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Similar to J Shankar Nwmg 2009 Final
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J Shankar Nwmg 2009 Final
- 1. Characterising the role and regulation of secreted proteins of Enterococcus faecalis Jayendra Shankar
- 3. The fsr Quorum Sensing TCS
- 5. GelE Temporally Regulates the Excreted Protein profile, particularly SalB Temporal regulation of excreted proteins of E. faecalis OG1RF and its isogenic gelE and fsrB mutants separated on a 11% gel. Samples were collected at mid-log (3h), late-log (5h), stationary (8h) and overnight (ON) phases of growth. M = Biorad Broad range marker
- 7. SalB is a Peptidoglycan Hydrolase with Unknown Specificity Left: Figure showing the separation of dilutions of purified SalB ( μ g) on a 11% gel stained with coomassie blue Right: Figure showing a 11% renaturing gel with 10 OD units of E. faecalis purified peptidoglycan stained with 0.1% methylene blue; Zones of clearing indicate SalB activity M=Page ruler, prestained marker
- 8. Functional analysis – HPLC for the identification of bond specificity Mutanolysin Mutanolysin + SalB Figure showing the HPLC separation of reduced mutanolysin or mutanolysin + SalB digested E. faecalis OG1RF peptidoglycan on a linear 0-45% gradient over 65 minutes
- 10. Functional analysis – HPLC for the identification of bond specificity Mutanolysin treated OG1RF salB peptidoglycan Mutanolysin + heat treated SalB
- 14. SalB Deletion Causes Morphological Changes OG1RF OG1RF gelE OG1RF gelE/salB OG1RF salB
- 15. SalB Deletion Causes Septation Defects OG1RF OG1RF gelE OG1RF gelE/salB OG1RF salB
- 16. SalB Deletion Results in a Hyper-Lytic Phenotype A B Fig 6: Autolysis assays for E. faecalis OG1RF ( ) and its isogenic mutants OG1RF gelE ( ), OG1RF fsrB ( ), OG1RF salB (▲), and double mutants OG1RF gelE/salB ( ) and OG1RF fsrB/salB ( ) conducted in 0.1mgml -1 Penicillin G ( A ) or 0.1% (v/v) Triton X-100 ( B ) induced stress. Exponentially growing cells were harvested, washed in PBS and resuspended in PBS containing 0.1mgml -1 Penicillin G or 0.1% v/v Triton X 100. Optical density at 600nm was collected hourly and turbidity was plotted as a percentage survival graph versus time.
- 19. With thanks to: Bacterial Pathogenesis Group: Dr. Mal Horsburgh Dr. John Kenny Ben Libberton Rachel Walker Proteomics: Dr. Deborah Ward Mark Prescott HPLC: Dr. Mark Wilkinson Confocal imaging and Flow Cytometry: Dr. Dave Spiller TEM: Allison Beckett Dr. Ian Prior And all members of LabH
Editor's Notes
- HI IM Jay and Im here to talk about the characterisation of the E. faecalis excretome and in particular the role and regulation of SalB, a putative autolysin. Enterococci are medically relevant particularly due to their capability to transfer antibiotic resitance, including Vancomycin resistance to other, more pathogenic bacteria including Staphylococci.
- Both cell wall associated and excreted virulence determinants of Enterococci are being characterised. The role of its physiology becomes important to better understanding this organism. The specific aims of the project was to characterise the excreted protein profile of E. faecalis OG1RF, a quorum sensing replete strain that produces a protease GelE, a known virulence agent.
- We are concerned with the quorum sensing fsr Two component system that regulates the expression of proteases Gelatinase (GelE ) and serine protease (SprE) The current model described for the operon is the accumulation of GBAP (gelatinase biosynthesis activating pheromone), which is an autoinducing peptide. The product of fsrD gene is detected by cell wall associated FsrC (Histidine kinase) which phosphosphorylates FsrA (Response regulator) which in turn regulates gene expression, particularly GelE and SprE, but also other genes. GelE, is involved in increasing autolysis, reduces aggregation and degrades polymerised fibrin. Its contribution to virulence has been established in animal models.
- 2D analysis of stationary phase proteins revealed a dominance of GelE and SprE. A total of 54 spots were excised and 44 were ascribed identifications based on matches to the MASCOT database of tryptic fragments of excised protein spots analysed by MALDI-TOF. The red arrows indicate proteins that we were unable to identify. Few obvious potential virulence determinants, sulphatase domain protein may aid in colonisation of mucosal surfaces, but most others are concerned with cell wall biosynthesis and stress response.
- Note: Temporal regulation due to protease activity(?) in OG1RF To study the effect of GelE on the regulation of excreted proteins of OG1RF, excreted proteins from the wildtype, a gelE mutant and and fsrB mutant were compared. Where the wildtype expresses a shift in profile into stationary phase, the mutants lacking GelE do not show this shift. Among the bands constitutively expressed, SalB was identified by Rachel Walker.
- And that was how we got interested in SalB. Previously, work has been published on SalB. It was recognised as a stress related protein, potentially conferring stress relief as it was identified on 2-D gels of proteins differentially expressed in E. faecalis JH2-2 (an fsr lacking strain) that were stressed by bile salt and glucose starvation when compared to the wild type. It was also identified by an American group as a homolouge of SagA of E. faecium (which binds ECM molecules and contributes to virulence) and studies indicate that SalB performs similar function in E. faecalis, potentially contributing to virulence The Deletion of SalB in JH22 was also noted to cause severe growth defects and morphological changes, particularly sepatation anamolies as studied by TEM. These authors suggest a role of SalB in stress relief by some cell wall modification process.
- Purified SalB was applied to an SDSPAGE gel with 10-OD units worth of E. faecalis peptidoglycan. Following separation, the proteins were renatured with triton and Mg2+ and the peptidoglycan was stained with Methylene blue. Zones of clearing indicate activity of SalB on the PG. Explain figure
- Purified peptidoglycan was digested with mutanolysin (a muramidase) and mutanolysin with SalB overnight. Digested muropeptides were reduced in the presence of Sodium borohydride and separated by HPLC over 65 minutes on a 45% linear gradient As is evident (picture) additional peaks were seen on the mutanolysin + salB fraction, and these were assumed to be as a result of SalB action
- Masses were calculated from monomers and dimers as a possible result for muramidase and endopeptidase activity. The unique fractions were collected and analysed by MS, however, we only identified SalB fragments. Before we got ahead of ourselves assuming that it was PG binding regions, we decided to make sure that it was not fractions of SalB from degradation. The following experiments were designed and conducted (from slide) However, Heat treated SalB also produced the same effect indicating that these peaks were probably SalB degrading overnight in the presence of Mutanolysin. (as confirmed by SDS-PAGE). At this point, no direct action on PG could be assigned to SalB
- As represented here, we were unable to pick up any peptidoglycan modification caused by the deletion of the salB gene in the mutant. Also the heat treatment of SalB overexpressed protein continued to produe a profile similar to un-heated sample indicating that the additional peaks observed were probably not due to SalB activity on peptidoglycan
- To characterise the effect of SalB on the physiology of E. faecalis, SalB deletions were constructed in E. faecalis OG1RF and also in the gelE and fsrB mutants. Additionally, sal B was complemented in the knockout strain.
- 2D DIGE was performed. Flouro labelled proteins from OG1RF and OG1RF salB were applied to the same gel. After separation, they were excited at appropriate wavelengths and images were overlaid.
- The effect of SalB on protein expression was analysed by 2D SDSPAGE 32 spots differentially expressed in the mutant compared to the wildtype. 32 spots were excised and 25 were identified. Red arrows indicate spots we were unable to ascribe an id to OF interest are spots A, C, F and V which are related to stress management. The cell seems to be induced into a stressed state when SalB activity is removed. However, our method for growth includes shaking which introduces O2 stress.
- Morphologies of the various strains were analysed by LSM under 100x after the cells were stained with BACLIGHT live dead kit from invitrogen. Greeen cells are live and red cells are dead. A chaining phenotype is seen in the fsrB and gelE deletions. This has been described before. However, a “clumping” phenotype is observed in the SalB deletion mutant. The double mutant shows an exacerbated condition of clumping and chaining. Note the varying scale bars.
- Additionally septum formation was studied using TEM. Again, SalB deletion in the wildtype or the OG1RF gelE background show abnormal septum formation and the condition is exacerbated in the double mutant.
- In induced autolysis experiments using cell wall active Penicillin G and surfactant triton , salB deletion causes the cell to show increased lysis either in the wildtype or the GelE mutant. IN both cases, the deletion of salB leads to increased susceptibility to PenG and triton. However, the GelE mutant exhibits a classical autolysin deficient response – increased susceptibility.