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

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.

Pathway of Cyclic Di-AMP

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? 目前發現肺炎鏈球菌有一個合成CDA的dacA基因，這個基因會encode雙腺核苷酸環化酶，還有兩個可以分解cda的基因pde1,pde2，encode cda phosphodiesterase 所以作者想要知道肺炎鏈球菌當中是否有能和cda結合的蛋白，並且知道這個蛋白會有甚麼的調控

Synthesize c-di-AMP and bind to c-di-AMP 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 DisA Non c-di-AMP receptor Synthesize c-di-AMP and bind to c-di-AMP DisA BSA 作者要找出肺炎鏈球菌當中能和CDA結合的蛋白 c-di-AMP

Test of the Column 9M urea input是control 就是過column之前兩個蛋白質的混和溶液

Purification of c-di-AMP binding proteins in S. pneumoniae 9M urea CabP ~21 kDa 破菌 過column Elute到最後可以看到大概在21kD的位置有比較明顯的band 因為這個蛋白可和CDA結合 所以作者將它命名為CabP CabP : C-di-AMP Binding Protein

Overexpression of purified CabP pST2788 E.Coli BL21 Induced by IPTG Cell lysis Ni column Elution His-tagged CabP ~28 kDa

Gel filtration sample 將CabP去做gel filtration

Gel filtration of CabP 280 nm ~210 kDa Octamerized His-tagged CabP 他在水溶液中會形成八聚體 The estimated molecular mass of ~210 kDa is equivalent to a calculated molecular mass of ~208 kDa

Electrophoretic mobility shift assay (EMSA) CabP―32P-labeled c-di-AMP complex 32P-labeled c-di-AMP PAGE protein (probe) Retarded band 接下來 作者想要更進一步知道CabP和CDA之間的interaction，他們利用emsa去了解他們之間的作用 首先 先將CDA的磷酸跟去做放射性標記，然後這個探針會和溶液中的能跟CDA結合的蛋白結合，然後去跑PAGE在轉印到PVDF膜上去做放射顯影，會看到complex跑得比較慢，有放射性標記的cda跑得比較快

Interaction between CabP and c-di-AMP PdxR是已知不會和CDA結合的蛋白，作為negative control 作者將這兩個蛋白先去跑page去確認分子量大小，再轉到膜上加入有放射性標記的CDA，發現有放射性訊號。代表cda可以和cabp結合 DisA是已知會和CDA結合的蛋白，從這張DATA可以看到CABP和DISA都可以和CDA結合，而PDXR沒辦法和CDA結合 所以沒有看到complex Mobility of 32P-labeled c-di-AMP in the absence or presence of various concentrations of purified CabP

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) 用過量的unlabeled nt去競爭labeled cda CabP binds c-di-AMP specifically

CabP shares 33% identity in amino acid sequence with S.aureus KtrA Sequence identity : 33% CabP : ortholog of S. aureus KtrA (S. pneumoniae) Transmembrane protein (S. aureus) 因為cabp和cda有專一性的結合 所以作者針對cabp這個蛋白去做更進一步的分析 作者去做gene alignment 發現 和KtrA的Sequence相似性高 而且KtrA也會和CDA專一性結合 作者在肺炎鏈球菌中找到KtrB的homolog SPD_0076 因為Ktra和Ktrb都是和鉀離子吸收有關的蛋白 所以作者想要知道cabp和0076跟鉀離子的吸收的關係 SPD_0076 (S. pneumoniae) c-di-AMP receptor (S. aureus)

Genetic organizations of the Trk proteins and the deleted regions in the mutant strains 作者發現CabP和0076有Trk domain，所以找出肺炎鏈球菌中其它有Trk domain的蛋白，而有Trk domain的蛋白跟ktr protein都跟鉀離子的吸收有相關 分別進行突變 去看哪些基因對鉀離子的吸收比較重要 SPD_0429是和SPD_0076很像的蛋白，SPD_0430是和CabP很像的蛋白

Growth of mutants in CDM with various concentrations of potassium S. pneumoniae CDM : chemically defined medium ST2798 將不同的SP突變株養在含有不同濃度的KCl的medium中去看sp mutant的生長情況 SPD_0076、cabP are more important for potassium uptake

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) 0076和cabp的互補實驗 右邊的表格是左邊的量化圖 0076和cabp是對於肺炎鏈球桿菌很重要的兩個蛋白

Bacterial two-hybrid analysis E.coli BTH101(Δcya) cya : adenylate cyclase Blue transcriptional activator 因為發現cap和0076是兩個對鉀離子吸收很重要的蛋白，而且這兩個蛋白是ktra ktrb的homolog，之前有講到說ktra ktrb這兩個蛋白會有interaction去影響鉀離子的吸收，所以作者利用雙雜交系統去看cabp和0076之間的interaction X-Gal β˗Galactosidase

Interaction between CabP and SPD_0076 E.coli BTH101 Zip positive c ST2845 negative c zip : leucine zipper motif disA : diadenylate cyclase

Growth of the WT and ST2734 in CDM with indicated concentrations of KCl pde1,pde2 : c-di-AMP phosphodiesterase ST2734 (Δpde1, Δpde2) WT [KCl] [KCl] 在肺炎鏈球俊當中，看CDA對於這兩個蛋白在肺炎鏈球菌中有甚麼影響 S. pneumoniae

C-di-AMP impairs potassium uptake by CabP-SPD_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

C-di-AMP binding protein, CabP Transmembrane protein, SPD_0076 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