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General enquiries on this form should be made to: Defra, Science Directorate, Management Support and Finance Team, Telephone No. 020 7238 1612 E-mail: [email protected] SID 5 Research Project Final Report Note In line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects. 1. Defra Project code 2. Project title This form is in Word format and the boxes may be expanded or reduced, as appropriate. 3. ACCESS TO INFORMATION The information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors. 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 6. It is Defra’s intention to publish this form. Please confirm your agreement to do so. ................................................................................... YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow. Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer. In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. (b) If you have answered NO, please explain why the Final report should not be released into public domain 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) Page 13 of 16 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) Page 14 of 16 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) SID 5 (Rev. 3/06) Page 15 of 16 SID 5 (Rev. 3/06) Page 16 of 16