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Journal of Bacteriology, August 2001, p. 4451-4458, Vol. 183, No. 15
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.15.4451-4458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Type IV Fimbrial Subunit Gene (fimA) of
Dichelobacter nodosus Is Essential for Virulence,
Protease Secretion, and Natural Competence
Ruth M.
Kennan,1,*
Om P.
Dhungyel,2
Richard J.
Whittington,3
John R.
Egerton,2 and
Julian
I.
Rood1
Bacterial Pathogenesis Research Group, Department of
Microbiology, Monash University, Victoria 3800,1
Department of Veterinary Clinical Sciences, The University of
Sydney, Camden, New South Wales 2570,2 and
Microbiology and Immunology Section, Elizabeth Macarthur
Agricultural Institute, NSW Agriculture, Menangle, New South Wales
2568,3 Australia
Received 20 March 2001/Accepted 15 May 2001
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ABSTRACT |
Dichelobacter nodosus is the essential causative agent
of footrot in sheep. The major D. nodosus-encoded
virulence factors that have been implicated in the disease are type
IV fimbriae and extracellular proteases. To
examine the role of the fimbriae in virulence, allelic
exchange was used to insertionally inactivate the fimA
gene, which encodes the fimbrial subunit protein, from the
virulent type G D. nodosus strain VCS1703A. Detailed
analysis of two independently derived fimA mutants
revealed that they no longer produced the fimbrial subunit protein
or intact fimbriae and did not exhibit twitching motility. In
addition, these mutants were no longer capable of
undergoing natural transformation and did not secrete
wild-type levels of extracellular proteases. These effects were not due
to polar effects on the downstream fimB gene because
insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a
sheep pen trial conducted under controlled environmental conditions
showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time
that molecular genetics has been used to determine the role of
virulence genes in this slow growing anaerobic bacterium.
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INTRODUCTION |
Footrot is a highly contagious
disease of the feet of sheep and is characterized by the separation of
the keratinous hoof from the underlying epidermal tissue, resulting in
severe lameness and loss of body condition (11, 39). The
consequences of the disease are very significant for the wool and sheep
meat industries, and footrot is among the most significant ovine
bacterial diseases, causing economic losses in most producer countries.
The disease is dependent on a mixed bacterial infection, but the
essential causative agent is Dichelobacter nodosus, a
slow-growing, anaerobic, gram-negative rod (2, 37).
D. nodosus exhibits a spectrum of virulence ranging from
virulent strains, which lead to severe underrunning of the horn of the
hoof, to benign strains, which cause a self-limiting interdigital
dermatitis (37).
Little is known about the pathogenesis of ovine footrot,
although the polar type IV fimbriae (12) and extracellular
proteases (23) of D. nodosus have been
traditionally considered virulence factors (2). In
addition, the vap and vrl genomic
islands have been shown to be preferentially associated with
virulent isolates (2). Analysis of the role that these
proposed virulence factors play in the disease process has been
hampered by the lack of a genetic system in D. nodosus.
However, we recently reported the successful transformation of several
D. nodosus strains (22). In these
experiments, a tetracycline resistance gene, tet(M), which was present on a suicide plasmid, was inserted into the chromosome by double-reciprocal crossover events. These studies have
provided the essential tools required to enable the use of reverse
genetics to examine the pathogenic role of the putative virulence
factors of D. nodosus.
The fimbriae of D. nodosus are classified as type IV
because of their highly conserved amino-terminal region, polar
location, association with twitching motility, and the presence of an
N-methylphenylalanine residue as the N-terminal amino acid
(41). D. nodosus fimbriae are highly
immunogenic, with agglutination reactions involving the fimbrial
antigens providing the basis of the classification of D. nodosus into nine major serogroups designated A to I
(4). Vaccination of sheep with whole cells of
D. nodosus or with purified fimbriae protects against
the disease, although this protection is serogroup specific (8,
12, 13). Multivalent recombinant fimbrial vaccines have been
prepared by overexpression of each of the nine fimbrial subunit genes
in Pseudomonas aeruginosa (9). However,
antigenic competition has limited the application of these vaccines
(20, 21, 33) so that the most effective strategies are
based on univalent and divalent formulations.
The fimA genes encoding the major fimbrial subunit from each
of the nine major serogroups have been sequenced, which has allowed the
division of D. nodosus isolates into two major classes
based on the genetic organisation of the fimbrial gene region
(18, 28). In class I strains (serogroups A, B, C, E, F, G,
and I), the fimA gene is followed by fimB, which
encodes a potential 29.5-kDa membrane protein of unknown function but
which is postulated to play a role in the export of fimbrial subunits
to the cell surface (18). Strains of serogroups D and H
belong to class II and have three additional genes downstream of
fimA, namely, fimC, fimD, and fimZ
(18). The fimD gene is postulated to be a
functional homologue of fimB (18),
fimC has sequence similarity to traX from the F
plasmid (14), and fimZ may represent a
redundant fimbrial subunit (18).
In these studies we decided to determine the role of fimbriae in the
disease process by using allelic exchange to disrupt the
fimA and fimB genes of a class I strain and by
examining the virulence of the resultant mutants in a sheep virulence
trial. The results showed that the fimA gene was essential
for the production of type IV fimbriae and twitching motility and was
also required for natural transformation and protease secretion. When
tested in sheep, the fimA mutants were avirulent.
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MATERIALS AND METHODS |
Strains, plasmids, and growth conditions.
All D. nodosus strains were derivatives of the type G strain VCS1703A and
were routinely grown in a Coy anaerobic chamber (Coy Laboratory
Products Inc.) in an atmosphere of 10% H2, 10% CO2, and 80% N2 on Eugon (BBL) yeast extract
(EYE) agar with 5% horse blood, with the addition of 1 µg of
tetracycline or erythromycin per ml for the selection of transformants,
or in EYE broth as described previously (22).
D. nodosus strains for use in the sheep virulence trial
were grown on hoof agar (42). Escherichia coli
strains were derivatives of DH5
(Bethesda Research Laboratories) and
were grown in 2YT medium (35).
Transformation of D. nodosus cells.
Overnight 150-ml broth cultures of the required D. nodosus strain were harvested by centrifugation at
6,000 × g for 10 min, and the cell pellet was
resuspended in 1 ml of fresh EYE broth. Aliquots (100 µl) of this
cell suspension were then mixed with 5 µg of plasmid DNA in Tris-EDTA
buffer (35) and left at room temperature in an anaerobic
environment for 4 h. Two milliliters of EYE broth was then added
to the mixture, and the culture was incubated anaerobically overnight
at 37°C. The cultures were then plated onto EYE blood agar containing
the appropriate antibiotic to select for transformants or onto EYE
blood agar without antibiotics to check viability. The plates were
incubated at 37°C in an anaerobic environment for 7 days.
Molecular methods.
Unless otherwise stated, molecular
techniques were performed using standard procedures (35).
Capillary PCR analysis (22) was used to confirm that the
putative transformants were D. nodosus and that they
carried the tet(M) gene. DNA sequencing was performed using
an ABI 373A automated sequencing apparatus (Applied Biosystems).
Southern hybridization.
Southern hybridization was performed
as described previously (22). In addition to the 16S
rRNA-and tet(M)-specific probes, probes specific for
fimA and fimB were used. These probes were the
PCR products of primers 4142 and 5013 and primers 4944 and 4143, respectively (Table 1), using chromosomal DNA from strain VCS1703A as
the template.
RT-PCR.
D. nodosus cells were grown in EYE
broth, and RNA was extracted using TRIzol (Life Technologies) according
to the manufacturer's instructions. Reverse transcriptase (RT)
reactions were performed at 42°C for 1 h on 5 to 10 µg of
total RNA in 20-µl incubation mixtures that contained 5 mM
MgCl2, 1× RT buffer, 1 mM each deoxynucleoside triphosphate, 1 U of RNasin (Promega), 15 U of avian myeloblastosis virus RT (Promega), and 3 µM appropriate oligonucleotide primer (Table 1). cDNA was amplified in 25-µl
PCRs using a standard protocol and 5 µl of the RT reaction as the
template. PCR products were analyzed on 1% agarose gels and sequenced
to confirm that they were derived from the correct genes.
Immunoblot analysis.
Whole-cell extracts of D. nodosus strains were prepared by removing an EYE blood agar
culture of each strain into 1 ml of phosphate-buffered saline (PBS).
The cell suspensions were then centrifuged at 12,000 × g for 5 min, and the cell pellet was resuspended in 200 µl of
gel loading buffer and boiled for 5 min. Aliquots (5 µl) were then
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(12% acrylamide), and the gels were electroblotted to nitrocellulose
using a Mini Protean II apparatus (Bio-Rad). The blots were developed
using serogroup G-specific antifimbrial antiserum raised in rabbits,
sheep anti-rabbit horseradish peroxidase conjugate, and Renaissance
Western blot chemiluminesence reagent (NEN Life Sciences) according to
the manufacturer's instructions.
Electron microscopy.
Three-day EYE blood agar cultures of
each D. nodosus strain were resuspended in 1 ml of PBS.
Cell samples were attached to carbon-coated copper-rhodium grids,
negatively stained with phosphotungstic acid, and examined using a JEOL
JEM 100S transmission electron microscope.
Twitching motility assays.
Twitching motility assays were
performed on EYE containing 1% agar as previously described
(5). The cells were stab inoculated to the bottom of the
petri dish using a straight wire, and the plates were incubated
anaerobically at 37°C for 3 to 5 days. The medium was then compressed
by placing paper towels on top of the agar, under pressure, for 30 min.
The paper was then removed, and the plates were stained with 0.2%
(wt/vol) Coomassie brilliant blue and then destained in 7% (vol/vol)
acetic acid-33% (vol/vol) methanol. Cultures that exhibited twitching
motility showed large stained zones of growth emanating from the point
of inoculation.
Protease assays.
All D. nodosus strains were
initially tested by standard diagnostic methods involving growth and
elastase production on elastin agar plates (38) and the
determination of protease stability using the gelatin-gel assay
(30). Total protease activity was subsequently determined
using azocasein (Sigma) as the substrate. Briefly, 200 µl of a 44-h
culture supernatant or periplasmic extract was mixed with 200 µl of
assay buffer (0.02 M Tris-HCl, 5 mM CaCl2, 0.2 M NaCl [pH
8.0]) and 400 µl of 6% (wt/vol) azocasein. A 200-µl sample was
withdrawn immediately and mixed with 1 ml of 5% trichloroacetic acid
(TCA), this sample served as the reaction blank. The remainder of the
sample was incubated at 37°C for 8 h, at which time a further 200-µl sample was removed and mixed with 1 ml of TCA. The samples were centrifuged at 12,000 × g for 5 min, and the
absorbance of the supernatant at 334 nm was determined in a Varian DMS
100S UV-visible spectrometer. Standard curves were prepared by
incubating elastase (Sigma) with azocasein for several days to allow
complete digestion. The resultant mixture was precipitated with TCA in the same proportions as above and treated as a 100% hydrolysate. Dilutions of this mixture in TCA enabled a standard curve to be constructed. One unit of protease activity is defined as the amount of
enzyme required to digest 1 µg of azocasein in 1 min.
Periplasmic extracts were prepared by washing the cells from a 150-ml
broth culture (44 h) in 10 mM Tris-HCl (pH 8.0) after
centrifugation at
6,000 ×
g for 10 min at room temperature. The
cells
were resuspended in 30 ml of 20% sucrose-30 mM Tris- HCl
(pH 8.0)-1
mM sodium EDTA, incubated at room temperature for 10
min, and then
centrifuged as before. The cell pellet was resuspended
in 30 ml of
ice-cold deionized water, incubated on ice for 10
min, and centrifuged
at 6,000 ×
g for 10 min at 4°C, and the supernatant
containing the periplasmic proteins was then
removed.
Virulence testing in sheep.
Six-month-old Merino sheep that
were free of footrot were obtained from the University of Sydney
farm at Marulan in the southern highlands of New South Wales,
Australia. Each sheep was identified by a numbered ear tag. The sheep
were randomly allocated into five groups of 10 and were housed in an
animal house on concrete floors, each group in a separate room
maintained at 22°C. These experiments were carried out in a PC2
containment facility in accordance with the guidelines of the
Australian Genetic Manipulation Advisory Committee and the Elizabeth
Macarthur Agricultural Institute Animal Ethics Committee. The sheep
were fed lucerne hay and oats; water was provided ad libitum. The
groups of 10 animals were challenged blind with wild-type strain
VCS1703A, fimA mutants JIR3727 and JIR3728, and plain agar
(negative control) as previously described (11). Briefly,
the feet of the animals were predisposed to infection by keeping them
on wet foam mats for 4 days before the challenge, to facilitate
maceration of the skin. All animals were sampled for D. nodosus with a swab stick applied to the interdigital skin prior
to challenge. The sheep were then challenged by applying 4-day-old
cultures of the each strain on plugs of 2% hoof agar to the
interdigital skin and holding them in place with bandages for 4 days.
Each plug contained 8.4 × 105 to 9.5 × 105 CFU of D. nodosus or, for the negative
control, uninoculated agar. The mats were removed from the floor 1 week
after the start of the challenge, and the animals were again sampled
for D. nodosus.
All animals were examined, and their feet were scored for footrot
lesions at the start of the trial and then at weekly intervals.
A
standard lesion scoring method was used (
46); this method
was a modification of an earlier protocol (
10). The total
weighted
foot score (TWFS) was used to provide an unambiguous overall
score
for the animal, a score that included information from each of
the four feet. Blood samples were collected from the jugular vein
of
sheep at the start of the trial and each time the feet were
scored. The
serum was separated from the blood and stored at
20°C
until
required for enzyme-linked immunosorbent assay
(ELISA).
ELISA for Omp and FimA.
Serum samples taken from sheep
during the course of the trial were tested for D. nodosus-specific antibodies. The Omp antigen (44) and
fimbrial antigen (47) ELISAs were performed essentially as
previously described.
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RESULTS |
The fimA gene is essential for the production of type
IV fimbriae, twitching motility, natural transformation, and protease
secretion.
Previous studies showed that the tet(M) gene
conferred tetracycline resistance in D. nodosus and
could be used to select the progeny of double-crossover events
(22). Insertional inactivation with tet(M) was
therefore the method of choice for the isolation of fimA
mutants. To construct the fimA suicide plasmid pJIR1895 (Fig. 1), an 800-bp fragment containing
the first 120 nucleotides of fimA and part of
aroA was cloned into pBluescript SKII. A 3.1-kb EcoRI fragment from pVB101 that contained the
tet(M) cassette (22) was then added, and a
750-bp fragment containing the last 150 bp of fimA and the
first 600 bp of fimB was cloned downstream of
tet(M), resulting in a 230-bp deletion within
fimA, at the site where tet(M) was inserted. The
suicide vector therefore had 800 bp of D. nodosus DNA
located upstream of tet(M) and 750 bp downstream of
tet(M), sufficient for homologous recombination to occur.
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FIG. 1.
Diagrammatic representation of the construction of the
fimA and fimB mutants of D. nodosus strain VCS1703A by homologous recombination.
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The virulent type G strain VCS1703A was chosen for use in these studies
because it was naturally transformable. In fact, in
experiments
designed to optimize the transformation of
D. nodosus,
it was determined that all of the
D. nodosus strains
that were
previously thought to be transformable by electroporation
were
naturally transformable (R. Kennan and J. Rood, unpublished
results).
VCS1703A, isolated from an outbreak of virulent ovine
footrot,
was fimbriated and had all of the properties normally
associated
with virulent isolates of
D. nodosus
(
34) in that it was elastase
positive after 7 to 10 days
of incubation on elastin agar and
was positive in a gelatin-gel
protease stability test. In addition,
in preliminary pen virulence
trials it was shown to produce virulent
footrot in sheep (data not
shown).
Transformation of strain VCS1703A with the
fimA suicide
plasmid pJIR1895 produced several tetracycline-resistant colonies.
These derivatives were confirmed as
D. nodosus by use
of a species-specific
16S rRNA gene PCR test (
24) and
shown by PCR to contain the
tet(M) gene. Two independently
derived mutants JIR3727 and JIR3728
were selected for further
characterization.
Genomic DNA was isolated from VCS1703A, JIR3727, and JIR3728, digested
with
NruI, and examined in Southern hybridization
experiments
using
fimA- and
tet(M)-specific
probes. The results showed that
in the
fimA mutants the band
that hybridized with the
fimA probe
was approximately 3 kb
larger than that which hybridized from
VCS1703A. As expected, this band
also hybridized with the
tet(M)
probe, which did not
hybridize to the wild-type strain (data not
shown). Based on these
results, it was concluded that JIR3727
and JIR3728 were chromosomal
fimA
tet(M) mutants derived from
allelic exchange with the
insertionally inactivated suicide vector
pJIR1895.
To determine if the mutant strains still produced the fimbrial subunit
protein, immunoblot analyses were performed with serogroup
G-specific
antiserum, using whole-cell extracts of the wild-type
and mutant
strains. The results (Fig.
2) showed that
although
a strong type G-reactive 17-kDa FimA band was observed in the
wild-type strain, no such band was observed in extracts of the
mutants.
These results indicated that the type G fimbrial subunits
were no
longer being produced by the
fimA mutants.
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FIG. 2.
Western immunoblot of whole-cell extracts of wild-type
strains and fimA and fimB mutants. Whole-cell
extracts were separated on 12% polyacrylamide gels, electroblotted to
nitrocellulose membranes, and developed with type G-specific fimbrial
antiserum. The arrowhead marks the position of the fimbrial subunit.
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Transmission electron microscopy was used to confirm that type IV
fimbriae were no longer produced by the
fimA mutants. The
results (Fig.
3) showed that although
type IV fimbriae were present
on the wild-type strain, there were no
fimbriae on either of the
mutants. In other type IV fimbriate bacteria,
the fimbriae impart
an unusual type of motility known as twitching
motility (
17).
It has been known for many years that
D. nodosus cells also exhibit
twitching motility
(
6). An agar stab twitching motility assay
(
5) was used to examine the effect of the
fimA
mutation on
the twitching motility process. Unlike the wild-type
strain, neither
of the
fimA mutants exhibited the spreading
zones typical of twitching
motility (Fig.
4). These experiments clearly showed that
the
fimA-encoded
fimbrial subunit was essential for the
production of biologically
functional type IV fimbriae.
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FIG. 3.
Transmission electron micrographs of D. nodosus cells. Wild-type strain VCS1703A (A), fimA
mutant JIR3727 (B), fimA mutant JIR3728 (C), and
fimB mutant JIR3737 (D) were grown on EYE blood agar for 3 days, removed with PBS, and negatively stained. Bars represent 1 µm.
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FIG. 4.
Twitching motility assays. Cultures were stab inoculated
to the bottom of 1% agar plates and incubated at 37°C for 3 to 5 days. The agar was then compressed and stained with Coomassie brilliant
blue to reveal the zones of twitching motility. The size of the dark
zone around the point of inoculation is indicative of the extent of
twitching motility. Representative assays of wild-type strain VCS1703A,
fimA mutant JIR3727, and fimB mutants JIR3737 and
JIR3739 are shown. The profile obtained with fimA mutant
JIR3728 was identical to that of JIR3727.
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Type IV fimbriae have been shown to be essential for natural
transformation in several bacterial species (
15,
16,
40).
To see if mutation of the
fimA gene affected natural
transformation
in
D. nodosus we performed a series of
transformation experiments
with strains VCS1703A, JIR3727, and JIR3728.
These studies utilized
the suicide vector pJIR1836, which contains the
erythromycin resistance
gene
erm(B) flanked by the
D. nodosus rrnA promoter and terminator.
Natural
transformation with strain VCS1703A consistently produced
erythromycin-resistant transformants that resulted from allelic
exchange between pJIR1836 and one of the three chromosomal rRNA
operons. However, despite repeated attempts, no transformants
were
obtained with either of the
fimA mutants. It was concluded
that the FimA fimbrial subunit was essential for natural transformation
in
D. nodosus.
These results had implications for the subsequent sheep virulence
studies. Normally, the genetic analysis of putative virulence
genes
involves not only the virulence testing of mutant strains
but the
complementation of the chromosomal mutants by a wild-type
virulence
gene and subsequent virulence testing. Accordingly,
since recombinant
plasmids could not be introduced into the
fimA mutants by
natural transformation, attempts were made to electroporate
these
strains. Unfortunately, despite repeated attempts with different
experimental conditions, it was not possible to successfully transform
these strains, even by using electroporation. Note that we have
also
been unable to introduce DNA into
D. nodosus by
conjugation.
Therefore, for technical reasons it was not possible to
complement
the original
fimA mutants. Accordingly, all of
the subsequent
virulence studies were carried out on both of the
independently
derived
fimA mutants.
Previous studies showed that expression of the
fimB gene,
which is located immediately downstream of
fimA, results
from readthrough
transcription from the
fimA promoter,
despite the fact that there
is a terminator located between these genes
(
18). To confirm
that the effects we were observing with
the
fimA mutants were
due only to the inactivation of
fimA and not to a polar effect
on
fimB, RT-PCR
reactions were performed. The primers used in
the RT reactions were
located within the deleted region of
fimA (primer
13240) and within
fimB (primer 13242). A
fimA-specific
transcript was detected only in VCS1703A
as expected (data not
shown). By contrast, a
fimB-specific
transcript was detected in
both of the
fimA mutants as well
as the wild-type (data not shown),
indicating that
fimB-specific mRNA was being produced and that
the
fimB gene was still expressed in the
fimA
mutants. Sequence
analysis confirmed that the RT-PCR products observed
in the
fimB reactions were derived from the
fimB gene.
In
P. aeruginosa, mutation of the fimbrial subunit gene
pilA results in reduced ability to secrete extracellular
proteins
such as elastase (
27). Therefore, routine
diagnostic footrot
protease tests such as the elastase test and the
gelatin-gel protease
stability test were performed on the wild-type
VCS1703A and the
fimA mutants JIR3727 and JIR3728. The
results showed that VCS1703A
was elastase positive after 7 days of
incubation and gelatin-gel
positive, whereas the two
fimA
mutants were negative in both of
these tests, suggesting that they no
longer produced wild-type
levels of extracellular protease. To confirm
that the protease
genes were still being transcribed in the
fimA mutants, RT-PCR
experiments were performed on
each of the protease genes
aprV2, aprV5, and
bprV, using separate RT primers that were specific
for each
protease gene. RT-PCR products specific for each protease
gene were
obtained in these experiments (data not shown), providing
evidence that
all three protease genes were transcribed in the
fimA
mutants. Quantitative protease assays subsequently were carried
out on culture supernatants (44 h) and periplasmic extracts of
all
three strains, using azocasein as the substrate. The results
confirmed the initial observations; the
fimA mutants had
lower
levels of protease activity in the supernatant than the wild type
(Fig.
5), and these levels were
significantly different from the
wild-type level (
P < 0.05). Periplasmic extracts of the
fimA mutants
had
higher levels of activity than the corresponding wild-type
extracts,
but these differences were not statistically significant.
It was
concluded that extracellular proteases are still synthesized
in the
fimA mutants but are not secreted efficiently.
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FIG. 5.
Protease activity of wild-type strain VCS1703A,
fimA mutants JIR3727 and JIR3728, and fimB mutant
JIR3737. Total protease activity was measured on culture supernatants
(A) and periplasmic extracts (B) of each of the strains, using
azocasein as the substrate.
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The fimB gene is not essential for fimbrial
biogenesis.
To examine the role of the fimB gene in
fimbrial biogenesis, we decided to construct fimB mutants by
allelic exchange. To construct the fimB suicide plasmid
pJIR2025 (Fig. 1), a 1-kb PCR fragment, which was derived from VCS1703A
chromosomal DNA with primers 4142 and 5013, was blunt-end cloned into
pT7Blue. This fragment contained part of aroA, all of
fimA, and the first 114 bp of fimB. The
tet(M) cassette from pVB101 was cloned into the EcoRI site of pBluescript SKII, and a second PCR fragment,
which was derived from the primers 5772 and 4143 and contained the last 350 bp of fimB and the first 360 bp of clpB, was
cloned into the SmaI site downstream of tet(M) so
that fimB was closest to tet(M). The complete
tet(M)-fimB-clpB fragment was then directionally cloned into the pT7Blue derivative with XbaI and
XhoI so that the tet(M) gene was located next to
the first 114 bp of fimB, thereby creating a 310-bp
fimB deletion where tet(M) was inserted. In this
construct, pJIR2025, there is 1 kb of D. nodosus DNA
upstream of tet(M) and 750 bp downstream.
Transformation of VCS1703A with pJIR2025 produced several
tetracycline-resistant colonies that as before were confirmed as
D. nodosus and shown to contain the
tet(M)
gene. Two independently
derived transformants, JIR3737 and
JIR3739, were selected for
further characterization using the
same approach as used for the
fimA mutants.
Southern hybridization analysis of
NruI-digested
genomic DNA produced two hybridizing bands with the
fimB probe, due to the
presence of a
NruI site
within
fimB. However, the smaller, approximately
2-kb band
present in the wild-type strain VCS1703A was no longer
present in
JIR3737 or JIR3739 but was replaced by an approximately
5-kb band,
which also hybridized to the
tet(M) probe (data not
shown),
as predicted for an insertionally inactivated
fimB gene.
To
confirm that we had disrupted the
fimB gene, RT-PCR was
performed
with primer 15202, which is located within the deleted region
of
fimB, and primer 4944. A product was obtained only with
the
wild-type strain (data not shown), confirming that the
fimB gene
was not transcribed in the
mutants.
Immunoblotting of whole-cell extracts with serogroup G-specific
antiserum (Fig.
2) showed that type G-specific fimbrial subunits
were
still produced by the
fimB mutants. Transmission electron
microscopy (Fig.
3D) and twitching motility assays (Fig.
4) showed
that
the
fimB mutants produced normal type IV fimbriae that were
still capable of imparting twitching motility. In addition, both
of the
fimB mutants could be transformed with pJIR1836 to
erythromycin
resistance, indicating that these mutants were still
naturally
competent. Finally, the
fimB mutants were positive
at 7 days in
the elastase test and were positive in the gelatin-gel
test, which
indicates that they were secreting functional extracellular
proteases,
as confirmed by the protease assays (Fig.
5). Note that in
these
experiments the wild-type strain VCS1703A did not grow quite as
well as the mutants, which may account for the
fimB mutant
having
somewhat higher levels of protease activity in the supernatant.
Nonetheless, by all of the available phenotypic parameters, the
fimB mutants were effectively the same as the wild-type
strain.
The fimA gene is an essential virulence gene in ovine
footrot.
Wild-type strain VCS1703A and fimA mutants
JIR3727 and JIR3728 were virulence tested in sheep in a controlled
environment using a standardized method. In summary, groups of 10 sheep
were challenged by applying agar cultures of D. nodosus
to the interdigital skin of all four feet. Over a 2- to 3-week period,
each foot was quantitatively scored on a scale of 0 to 4 for the
presence of footrot lesions, and the values were converted to a
TWFS, which provides an objective summary of the footrot lesions
within a sheep (10, 46). Culturing of foot swabs from the
sheep prior to challenge showed them to be free of D. nodosus, while culturing of foot swabs taken 2 weeks after
challenge produced D. nodosus isolates only from those
sheep challenged with VCS1703A. Characterization of these isolates by
serogrouping, elastase, gelatin-gel, and Omp PCR-restriction fragment
length polymorphism testing showed them to be identical to VCS1703A.
The results of the virulence trials (Fig.
6) clearly showed that only the sheep
challenged with the wild-type strain contracted
virulent footrot.
There was no sign of footrot in either of the
groups challenged
with the
fimA mutants or in the negative control
group,
which received no
D. nodosus cells. These data provide
unequivocal evidence that mutation of the
fimA gene
eliminates
the ability to cause ovine footrot. Note that because of
the severity
of the lesions in the sheep infected with the wild-type
strain,
for animal ethics reasons these animals had to be treated with
antibiotics to eliminate the infection.
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|
FIG. 6.
TWFS and ELISA responses. Sheep were challenged with
wild-type strain VCS1703A and fimA mutants JIR3727 and
JIR3728 in a blind pen trial. Feet were scored for footrot lesions,
(black bars), and blood samples were taken before challenge (week 0)
and 2 and 3 weeks after challenge for Omp ELISA (grey bars) and
fimbrial ELISA (hatched bars).
|
|
Serum samples collected in the course of the sheep trial were analyzed
by ELISA for responses to the
D. nodosus Omp and
fimbrial
antigens. The sheep challenged with the
fimA
mutants produced
no response to either antigen, indicating that they
had remained
uninfected. The sheep challenged with the wild-type strain
had
a limited immune response to both antigens (Fig.
6). These
responses
were typical of those seen in sheep 3 weeks postinfection,
and
it is anticipated that they would have increased further if the
sheep had not been treated with antibiotics, as the humoral responses
are lesion dependent (
45).
In a separate and smaller virulence trial, the virulence of the
fimB mutant JIR3737 was tested in a similar manner. The
results
showed that this strain still had the ability to cause virulent
footrot, since more than 10% of the sheep had a TWFS of greater
than or equal to 9. However, the average TWFS of 9.4 was lower
than
that observed with the wild-type strain (TWFS of 36.5), suggesting
that
the
fimB mutant may be slightly attenuated. Further
virulence
trials would be required to verify this hypothesis. However,
since
this strain clearly causes virulent footrot, these
experiments
could not be justified on animal ethics
grounds.
| |
DISCUSSION |
The type IV fimbriae produced by D. nodosus are
regarded as a major virulence factor, as they are highly immunogenic
and vaccination with whole cells, purified native fimbriae, or
recombinant fimbriae protects against disease (9, 12).
We therefore chose to disrupt the fimA fimbrial subunit
gene and to examine the effect of this disruption on fimbrial
biogenesis and the disease process in sheep. This study represents the
first time that genetically defined mutations in putative virulence
genes have been constructed and analyzed in this important sheep pathogen.
The results presented here provide evidence that the fimbrial subunit
gene is essential for the virulence of D. nodosus in sheep. Virulence testing of two fimA mutants showed they
were avirulent, whereas the wild-type strain from which they were
derived produced virulent footrot in the same trial, which was
conducted under blind conditions. There was no serological response in
the sheep infected with the mutants, and no D. nodosus
cells were isolated from their feet at the end of the trial, indicating
that the mutants did not colonize the ovine hoof. The simplest and most
likely explanation for these results is that colonization of the
interdigital skin and subsequent penetration of the stratum corneum
requires the adhesive activity of type IV fimbriae. However, since
these mutants were also altered in their ability to secrete extracellular proteases and other, as yet unknown extracellular proteins, we cannot rule out the possibility that these factors are the
major determinants involved in colonization or penetration.
The type II secretion machinery that is responsible for the secretion
of many extracellular enzymes and toxins in gram-negative bacteria has several genes that are homologous to type IV fimbrial biogenesis genes (31). Examples include
pulGHIJ of Klebsiella oxytoca
(32), xcpTUVW of P. aeruginosa
(1), and outGHIJ of Erwinia
chrysanthemi (26). P. aeruginosa mutants
in pilA, which encodes the major pilin subunit, are also
defective in protein secretion, leading to the conclusion that the
biogenesis of type IV pili (or fimbriae) and protein secretion pathways
are shared in this organism (27). The results presented in
this paper provide evidence that these pathways are also shared in
D. nodosus. Both of the fimA mutants
produced little or no extracellular protease or elastase activity, yet
in these mutants all three protease genes were still being transcribed
and protease activity was detected in periplasmic extracts. It has
recently been postulated that type IV fimbriae function in secretion by
acting as a piston that pushes secreted proteins through the secretion
pore in the outer membrane during extension and retraction of the
fimbriae (36). Extension and retraction of the type IV
pilus of Neisseria gonorrhoeae has been demonstrated as the
process that imparts twitching motility to the bacterial cells
(29). The pilT gene of N. gonorrhoeae has been shown to be essential for twitching motility,
but not fimbrial biogenesis (48), with pilT
mutants still producing fimbriae but not exhibiting twitching motility.
Therefore, if in D. nodosus the fimbriae were to
function as a piston to push out secreted proteins, this would explain
the reduced protease secretion observed in the fimA mutants
but still account for the small level of protease activity observed in
culture supernatants and the apparent accumulation of proteases in the
periplasm. A pilT homologue is yet to be identified in
D. nodosus, but the identification and mutation of such
a homologue may provide a better understanding of the relationship
between fimbriae, twitching motility, protease secretion, and virulence.
The only method by which we are able to introduce recombinant DNA
molecules into D. nodosus cells is by a natural
transformation process that leads to recombination onto the chromosome.
However, since the fimA mutants were no longer naturally
transformable, we were unable to complement these mutants to ensure
that the phenotypic changes we observed were attributable solely to the disruption of the fimA gene. Two distinct experiments were
undertaken to reduce the probability that these changes were not due to
the inability to produce the FimA subunit. First, all of the biological tests, including the sheep virulence experiments, were carried out on
two independently derived fimA mutants. Identical results were obtained with these mutants, suggesting that it was unlikely that
the phenotypic effects resulted from secondary mutations elsewhere on
the chromosome. Second, the only gene that could have been affected by
a polar fimA mutation was fimB. The RT-PCR results showed that the fimB gene was expressed in the
fimA mutants. In addition, the isolation and analysis of
independent fimB mutants showed that mutation of
fimB had no effect on fimbrial biogenesis, twitching
motility, natural transformation, or protease secretion. The virulence
tests confirmed that a fimB mutant could still cause virulent footrot in sheep. Accordingly, it was concluded that the
phenotypic effects observed in the fimA mutants were the
direct result of the inability of the mutants to produce the FimA
protein rather than coincident mutations or polar effects on
fimB.
Functional type IV fimbrial subunit genes are essential for the natural
transformation of N. gonorrhoeae (15),
Legionella pneumophila (40), and
Pseudomonas stutzeri (16). In addition, other
bacteria that are naturally competent possess type IV pilus-related import systems but do not produce fimbriae (3, 25, 43). Our finding that the fimbrial subunits of D. nodosus
are essential for natural transformation is in agreement with the
observation that the D. nodosus fimA gene can
complement a pilA mutant of P. stutzeri and
restore pilus production and natural competence (16).
Although it has been known for some time that there are common
components in the type IV fimbriae assembly apparatus, DNA uptake
systems, and type II secretion systems (7, 19), it appears
that this is the first report of a bacterium using the same system for
all three processes. Identification of further homologues of genes
involved in these three processes in D. nodosus will
lead to a better understanding of the extent to which these three
systems are shared.
| |
ACKNOWLEDGMENTS |
We sincerely thank the technical staff of EMAI for assistance
with the very complex sheep virulence trials and Khim Hoe for help with
electron microscopy.
The project was supported by a grant to J.I.R. from the Australian
Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Bacterial
Pathogenesis Research Group, Department of Microbiology, Monash
University, Victoria 3800, Australia. Phone: 613 9905 4808. Fax: 613 9905 4811. E-mail:ruth.kennan{at}med.monash.edu.au.
| |
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Journal of Bacteriology, August 2001, p. 4451-4458, Vol. 183, No. 15
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.15.4451-4458.2001
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Abstract
Introduction
