Towards the development of a rapid diagnostic test for

Buruli ulcer: Identification of Mycobacterium ulcerans

specific antigensSacha J. Pidot, Jessica L. Porter

John K. Davies, Paul D. R. Johnson & Timothy P. Stinear

What do we want to achieve?What do we want to achieve?

Positive Negative

www.mirates.nl

www.cellestis.com

What do we want to achieve?What do we want to achieve?

Project aimsProject aims

• To develop immunodiagnostic tools for rapid identification of M. ulcerans (MU) infection

– Identification of MU specific gene sequences– Cloning and overexpression of these sequences– Testing by Western blotting

• Addresses a specific WHO Buruli Ulcer research priority

M. ulcerans M. ulcerans antigen identificationantigen identification• Need to identify antigens specific to

M. ulcerans (MU)• Compare MU genome sequence to other

mycobacterial genomes

M. ulcerans M. ulcerans antigen identificationantigen identification

• Classes of MU specific proteins

– Membrane proteins– Secreted proteins– Hypothetical proteins– Unique / Conserved– Mycolactone PKS

domains

• Total of 41 potential antigens identified

Determination of MU specific genesDetermination of MU specific genes

• Are identified genes really MU specific?

• High genetic similarity between MU and M. marinum

• Sequenced M. marinum ‘M’ strain is lab adapted

• Are these genes present in non-sequenced isolates?

• Tested 27 diverse M. marinum strains for presence of MU genes by PCR

Determination of MU specific genesDetermination of MU specific genesCertain genes found in >90% of tested M. marinum

strains - deletions in sequenced ‘M’ strainPercentage of M. marinum strains containing MU genes

0

10

20

30

40

50

60

70

80

90

100

MU29372 MU74349 MU520667 MU538935 MU540676 MU1214321 MU2891760 MU3579607 MU4686564 MU4690164

• 10 genes found to be present in tested M. marinum strains

• Removed non-MU specific genes from list of potential antigens

• Total of 31 potential antigens• Do all MU strains contain these 31 genes?• Tested 14 diverse MU strains by PCR

Determination of MU specific genesDetermination of MU specific genes

Percentage of MU strains containing MU unique genes (14 strains)

0

10

20

30

40

50

60

70

80

90

MUP13

MUP14

MUP15

MUP17

MUP57

MUP64

MUP68

MUP74

MUP76

MU527

240

MU527

247

MU530

424

MU105

0519

MU105

0554

MU105

2616

MU316

1439

MU358

9715

Variable distribution of MU specific genes across strains

Expression of recombinant antigensExpression of recombinant antigens

+ inducer

Lysis

Purification

Reactivity of human sera against Reactivity of human sera against MU antigensMU antigens

6xHis

MU

P15

Ag8

5A

MU

P45

Patient

(pooled)

Exposed

(pooled)

Control

(pooled)

MU

P15

Ag8

5A

MU

P45

MU

P15

Ag8

5A

MU

P45

MU

P15

Ag8

5A

MU

P45

64

22

36

50

22

36

5064

Agy

99

Agy

99

Cha

nt

Cha

nt

Cha

nt

Agy

99

Patient

(poole

d)

Exposed (p

ooled

)

Control

(pooled

)

Antigens tested so farAntigens tested so far

• 19 proteins tested so far - no serum reactivity to recombinant proteins observed to date

MUP13 Possible conserved membrane protein Dehydratase PKS domainMUP14 Putative integral membrane protein Ketoreductase A PKS domainMUP15 Possible secreted protein Ketoreductase B PKS domainMUP17 Possible conserved transmembrane protein Acyltransferase acetate 1 PKS domainMUP45 Probable beta-ketoacyl synthase-like protein Acyltransferase acetate 2 PKS domainMUP57 Possible lipoprotein Acyltransferase propionate PKS domainMUP64 Possible conserved membrane protein Enoylreductase PKS domainMUP68 Conserved membrane protein Acyl Carrier Protein 1 PKS domainMUP74 Possible membrane protein Acyl Carrier Protein 2 PKS domainMUP76 Possible membrane protein Acyl Carrier Protein 3 PKS domainMU527240 Unique hypothetical protein Ketosynthase core PKS domainMU527247 Unique hypothetical protein Ketosynthase alternate PKS domainMU530424 Unique hypothetical protein hsp65 GroEL2MU1050519 Unique hypothetical proteinMU1050554 Conserved membrane proteinMU1052616 Unique hypothetical membrane proteinMU3589715 Zinc metalloproteaseAg85A Part of known M.tb antigen complex

Future DirectionsFuture Directions• Proteomic approach

• 2D gel electrophoresis and Western blotting

• Find proteins specifically recognised by patient sera, then identify by MassSpectroscopy

TT--cell antigenscell antigens

• T-cells - alternate arm of immune system• Destroy infected cells• Recognise antigens presented by infected

cells• Important in immunity to

other mycobacterial diseases (TB, leprosy)

• Are any of these antigens T-cell stimulators?

TT--cell antigenscell antigens

• Current case-control study in Victoria

– Obtain patient blood– Separate PBMCs– Stimulate using

purified MU antigens– Cytokine production /

Proliferation

AcknowledgementsAcknowledgements• Davies Lab• Tim Stinear & John Davies• May Smith, Travis Gooding, Frances

Oppedisano – Royal Childrens’ Hospital, Victoria, Australia

Variable distribution of MU specific genes across strains

0

10

20

30

40

50

60

70

80

90

100

MUP13

MUP14

MUP15

MUP17

MUP57

MUP64

MUP68

MUP74

MUP76

MU527

240

MU527

247

MU530

424

MU105

0519

MU105

0554

MU105

2616

MU316

1439

MU358

9715

% of strains

% of african strains