Characterising the role and Characterising the role and regulation of secreted proteins regulation of secreted proteins of of Enterococcus faecalisEnterococcus faecalis

Jayendra Shankar

BackgroundBackground E. faecalis is an increasing clinical menace, causing

wound infections, endocarditis and urinary tract infections.

Multiply antibiotic resistant strains are typical – notoriety for transfer of Tn1545 conferring van resistance to S. aureus.

Aims1. Characterise E. faecalis excreted proteome to better understand the virulence repertoire.

2. Investigate regulation of excreted proteins.

The The fsrfsr Quorum Sensing TCS Quorum Sensing TCS

Excreted Proteins of OG1RF – 2D Excreted Proteins of OG1RF – 2D PAGE at 8hPAGE at 8h

• 54 spots excised; trypsin digested -> MALDI-TOF -> MASCOT database• 44 identified•Red arrows – proteins not identified• Heavy abundance of GelE (9) and SprE (7)

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GelE Temporally Regulates the Excreted GelE Temporally Regulates the Excreted Protein profile, particularly SalBProtein 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

And so we got interested with And so we got interested with salBsalB......

Expressed in stressed E. faecalis JH22 (an fsr lacking strain), especially bile salt stress

Homologous to SagA of E. faecium and found to adhere ECM molecules; a potential role in infection (Sag-A Like protein B)

Deletion of salB in JH22 causes septation anamolies; a role in stress protection by cell wall modification was proposed

SalB is a Peptidoglycan Hydrolase with SalB is a Peptidoglycan Hydrolase with Unknown SpecificityUnknown Specificity

Left: Figure showing the separation of dilutions of purified SalB (μg) on a 11% gel stained with coomassie blueRight: 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 activityM=Page ruler, prestained marker

Functional analysis – HPLC for the Functional analysis – HPLC for the identification of bond specificityidentification of bond specificity

Minutes

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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

Analysis by MS of HPLC peaksAnalysis by MS of HPLC peaks Calculated masses of various fractions of

momneric and dimeric units of PG Identified 2 peptides : both were SalB derived Series of tests to determine if unique peaks

were indeed SalB fragments and not peptidoglycan fractions as a result of SalB action:◦ SalB + mutanolysin◦ Heat treated SalB + mutanolysin◦ PG from OG1RF salB digested with mutanolysin

Functional analysis – HPLC for the Functional analysis – HPLC for the identification of bond specificityidentification of bond specificity

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Mutanolysin treated OG1RF salB peptidoglycan

Mutanolysin + heat treated SalB

Strains Created for our StudyStrains Created for our Study

OG1RF salB :: pTex4577 (KanR)

Complemented strain OG1RF salB pAT18 (salB) (ErmR)

To determine the role of gelE, we also created a gelE/salB and an fsrB/salB double mutants

Comparison of Excreted Proteins of Comparison of Excreted Proteins of OG1RF and OG1RF OG1RF and OG1RF salBsalB

• OG1RF wild type – blue• OG1RF salB - red

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SalB Deletion Induces a Stressed State SalB Deletion Induces a Stressed State in the Cellin the Cell

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•32 spots excised•25 identified•Spots of interest:

• A- Hsp 70• C- GroEL• F- NADH peroxidase• V- SOD

SalB Deletion Causes Morphological SalB Deletion Causes Morphological ChangesChanges

OG1RF OG1RFgelE

OG1RF gelE/salB

OG1RF salB

SalB Deletion Causes Septation DefectsSalB Deletion Causes Septation Defects

OG1RF OG1RFgelE

OG1RF gelE/salB

OG1RF salB

SalB Deletion Results in a Hyper-Lytic SalB Deletion Results in a Hyper-Lytic PhenotypePhenotype

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.

SummarySummary

The E. faecalis excreted proteome is regulated temporally by Fsr, the density-dependent two-component signalling system.

GelE post-translationally regulates the excreted proteome and a mutant lacking gelE expresses SalB constitutively.

Deletion of salB function causes a stressed state and alters the excreted protein profile.

SalB has peptidoglycan hydrolase activity, however its specificity could not be determined.

E. faecalis OG1RF salB shows alterations in cell morphology, with septation anomalies, as has been reported previously for strain JH2-2, a natural Fsr mutant.

SummarySummary

The OG1RF gelE salB double mutant strain displays exacerbated morphological defects with severe septation anomalies and defective cell separation resulting in clumping.

Protein localisation studies are currently being performed to explore the specific function of SalB in peptidoglycan hydrolysis and cell morphogenesis. SalB contains a coiled-coil domain and might thus interact with a partner to form a complex. This will be explored in future studies

With thanks to:With thanks to:Bacterial Pathogenesis Group:Dr. Mal HorsburghDr. John KennyBen LibbertonRachel Walker

Proteomics:Dr. Deborah WardMark Prescott

HPLC:Dr. Mark Wilkinson

Confocal imaging and Flow Cytometry:Dr. Dave Spiller

TEM:Allison BeckettDr. Ian Prior

And all members of LabH