Structure And Function Of Pneumococcal Peptidoglycan Hydrolase Lytb And Drug Resistant Protein Sp0010

Ⅰ. Structure and function of pneumococcal peptidoglycan hydrolase LytB Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the1.65A crystal structure of the catalytic domain (residues Lys375-Asp658) of LytB (termed LytBCAT), excluding the choline-binding domain. LytBCAT consists of three structurally independent modules:SH3b, WW and GH73. These modules form a "T-shaped" pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan (PG). Structural comparison and simulation revealed that the GH73module of LytB harbors the active site, including the catalytic residue Glu564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of specific substrate of LytB in the immature PG. Combined with in vitro cell-dispersing and in vivo cell-separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases. Ⅱ. Structure and function of pneumococcal drug resistant protein Sp0010The high mortality of pneumococcal diseases is due to the emergence of multidrug resistant Streptococcus pneumoniae. β-Lactamase is the leading cause of Gram-negative and Gram-positive bacterial resistance to β-lactam antibiotics. However, no β-lactamase had been identified in S. pneumoniae. The research of our collaborator showed that knocking out of the putitive β-lactamase gene sp0010of TIGR4leads to multi antibiotic susceptibility of S. pneumoniae. Here we have overexpressed and purified the protein Sp0010, and further collected a diffiraction data of the apo-form at2.5A. Furthermore, we found that Sp0010can bind to pneumcoccal peptidoglycan. The study of function and structure will help us to reveal the molecular mechanism of the multidrug resistance of Sp0010.