Study On The Function Of SPD₁590 In Streptococcus Pneumoniae

SPD₁590 is overexpressed in piuABCD/piaABCD/pitABCD triple mutant strain,but its specific biological function is unknown.To explore the role of SPD₁590 in iron transportation and virulence of S.pneumoniae,the recombinant vector pBAD/HisA-1590 was constructed by molecular cloning technique.The SPD₁590 with high purity was obtained by affinity chromatography.Subsequently,native gel electrophoresis,UV/Visible spectrophotometry,fluorescence spectrophotometer,and protease K digestion were used to study the binding ability of SPD₁590 with hemin and conformational changes.The survival curves of silkworms were drawn to explore the effect of SPD₁590 on the virulence of S.pneumoniae.In addition,the expression levels of inflammatory factors in A549 cells after WT and 1590 mutant strain infection were determined with qRT-PCR.Furthermore,iTRAQ-based quantitative proteomics technology was used to deeply find out the biological functions of SPD₁590.The SPD₁590 with high purity and yield was obtained by cloning,expression and purification,and verified by western blot.The results based on UV/Vis spectra and fluorescence spectra showed that the SPD₁590 could bind to hemin with the dissociation constant of 2.4 ± 0.05 ?M.Hemin binding increased the resistance capability of SPD₁590 to enzymatic cleavage,but did not induce any significant changes of secondary structure.qRT-PCR results suggested that the expression of pro-inflammatory cytokines in ?1590 treated A549 cells were evidently decreased compared to that in WT treated A549 cells.In addition,iTRAQ-based quantitative proteomics data showed that the differentially expressed proteins mainly concentrated in the ABC transport system,glucose,pyrimidine metabolism and two component systems.Besides,virulence factors of S.pneumoniae,such as pspA?binding pneumococcal surface protein A?and the lytA?pneumococcal autolysin,autolysin?,and proteins associated with biofilm formation including luxS and cbpD were down-regulated in 1590 mutant strain.SPD₁590 is a hemin-binding protein in S.pneumoniae.Hemin binding induces a conformational change of SPD₁590.SPD₁590 is possibly directly involved in iron transportation or affects the expression levels of other iron transporters,proteins involved in glucose metabolic pathways,virulence factors,and thus changes the virulence of S.pneumoniae.