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SID 5
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Research Project Final Report
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SID 5 (Rev. 3/06)
Project identification
OD1716
The rational selection of candidate antigens for inclusion
in vaccines against bovine mastitis caused by S. uberis
Contractor
organisation(s)
Oxford University, Nuffield Department
of Clinical Laboratory Sciences
John Radcliffe Hospital
Headley Way, Headington
Oxford
Oxon
OX3 9DU
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 16
£
298142.5
start date ................
02 January 2006
end date .................
30 June 2007
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Please confirm your agreement to do so. ................................................................................... YES
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Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
The bacterium Streptococcus uberis is a common cause of intramammary infection in dairy
cattle and is a leading cause of bovine mastitis worldwide. In the UK it has recently been shown
that S. uberis is the most common cause of clinical mastitis. The ability of the organism to grow
in milk has been shown to be essential for infection and disease. Investigation of the processes
underlying growth of S. uberis in milk and means by which these may be prevented could lead
directly to the development of vaccines against this organism.
This project investigated the hypothesis that one or more of seven enzymes (proteases) capable
of degrading proteins to peptides and amino acids was required to enable growth of S.uberis in
bovine milk and thus that antibodies directed against it may be able to inhibit the activity and
prevent growth of Streptococcus uberis in bovine milk.
Seven genes form S. uberis considered to encode proteases that were likely to be exported
outside the cell were identified from the recently completed and annotated genome sequence of
S. uberis. These genes were cloned in such a manner that the encoded protein was produced
with an additional six amino acids (HHHHHH) at the start of the sequence. This HHHHHH region
of the protein enabled each to be purified. The purified proteins were used to produce high titre
antiserum. Antibodies were purified from each serum and added to bovine milk to determine the
ability of each to inhibit bacterial growth. In addition, the purified antibodies were combined to
determine the ability of any combination of antibodies to inhibit bacterial growth. None of the
antibody preparations either alone or in combination with others was able to inhibit bacterial
growth. These data imply that either that the particular proteases were not essential for growth or
that the antibodies were not capable of inhibiting their function.
To complement these studies mutant strains of S. uberis carrying lesions within the genes of
interest and thus not able to produce the relevant gene product were isolated from a pool of
mutants held in this laboratory. Mutant strains were isolated that failed to produce 5 of the seven
proteins under investigation. In one other case, a mutant strain could not be isolated (possible
due the requirement of this gene product for the viability of the bacterium). In the final case, a
mutant strain that produced a truncated from of the protease (that may retain its enzymatic
activity) was isolated. Investigation of the ability of these strains to grow in bovine milk revealed
SID 5 (Rev. 3/06)
Page 2 of 16
that all mutant strains grew at a rate and to a final cell density similar to that of the wild type
(genetically intact) strain.
Consequently, it is concluded that of the seven proteases under investigation that five definitely
do not play an essential role in the growth of S. uberis in milk. It would also appear unlikely that
the other two enzymes play a role in this process although the data supporting this would require
a demonstration that the antisera produced to these proteins was able to inhibit their biological
function.
In conclusion, there is no evidence to support the inclusion of any of the proteases in vaccines
aimed at inhibiting growth of S. uberis in the bovine mammary gland. In another active project
within the group several of the proteins investigated in this project have been implicated in the
process of pathogenesis and their role in this will be investigated in the newly funded
(Government Partnership Award; BBSRC-GPA) CEDFAS project on bovine mastitis caused by
S. uberis.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
SID 5 (Rev. 3/06)
Page 3 of 16
FINAL REPORT: The rational selection of candidate antigens for inclusion in vaccines against
bovine mastitis caused by S. uberis
1.
Scientific Objectives
The aim of this project was to assess the ability of secreted proteins to induce growth-inhibiting
responses and thus investigate their potential as candidates for inclusion in a growth inhibiting vaccine
against bovine mastitis caused by S. uberis. This was to be achieved by completing the following
objectives.
1.
Determine the capability of externally located gene products to induce immunoglobulin
responses that are inhibitory to growth in milk
1.1
Clone and express 7 proteins corresponding to proteases encoded by S. uberis
1.2
Purify at least 200μg of each protein for antibody production
1.3
Purify 100 mg of immunoglobulin (IgG) from antiserum
1.4
Compare the ability of wild type S. uberis to grow in bovine milk in the presence/absence
of immunoglobulin directed against secreted proteases
2.
Isolate and functionally characterise with respect to growth in milk mutants carrying lesions
within each putative protease gene
2. Extent to which Objectives have been met
Objectives 1.1 - 1.4 have been met in full. Objective 2, with the exception of the production and
analysis of a mutant lacking the gene product encoded by sub1508 of S. uberis 0140J
(http://www.sanger.ac.uk/Projects/S_uberis/), was met in full.
3.
Scientific report
Background
Earlier investigations revealed that S. uberis was auxotrophic for a number of amino acids that are not
present either as free or short chain peptides in bovine milk. The only access to such amino
acids/peptides for bacterial growth is by degradation of host proteins. Earlier investigations focussed on
the presence of a plasminogen activator, PauA, which in the absence of demonstrable protease activity
was considered to be responsible for hydrolysis of host proteins. A vaccine based on culture
supernatants containing PauA was shown to be effective in prevention of disease. However, mutants
lacking the ability to activate plasminogen were able to grow in bovine milk and were equally virulent as
the isogenic wild type strain, 0140J. This indicated that PauA alone was not responsible for release of
essential amino acids for growth and that in isolation it was unlikely to make an effective vaccine.
Completion of the S. uberis genome has permitted the identification of genes encoding activities that
were previously cryptic including 7 genes that show functional homology to secreted proteases. Any or
all of these may be involved in the release of amino acids from host proteins to permit bacterial growth.
Earlier analysis of the mutant bank for strains that fail to grow in milk did not reveal mutants containing
lesions in these genes, however, functional redundancy may require that more than one gene is
inactivated to inhibit growth substantially. Furthermore, the selection procedure used to identify mutants
was based primarily on detection of acid production from growing cultures and typically required the
total absence of growth rather than a minor perturbation as may be detected if multiple gene products
with overlapping functions were present
This project aimed to determine which of these proteases may have function with respect to bacterial
growth in milk and determine the ability of each to raise a growth inhibiting immune response. Any
such candidates would greatly facilitate the commercial development of effective vaccines against this
disease.
SID 5 (Rev. 3/06)
Page 4 of 16
Methodology & Results
Objective 1.1: Clone and express 7 proteins corresponding to proteases encoded by S. uberis
Seven open reading frames within the completed genome of S. uberis (sub1154, 0826, 1868, 1738,
0350, 1508 &1370) were predicted to encode products putatively identified as proteases that are likely
to be secreted or located at the outer surfaces of the bacterial cell.
Table 1: Proteases identified in S. uberis that contain secretion signal sequences
Open Reading Frame
Likely function*
sub1154
Synonym in current
report
DC1
sub0826
DC2
Serine protease
sub1868
DC3
Serine protease
sub1738
DC4
Dipeptidase
sub0350
DC5
Carboxypeptidase
sub1508
DC6
D,D-Carboxypeptidase
sub1370
DC23
Metallo-protease
Serine protease
Molecular weight **
(predicted cleavage
of signal peptide) ***
124 kDa
(aa 33-34)
164 kDa
(aa 38-39)
37 kDa
(aa 51-52)
53 kDa
(aa 23-24)
42 kDa
(aa 24-25)
24 kDa
(aa 32-33)
113 kDa
(aa 37-38)
* As predicted by Pfam (http://www.sanger.ac.uk/Software/Pfam/)
** Molecular weight of mature protein sequence (ie lacking secretion signal peptide)
*** As predicted by Signal P V3.0 (http://www.cbs.dtu.dk/services/SignalP/)
The mature sequence of each (i.e. lacking the proposed N terminal signal sequence was amplified by
PCR and the resulting gene fragment cloned in plasmid (pQE-1; Qiagen) expression vectors in E. coli
(M15 pREP4). Cloned DNA was sequenced to ensure the correct sequence and context of each open
reading frame. Over-expression of the target protein was achieved by growth to late log phase and
subsequent induction of expression using IPTG. Harvesting of induced bacterial cells by centrifugation
then permitted the isolation and purification of the recombinant gene product. Briefly, recombinant
strains of E. coli were grown in LB media and proteins expressed following induction with IPTG.
Recombinant protein expression was not optimised, but in each case the recombinant proteins were
readily detected following induction for 2 h with IPTG by SDS PAGE separation of whole cell lysates.
In three cases, DC1, DC2 and DC23, following growth of bacterial cultures and induction at 37oC
proteins appeared degraded. To overcome this, cultures were grown at 20oC, and cultures
subsequently induced at this reduced temperature. In each case this substantially increased the
proportion of intact recombinant protein that could be detected.
SID 5 (Rev. 3/06)
Page 5 of 16
Objective 1.2: Purify at least 200μg of each protein for antibody production
Recombinant protein was purified from cultures expressing the cloned gene fragment using affinity
purification. Briefly, cells were collected from induced cultures and lysed by a combination of enzymatic
(lysosyme 0.4 mg/ml) and chemical (Cell Lytic; Sigma) means. The recombinant protein was
immobilised on a Ni containing resin (His-Select; Sigma) and eluted in line with the supplier’s
instructions using imidazole. The purity of the protein was determined by SDS PAGE.
For proteins DC3, DC4, DC5, and DC6 cultures were grown overnight in LB media (containing
Ampicillin 50μg/ml (Amp) and Kanamycin 25μg/ml (Kan)) at 37oC. Each recombinant strain was sub
cultured into 10 volumes of the same medium and grown at 37oC with aeration (shaking 250 rpm) for
3h. Recombinant protein expression was subsequently induced by the addition of IPTG (1mM) and
cells harvested after 2 h.
In order to reduce protein degradation, proteins DC1, DC2 and DC23 were extracted from cultures
induced at 20oC. In brief, overnight cultures grown at 37oC in LB (Amp, Kan) were sub cultured in static
conditions in 5 volumes of the same medium at 20oC for 2h (DC1 and DC2) or 4.5h (DC23). Protein
expression was induced by the addition of IPTG (0.2mM) and cells harvested after 2 h. For these
proteins it was also necessary that all subsequent protein purification steps were conducted in the
presence of protease inhibitors (Complete-EDTA free; Roche).
Proteins were purified (Table 2) from either soluble preparations or from insoluble preparations
(inclusion bodies; solubilised in the presence of 8M Urea). Purified proteins were dialysed twice against
200 volumes of 1/10 PBS. Protein concentration was determined by UV spectophotometic analysis
using the calculated molar extinction co-efficient for the particular recombinant protein (as determined
using ProtParam: http://www.expasy.org/tools/protparam.html) and/or the level of intact target protein
determined by titration of samples and analysis by SDS PAGE using Coomassie-blue based detection
systems (Instant blue; Novexin and Gelcode blue; Pierce); using the detection limits supplied by the
manufacturer of each staining system. Proteins were stored as freeze dried preparations at -20oC.
Table 2: Recombinant proteins purified
Protein
Approximate
Culture volume
Soluble/
Inclusion bodies
Quantity produced
DC1
1600 ml
Soluble
960 μg
DC2*
400 ml
Soluble
DC3
384 ml
Soluble
180 μg (full length)
960 μg (fragmented)
360 μg
DC4
350 ml
Soluble
750 μg
DC5
960 ml
Insoluble
210 μg
DC6
384 ml
Inclusion
1440 μg
DC23
800 ml
Soluble
320 μg
* Protein fragments (all >60 kDa) bound his-select Ni affinity resin implying presence of intact 6XHis
and further suggesting that each represented C-terminal deletion of the recombinant protein.
Freeze dried proteins (5-10 aliquots of between 28-140 μg) were supplied to Davids Biotechnologie
(Germany) for serum production in rabbits. Anti-serum (>50 ml in each case) was supplied filter
sterilised and containing 0.02% sodium azide as a preservative.
SID 5 (Rev. 3/06)
Page 6 of 16
Objective 1.3: Purify 100 mg of immunoglobulin (IgG) from antiserum
Activity of each antiserum was determined by titration ELISA against the immunising antigen. Briefly,
antigen (10 ng/well) was applied to Micro-titre trays (Maxisorb Immunoplate; Nunc) at pH 9.6 (1M
Carbonate buffer) and allowed to interact overnight at 4oC. Unbound antigen was removed and plates
washed (x3) in PBS containing tween-20 at a final concentration of 0.1% (PBST). Remaining antigen
binding sites were blocked by incubation in PBST containing 1% Marvel (PBSTM) at 37oC for 90 min.
Primary antibody was applied as dilutions in PBSTM and incubated at 37oC for 60 min after which
unbound material was removed and plates washed (x3) in PBST. Secondary antibody (goat anti-rabbit
IgG conjugated to horseradish peroxidase (HRP); Southern Biotech) was applied at a dilution of 1/2000
in PBSTM and allowed to react at 37oC for 60 min. Unbound material was removed and plates washed
(x3) in PBST and dried at room temperature. Bound HRP was detected by addition of TMB liquid
substrate system for ELISA (Sigma) and the reaction stopped after 2 min by addition of Stop reagent
for TMB substrate (Sigma). Absorbance was measured in each well at 450nm using an microtitre plate
reader (Anthos) and data was transferred to MS-Excel using the Stingray software (Dazdaq).
The level of specific IgG was estimated by determination of the dilution resulting in A450nm of 1.0 and
by the determination of the dilution at which A450nm was equal to that in the absence of primary IgG
(end-point titration).
Table 3: Detection of IgG against specific antigen in produced antisera
Protein
Serum dilution
at end point
Serum dilution
at A450nm=1.0
DC1
>1/2,000,000
1/128,000
DC2
>1/1,024,000
1/32,000
DC3
>1/20,000,000
1/2,560,000
DC4
1/1,024,000
1/50,000
DC5
1/128,000
1/4000
DC6
>1/2,000,000
1/750,000
DC23
1/2,048,000
1/16,000
In no instance was any reaction detected in serum obtained from rabbits prior to immunisation.
With the exception of anti sera against DC5, the specificity of IgG was confirmed by detection of
specific proteins of the expected molecular weights within cell extracts and or concentrated culture
supernatants of S. uberis grown in either Todd Hewitt broth or Brain Heart infusion broth. DC5 was not
detected in any system indicating that this product was possibly not produced by the bacterium under
these conditions.
IgG was isolated by affinity chromatography using Hi-Trap Protein-G affinity columns (G.E. Healthcare)
according to the suppliers protocols. In brief, 10 ml aliquots of serum were mixed with an equal volume
SID 5 (Rev. 3/06)
Page 7 of 16
of 2x PBS and applied to the column. Unbound material was washed through the column with 10
column volumes of PBS and IgG eluted with 10 column volumes of Glycine HCl (pH2.8; 0.1M). Effluent
was collected in 4ml fractions into 0.3ml Tris HCl (pH9.2; 1M).
Eluted protein was detected by measuring absorbance at 280nm and fractions with A280nm >1.0 were
pooled and protein quantified by measurement of absorbance of a 1/10 dilution (protein (mg/ml) =
[A280nm x10] /1.4*; * Molar extinction co-efficient of IgG). The mean yield of IgG isolated from 20 ml
of serum was 117mg, ranging from 70 mg/20 ml for anti-serum against DC5 to 136.5 mg/20ml for
antiserum against DC3.
Purified IgG was dialysed against 400 volumes of purified water, concentrated by centrifugal ultrafiltration (10 kDa exclusion) using centrifugal filter devices (Amicon Ultra, Millipore) and stored as 5 mg
aliquots at -20oC.
Objective 1.4: Compare the ability of wild type S. uberis to grow in bovine milk in the
presence/absence of immunoglobulin directed against secreted proteases
S. uberis 0140J was grown in duplicate milk samples from three different animals obtained at 39 (cow
1441), 36 (cow 1525) and 18 (cow 3193) days post calving. Each milk was supplemented with IgG (5
mg) directed at one of the recombinant proteins or distilled water in control samples. In no instance was
any perturbation of growth rate or yield detected. Bacterial yields at 24h post inoculation in the
absence of IgG were (cfu/ml log10) 6.89 and 6.97 (cow 1441), 7.68 and 7.66 (cow 1525), 7.88 and 7.64
(cow 3193).
Fig 1: Growth of S. uberis in the presence of immunoglobulin directed at individual
proteases
A
Growth in milk from cow number 1441
Log cfu/ml
8
6
4
2
0
0
3
6
9
12
24
Time (h)
SID 5 (Rev. 3/06)
None
None
DC1
DC1
DC2
DC2
DC3
DC4
DC5
DC5
DC6
DC6
DC23
DC23
Page 8 of 16
DC3
DC4
Fig 1B
Growth in milk from cow number 1525
Log cfu/ml
10
8
6
4
2
0
0
6
12
24
Time (h)
None
None
DC1
DC1
DC2
DC2
DC3
DC4
DC5
DC5
DC6
DC6
DC23
DC23
DC3
DC4
Fig 1C
Growth in milk from cow number 3193
Log cfu/ml
10
8
6
4
2
0
0
6
12
24
Time (h)
None
None
DC1
DC1
DC2
DC2
DC3
DC4
DC5
DC5
DC6
DC6
DC23
DC23
DC3
DC4
In addition S. uberis 0140J was grown in duplicate milk samples from the same cows in which IgG from
all of the recombinant proteins had been added, each at a concentration of 0.5 mg/ml. Addition of the
combined IgG to milk had no inhibitory effect on either growth rate or the final yield of S. uberis.
SID 5 (Rev. 3/06)
Page 9 of 16
Fig 2: Growth of S. uberis in bovine milk in the presence of pooled immunoglobulin
A
Growth in milk (1441) with pooled IgG
Log cfu/ml
8
6
4
2
0
0
6
12
24
12
24
12
24
Time (h)
IgG
IgG
None
None
Fig 2B
Growth in Milk (1525) with pooled IgG
Log cfu/ml
10
8
6
4
2
0
0
6
Time (h)
IgG
IgG
None
None
Fig 2C
Growth in milk (3193) with pooled IgG
Log cfu/ml
8
6
4
2
0
0
6
Time (h)
IgG
IgG
None
None
In two milk samples (cows 1441 and 3193) the presence of IgG appeared to enhance the rate of
growth of S. uberis; at 6 h and this effect was still evident in the cultures in the milk sample from cow
1441 at 12 h. This effect was not detected in cultures grown in the other milk sample from cow 3193. At
24 h cultures in the presence /absence of IgG had attained similar cell densities.
SID 5 (Rev. 3/06)
Page 10 of 16
Objective 2: Isolate and functionally characterise with respect to growth in milk mutants
carrying lesions within each putative protease gene
Genotypic selection was used to isolate insertion mutants (Table 4) from a bank of random mutants.
Strains carrying lesions in of the 6 of the 7 proteases (Table 1) were obtained. A mutant carrying an
insertion in the gene encoding DC6 (sub1508, a DD carboxypeptidase) was not detected within the
bank and may imply an essential function for this protease. The location of the insertion in each case
was determined by sequence analysis of the open reading frame. The failure to produce the protein of
interest was confirmed by immunoblotting the appropriate bacterial extract using antigen specific
antisera. In the case of DC1 (sub 1154) a truncated gene product from the mutant strain was detected
although this was located in the culture supernatant rather than attached to the cell wall. This
observation was consistent with the position of the insertion within this gene and the consequent loss of
the C-terminal portion of the protein, which encodes a motif that enables anchoring of this protein to the
bacterial cell. In the case of DC6, a mutant was detected that carried an insertion in close proximity to
sub1508 (31 bp before the start codon), however, immunoblotting revealed that this did not alter
expression of the protein from S. uberis.
Table 4: Location of mutations in isolated mutants
Gene & Gene product
Location of insertion relative
to start codon
(full sequence length)
sub 1154
DC1 Serine protease
2792 bp after ATG
(3435 bp)
sub 0826
DC2 Serine protease
657 bp after TTG
(4452 bp)
sub 1868
DC3 Serine protease
451 bp after ATG
(1203 bp)
sub 1738
DC4 Dipeptidase
755 bp after ATG
(1497 bp)
sub 0350
DC5 Carboxypeptidase
484 bp after ATG
(1203 bp)
sub 1508
DC6 DD-carboxypeptidase
31 bp before ATG
(747 bp)
sub1370
DC 23 Metalloprotease
281 after ATG
(3225 bp)
Each of the mutants was inoculated into bovine milks (as previously described) and their growth at
37oC compared with that of the wild type strain, 0140J. This experiment was conducted on two
occasions; in no instance did any of the mutants show any growth retardation. Each mutant strain grew
at a rate and attained a final cell density similar to that of the genetically intact organism. (Fig 3)
Fig 3 Growth of bacterial mutant strains (Table 4) in milk from three different animals
SID 5 (Rev. 3/06)
Page 11 of 16
Fig 3A Experiment 1
Mean Bacterial Growth in milk from three cows
(1441, 1525, 3193)
log cfu/ml
8
6
4
2
0
0
6
9
12
24
time points (h)
DC1
DC2
DC3
DC4
DC5
DC6
DC23
WT
Fig 3B Experiment 2
Mean Bacterial Growth in milk from three cows
(1441, 1525, 3193)
log cfu/ml
8
6
4
2
0
0
6
9
12
24
time points (h)
DC1
DC2
DC3
DC4
DC5
DC6
DC23
WT
4. Discussion of Results Obtained
All the proteases under investigation in the present project were stably cloned and expressed from E.
coli. In three cases (DC1, DC2 and DC3 the products of sub1154, 0826 and 1370, respectively) these
were expressed as highly active proteases. This resulted in their self cleavage following expression
and this initially hampered their purification. A combination of lower level expression following growth at
lower temperatures and inclusion of protease inhibitor cocktail permitted purification of intact protein in
sufficient quantity for this investigation. In the case of DC2 the purified product contained both intact
and cleaved protein. The cleaved protein was in excess of 60 kDa and bound to the Ni coupled affinity
resin indicating the presence of an intact N terminus. This implies that these fragments represented Cterminal deletions of this protein. As such these fragments would include the domain of the protein
predicted (by Pfam) to act as the active protease.
All proteins proved to be immunogenic in rabbits and high titre antisera was produced in all cases. An
investigation as to the ability of these sera to inhibit the protease against which they were produced
SID 5 (Rev. 3/06)
Page 12 of 16
was not undertaken during this study and this has some implications with regard to interpretation of
subsequently acquired data.
In no instance did any of the immunoglobulin preparations show evidence of activity capable of
preventing or slowing growth of S. uberis. This was the case whether immunoglobulin directed at
individual proteins or pools of immunoglobulin against all the investigated proteins were used. The
interpretation of these data is that none of the proteases either individually or in combination is capable
of inducing growth inhibitory responses. This may reflect either the absence of a role in the acquisition
of essential amino acids from bovine milk or the inability of the protein to induce IgG that is capable of
inhibiting function.
Mutants were isolated that carried insertions within 6 out of the 7 open reading frames investigated. In
the case of DC6 no insertion within the open reading frame was detected and an insertion in close
proximity to the start codon of this gene did not alter expression of the product.
The insertion detected in DC1 was sufficiently distant from the start codon that a truncated protein
product resulted. Unlike the situation with the wild-type strain this truncated protein was released into
the culture supernatant from where it could be detected by immunoblotting. This is consistent with data
from other projects that has demonstrated that this protein (in a larger form) is released into culture
supernatant in the absence of sortase activity. Together these data supply strong evidence that DC1 is
a sortase anchored protein and that the motif that is anchored to the bacterial cell is located in the Cterminal portion of the protein. Bioinformatic analysis (using Pfam) of the truncated product of DC1
indicated that such a product would contain the region of the protein considered to contain the protease
motif. Thus suggesting that although improperly anchored this protein may still retain its biochemical
activity. In the case of DC5, Immunoblotting failed to detect protein from either the wild type or the
mutant strain implying that this product was not expressed in the growth conditions used. In all other
cases (DC2, DC3, DC4, DC23) the protein was detected in the wild type strain, but was absent in the
appropriate mutant strains indicating that the mutation had totally disrupted gene function.
Mutant strains carrying a lesion within the appropriate open reading frames (Table 4) did not have
altered growth rates or yields compared to the wild-type (genetically intact strain). this would imply that
in the case of DC2, DC3, DC4, DC5 and DC23 (sub 0826, 1868, 1738, 0350, and 1370 respectively)
that the absence of the gene product did not alter the ability of the S. uberis to grow in bovine milk.
In the case of DC1 (sub 1154), a truncated gene product was apparent from the mutant strain and this
is likely to contain the protease motif predicted by Pfam, consequently this product may well retain
protease activity. This truncated product, however, lacks the C-terminal anchor region predicted by
Pfam and correspondingly this product was found in culture supernatants and did not remain
associated with the bacterial cell as was the case in the wild type strain. Therefore in this case the only
conclusion we can reach is that bacterial association of DC1 was not required for growth in bovine milk.
In the case of DC6 (sub 1508) insertion in this gene was detected within the mutant bank and
consequently we can not conclude from data obtained under Objective 2 that inactivation of this gene
product had any effect on growth in milk.
5. Main implications of the findings
In conclusion, this project provided strong evidence that none of the proteases DC2, DC3, DC4, DC5
and DC23 (sub 0826, 1868, 1738, 0350, and 1370 respectively) were responsible for the release of
growth stimulating peptides and amino acids from bovine milk. These gene-products were not essential
for growth of S. uberis in this medium. The presented data further suggests that neither DC1 nor DC6
are capable of raising immune responses capable of inhibiting growth of S. uberis in bovine milk. This
situation arises either from the absence of a role, in bacterial growth, for these proteins (this was not
fully tested due to the absence of a mutant for DC6 and the location of the specific insertion within
DC1) or from their inability to raise neutralising responses. Given the very high tire of IgG obtained
(Table 3) the latter would seem unlikely and we consider the most likely scenario the absence of a role
for these gene products.
SID 5 (Rev. 3/06)
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The practical implication of these data is that there is currently no evidence that any of the proteins
under investigation would lead to effective growth inhibiting vaccines against bovine mastitis due to S.
uberis.
6. Possible Future Work
A number of the proteins investigated during this project have been implicated in pathogenesis of
bovine mastitis in other project areas.
The serine protease, DC1, which shows homology to the C5a peptidase of other streptococci has been
shown to be anchored at the surface of the bacterium through the action of sortase (a transamidase
capable of covalently linking proteins to the cell wall). It has also been shown that a mutant strain
lacking sortase activity was avirulent in dairy cattle. The possible role of sortase anchored proteins is
part of an investigation that has recently been funded by BBSRC through a GPA (Government
Partnership Award) and the role of DC1 will be investigated in vivo as part of this project. Similarly, DC2
and DC23 have been shown to have altered location in the sortase mutant. Although the anchoring of
these proteins to the bacterial cell is less clear than is the case with DC1. The role of DC2 and DC23
will investigated in the newly funded project. The reagents (clones, antisera and mutant strains)
generated for DC1, DC2 and DC23 during the current project will prove a valuable resource for the
forthcoming investigations.
7. Recommended Action Resulting from this Research
Current evidence would indicate that none of the potential vaccine candidates investigated in the
current project should be considered likely candidates for inclusion in growth inhibiting vaccines against
bovine mastitis caused by S. uberis. Substantive evidence from further in vivo studies would be
required to alter this position. Such evidence may be acquired from the recently awarded grant in this
area.
SID 5 (Rev. 3/06)
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References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
There are no references specifically relating to this material however the group has published other
articles during the lifetime of this project and these are listed below.
Refereed Articles
1. T. Tomita, B. Meehan, N. Wongkattiya, J. Malmo, G. Pullinger, J. Leigh and M. Deighton (2007)
Identification of Streptococcus uberis Multilocus Sequence Types Highly Associated with Mastitis.
Journal of Applied and Environmental Microbiology (submitted)
2. M.G. Lopez-Benavides, J.H. Williamson, G.D. Pullinger, S.J. Lacy-Hulbert, R.T. Cursons and J.A.
Leigh (2007) Field Observations on the Variation of Streptococcus uberis Populations in a Pasturebased Dairy Farm. Journal of Dairy Science (In press)
3. S. Wilson, P. Norton, K. Haverson, J. Leigh, M. Bailey (2007) Early, microbially driven follicular
reactions in the neonatal piglet do not contribute to expansion of the immunoglobulin heavy-chain VDJ
repertoire. Veterinary Immunology and Immunopathology (In press)
4. S. Wilson, P. Norton, K. Haverson, J. Leigh, M. Bailey (2007) Interactions between Streptococcus suis
serotype 2 and cells of the myeloid lineage in the palatine tonsil of the pig. Veterinary Immunology and
Immunopathology. 117:116-23.
5. G.D. Pullinger, T.J. Coffey, M. C. Maiden and J. A. Leigh (2007) Multilocus sequence typing analysis
reveals similar populations of Streptococcus uberis are responsible for bovine intramammary
infections of short and long duration. Veterinary Microbiology. 119. 194-204
6. G. D. Pullinger, M. López-Benavides, T. J. Coffey, J. H. Williamson, R. T. Cursons, E. Summers, J. LacyHulbert, M. C. Maiden and J. A. Leigh (2006) Application of Streptococcus uberis MLST: analysis of the
population structure detected within environmental and bovine isolates from diverse geographical
locations Journal of Applied and Environmental Microbiology. 72. 1429-1436
7. T.J. Coffey, G. D. Pullinger, K. A. Jolley, S. M. Wilson, M. C. Maiden, and J. A. Leigh. (2006) First
insights into the evolution of Streptococcus uberis: a Multi Locus Sequence Typing (MLST) scheme
that enables interrogation of its population biology Journal of Applied and Environmental Microbiology
72. 1426-1428
Conference proceedings
8. J.A. Leigh (2006) Enhancing immunity to streptococci In proceedings of Pan American Congress on
Mastitis. INVITED CONTRIBUTION
9. J.A. Leigh, Coffey, T.J. Pullinger, G.D. Lopez-Benavides, Williamson, J.H. Lacy-Hulbert, S.J. (2006) What
can learn by investigation of bacterial populations In proceedings of Pan American Congress on Mastitis
INVITED CONTRIBUTION
10. M.G. Lopez-Benavides, Williamson, J.H, Pryor, S.M. Pullinger, G.M. Leigh, J.A. Lacy-Hulbert, S.J.
Cursons, R.T. (2006) New perspectives on Streptococcus uberis ecology in a pasture based system. In
proceedings of Pan American Congress on Mastitis INVITED CONTRIBUTION
Conference presentations
11. J A Leigh (2007) Modern molecular approaches to the discovery of vaccines candidates against S.
uberis. British Cattle Veterinary Association, Southport, UK
12. E. L. Denham, P. N. Ward, and J. A. Leigh (2006). Determination of the Genes Involved in the
Processing of Extracytoplasmic Proteins in Veterinary Streptococci. In Abstracts of ) ASM Conference on
Streptococcal Genetics St Malo, France (POSTER)
13. R. Crowley, J.A. Leigh, P.N. Ward, H.M. Lappin-Scott, L.D. Bowler (2006) Analysis of Differential Protein
Expression during Streptococcus uberis Biofilm Development. ASM Conference on Streptococcal
Genetics St Malo, France (POSTER)
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