Transcript Enzymes of the sulphate assimilation pathway induced during.

Enzymes of the sulfate assimilation pathway
induced during intracellular growth are novel
protective antigens of Mycobacterium tuberculosis.
Warwick Britton, Lisa Leotta, Erin Shanahan, Nick West,
Tom Leyh, Rachel Pinto, Jamie Triccas.
Centenary Institute &
Dept of Infectious Diseases & Immunology
University of Sydney
Antigen Discovery and TB vaccines
Screen for M. tb genes
expressed in host cells
Tests recognition of
novel antigens by
TB patients
Clone genes,
express proteins,
test vaccine
potential in
animal models
cytoplasm
Sulphur Assimilation Pathway
Sulpholipids
CysDNC
subI
Sulphate APS
PAPS
cysTWA
(ATP sulfurylase/Kinase)
Sulphate
Cell membrane
cysDNC
Serine
CysE (Serine
Acetyl transferase)
O-Acetyl-L-serine
(Sulfite
Reductase)
Sulphite
Sulphide
CysK1
SirA
(O-Acetly-L-Serine
Proteins
Sulfhydralase/OASS)
Fe-Sulphur clusters
Mycothiol
Coenzymes
Methionine
Cysteine
Pinto R, Triccas J et al, JID, 2013
M. tuberculosis Sulphate Activation
Complex (SAC)
Sulphate
ATP
Sulphurylase
(CysDN)
CysD
CysNC
The ‘SAC’
CysNC
APS
APS kinase
(CysC)
CysD
PAPS
Cys, Met
(proteins)
Pinto et al, Microbiology 2004
Pinto et al, J. Bact 2007
Sun et al, JBC, 2005
Triccas Lab (USyd)
Leyh lab (AECOM, New York)
A
B
cysD
cysN cysC
ATP
GTPase APS
Kinase
sulfurylase
Relative CysDNC
expression
CysDNC are expressed as an operon during intracellular
growth of M. tuberculosis & in vivo infection
6
5
4
3
2
1
0
D
8
6
4
2
in vivo
No Det
Det
Relative CysDNC
expression
CFU/mL (x107)
C
in culture
0
Days
Pinto R, Triccas J et al, JID, 2013
CysDNC stimulates T cell response in Mtb-infected
mice and in TB patients
A
100
60
*
*
*
*
40
20
0
200
160
B
*
120
80
*
Stimulation Index
Number of INF- producing cells
per 105 MLN
C
*
80
40
CFP
0
CysDNC
Ag85B
Protein
Protein
Pinto R, Triccas J et al, JID, 2013
DNA vaccines expressing CysD & CysNC induce
protective immunity against Mtb infection
B. Spleen
A. Lung
Log10CFU/Organ
*
6.5
*
5.0
4.5
6.0
4.0
5.5
3.5
5.0
3.0
4.5
2.5
4.0
2.0
Groups
Pinto R, Triccas J et al, JID, 2013
cytoplasm
Sulphur Assimilation Pathway
Sulpholipids
CysDNC
subI
Sulphate APS
PAPS
cysTWA
(ATP sulfurylase/Kinase)
Sulphate
Cell membrane
cysDNC
Serine
CysE (Serine
Acetyl transferase)
O-Acetyl-L-serine
(Sulfite
Reductase)
Sulphite
Sulphide
CysK1
SirA
(O-Acetly-L-Serine
Proteins
Sulfhydralase/OASS)
Fe-Sulfur clusters
Mycothiol
Coenzymes
Methionine
Cysteine
Pinto R, Triccas J et al, JID, 2013
CysH, SirA, CysK1 & CysE are expressed in Mtb-infected
macrophages & stimulate T cell responses in mice
A. mRNA in infected Mp
7
Number of INF- producing
cells per 105 MLN
Relative expression
8
B. T cell responses during Mtb infection
*
*
200
*
*
6
5
4
3
2
1
160
120
80
40
0
0
Transcript
Protein
Pinto R, Triccas J et al, JID, 2013
CysH, SirA, CysK1 & CysE elicit T cell responses
in Tuberculosis patients
*
*
*
*
CFP
CysH
SirA
CysK1
CysE
Stimulation Index
*
Protein
Pinto R, Triccas J et al, JID, 2013
CysD & CysN proteins in MPL/DDA induce protective
immunity against Mtb infection
B. Spleen
A. Lung
Log10 CFU/Organ
5.5
5.0
*
*


*
5.1
*
*
4.6


4.5
4.1
4.0
3.6
3.5
3.1
3.0
2.6
*
Groups
Pinto R, Triccas J et al, JID, 2013
CysD & CysN proteins in MPL/DDA boost BCG-induced
protective immunity against Mtb infection
Log10 CFU/Organ
A. Lung
*
*
B. Spleen
6.5
6.0
6.0
5.5
5.5
*
*
5.0
**
4.5
NS
5.0
4.0
4.5
3.5
4.0
3.0
3.5
2.5
Groups
Pinto R, Triccas J et al, JID, 2013
Components of SAP as TB vaccines
• Enzymes of sulphur assimilation pathway are
induced during intracellular growth of Mtb
• Proteins of sulphur assimilation pathway are potent T
cell antigens during human and murine TB infection
• CysD and CysN as DNA and protein vaccines induce
protective immunity against Mtb infection
• CysD and CysN as protein vaccines boost BCGinduced protective immunity against Mtb infection
CysD & CysN are strong candidate antigens for TB
subunit vaccines to boost BCG
Acknowledgments
University of Sydney
Infectious Disease & Immunology
Jamie Triccas
Rachel Pinto
Lisa Leotta
AECOM, New York
Tom Leyh
Centenary Institute
Mycobacterial Research
Warwick Britton
Nick West
Erin Shanahan
Funding:
NHMRC, Uni Sydney
ARC