of
IEF equipment in his laboratory. We also thank W. Dickhoff,
S. Lory, and L. Rhodes for critically reading the manu-
script and offering helpful suggestions. This research was
supported by a NSF/USDA-CSREES Microbial Genome
Grant (WNR-2004-00585) (www.nwfsc.noaa.gov/rs-genome)
and from the NOAA FCRPS Biological Opinion Implementa-
tion Project. Additional funding was provided by the NOAA
Fisheries Service.
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127
Editorial responsibility: David Bruno,
Aberdeen, UK
Submitted: May 2, 2007; Accepted: August 5, 2007
Proofs received from author(s): December 7, 2007