Structure And Function Of ABC Transporter Spr1677-78 And Choline-binding Protein CbpJ From Streptococcus Pneumoniae

(?)Preliminary study on Streptococcus pneumoniae ABC transporter Spr1677-78The compatible solutes regulate cell osmotic pressure to resist the loss of cellular water in all kindoms of life.The accumulation of compatible solutes is crucial for the cells to withstand sudden alterations of osmolality in external environment.Spr1677-78 is an ABC transporter which may be responsible for the transport of compatible solutes in S.pneumoniae.As we know,Spr1677 possesses a transmembrane domain followed by a C-terminal substrate-binding domain,whereas Spr1678 is an ATPase locating at the cytosol.In this study,we screened the detergents and Nanodisc assembly condition that are suitable for structural studies of Spr1677-78.Moreover,we constructed a series of truncated versions of Spr1677-78 and obtained proteins with high purity and homogeneity.Then we screened the putative substrate molocules of Spr1677-78 by gene knockout and phenotypic microarray experiments.Although these molecules have no direct interactions with the substrate-binding domain of Spr1677-78,they might share a core structure similar to the physiological substrate of Spr1677-78.In addition,we purified and crystallized the proteins of Spr1679,which is thought to be functionally related to the ABC transpoter Spr1677-78.(?)Functional insight into choline-binding protein CbpJ from Streptococcus pneumoniaeStreptococcus pneumoniae encodes various surface proteins,which can be classifiedinto three major groups distinguished by their mode of attachment to the host cell:the proteins harboring the LPxTG motif,lipoproteins and choline-binding proteins(CBPs).The CBPs generally consist of at least two domains:a highly conserved choline-binding domain(CBD)and a functional domain.The CBD can anchor the CBPs to the phosphocholine molecule on the cell wall by non-covalent interaction.S.pneumoniae TIGR4 CbpJ has been described as a putative adhesin.To detect its function,we constructed cbpJ knockout strain(KO)and found that it lost 90%adhesion ability compared with the wild-type strain.Furthermore,we deleted the coding regions of the N-domain(ANT)and the CBD(ACT),repectively.In vivo assasys showed that the ANT strain retains about 70%adhesion activity,whereas the ACT lost 90%adhesion activity,indicating that the CBD of CbpJ is essential for the adhesion of pneumococci to human lung epithelial cell A549.For the first time,the choline-binding domain was proved to be involved in helping pneumococcal adhesion to lung epithelial cells,in addition to anchoring on the bacterial cell wall.