Transcript Cyclic Di-AMP

Cyclic Di-AMP Impairs Potassium Uptake
Mediated by a Cyclic Di-AMP Binding Protein
in Streptococcus pneumoniae
JB Feb 2014 Vol.196 No.3 : p.614-623 Yinlan Bai, Jun Yang, Tiffany M. Zarrella
Advisor : Shan-ho Chou
Student : Tsai-Lun Chen
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Cyclic Di-AMP
• A second messenger in bacterial physiology and infections
- bacterial virulence
- bacterial growth
- biofilm formation
• Bacillus subtilis, Streptococcus pneumoniae, Staphylococcus aureus,
Mycobacterium tuberculosis,etc.
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Pathway of Cyclic Di-AMP
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Streptococcus pneumoniae
• Gram-positive pathogen
• Pneumonia, septicemia, sinusitis ,and meningitis
• Diadenylate cyclase (dacA)
C-di-AMP phosphodiesterase encoding genes (pde1 and pde2)
• Are there any proteins that can bind to c-di-AMP?
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Identification of a c-di-AMP binding protein in S. pneumoniae
• C-di-AMP affinity chromatography
→ 2’-AHC-c-diAMP-agarose resin
(2'- O- (6- Aminohexylcarbamoyl)- cyclic diadenosine monophosphate )
Test of the Column
BSA
Synthesize c-di-AMP
and bind to c-di-AMP
DisA
Non c-di-AMP receptor
BSA
c-di-AMP
DisA
BSA
DisA
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Test of the Column
9M urea
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Purification of c-di-AMP binding proteins
in S. pneumoniae
9M urea
CabP
~21 kDa
CabP : C-di-AMP Binding Protein
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Overexpression of purified CabP
cabP
Elution
pST2788
E.Coli BL21
Induced by IPTG
His-tagged CabP
~28 kDa
Cell lysis
Ni column
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Gel filtration
sample
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Gel filtration of CabP
280 nm
~210 kDa
Octamerized His-tagged CabP
The estimated molecular mass of ~210 kDa is equivalent to a
calculated molecular mass of ~208 kDa
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Electrophoretic mobility shift assay (EMSA)
protein
32P-labeled c-di-AMP
(probe)
PAGE
CabP―32P-labeled c-di-AMP complex
32P-labeled c-di-AMP
Retarded band
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Interaction between CabP and c-di-AMP
Mobility of 32P-labeled c-di-AMP in the absence or presence of various
concentrations of purified CabP
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Interaction between CabP and c-di-AMP
• Mobility of 32P-labeled c-di-AMP in the presence of purified CabP
and an excessive amount of each unlabeled nucleotide
(CabP-labeled c-di-AMP complex)
CabP binds c-di-AMP specifically
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• CabP shares 33% identity in amino acid sequence with S.aureus KtrA
Sequence identity : 33%
Transmembrane protein
(S. aureus)
c-di-AMP receptor
(S. aureus)
SPD_0076 (S. pneumoniae)
CabP : ortholog of S. aureus KtrA
(S. pneumoniae)
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Genetic organizations of the Trk proteins and the
deleted regions in the mutant strains
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Growth of mutants in CDM with various
concentrations of potassium
S. pneumoniae
CDM : chemically defined medium
ST2798
SPD_0076、cabP are more important
for potassium uptake
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Growth of the complemented SPD_0076 and cabP
mutants in the formulated CDM
ST2803 :
ST2797(ΔSPD_0076)+recombinant plasmid(SPD_0076)
ST2805 :
ST2796(ΔcabP)+recombinant plasmid(cabP)
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Bacterial two-hybrid analysis
E.coli BTH101(Δcya)
cya : adenylate cyclase
Blue
transcriptional activator
X-Gal
β˗Galactosidase
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Interaction between CabP and SPD_0076
E.coli BTH101
zip : leucine zipper motif
disA : diadenylate cyclase
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Growth of the WT and ST2734 in CDM with
indicated concentrations of KCl
pde1,pde2 : c-di-AMP phosphodiesterase
WT
[KCl]
ST2734 (Δpde1, Δpde2)
[KCl]
S. pneumoniae
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C-di-AMP impairs potassium uptake by CabPSPD_0076 complex
• Expression of SPD_0076, cabP, and M. tuberculosis disA(Mtb_disA) in LB2003
(LB2003 : potassium transport-deficient E. coli mutant)
low-potassium medium
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Conclusion
• Model of c-di-AMP controlled potassium uptake by S. pneumoniae
C-di-AMP binding protein, CabP
Transmembrane protein, SPD_0076
SPD_0076
c-di-AMP
CabP
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