Research On The Role And Mechanism Of The Spd0300 Gene Of Streptococcus Pneumoniae In Hyaluronic Acid Metabolism

Objective:Streptococcus pneumoniae（S.pn）is a major causative agent of community-acquired pneumonia,otitis media,bacteremia,and meningitis.The identification of in vivo inducible genes in S.pneumoniae is of great importance as these genes play a key role in its pathogenesis,and could serve as potential targets for the development of antibiotics and vaccines.In a previous study,we utilized In vivo expression technology（IVET）to identify spd0300 as a novel in vivo inducible gene in S.pneumoniae.Although the specific function of this gene remains unknown,it is likely to be a new virulence factor.Notably,spd0300 is located within an operon involved in hyaluronic acid metabolism.Therefore,this study represents the first analysis of the function of the spd0300 gene,with a focus on exploring its role in hyaluronic acid metabolism.Methods:We conducted the following studies to investigate the expression of spd0300 and its impact on the growth phenotype of Streptococcus pneumoniae:1)Through Q-PCR and WB analysis,we evaluated the expression of the spd0300 gene both in vivo and in vitro;2)Using subcellular fractionation and flow cytometry,we analyzed the subcellular localization of the spd0300 gene product;3)We constructed spd0300 mutant strains and explored its effect on the growth phenotype of S.pneumoniae by observing colony morphology,performing Gram staining,and analyzing growth curves,among other methods.In terms of identifying the function of spd0300 gene products,we conducted the following studies:1)Preliminary bioinformatics analysis of the spd0300 gene product was carried out using BLAST alignment,conserved domain analysis,transmembrane structure prediction,and signal peptide sequence prediction;2)We analyzed whether the spd0300 gene product had relevant protease functional activity using biochemical detection;3)We explored the overall protein expression of S.pneumoniae affected by spd0300 deficiency through quantitative proteomics analysis;4)The mechanism by which spd0300 affects regR expression was investigated by constructing spd0300-related mutant strains and performing Q-PCR,WB,and luciferase reporter assays;5)We conducted a DNA pulldown experiment to enrich the trans-acting factors that could potentially interact with the cis-regulatory elements contained in spd0300;6)We verified the interaction between related transcription factors and the cisregulatory elements contained in spd0300 through EMSA experiments.Results:SPD₀300 is located on a hyaluronic acid metabolism-related operon and can be induced by hyaluronic acid.Under the condition of using only hyaluronic acid as the sole carbon source,we were able to detect the expression of SPD₀300 protein in vitro.Through subcellular fractionation analysis,we discovered that SPD₀300 is an intracellular protein located in the cytoplasm.Although SPD₀300 protein does not exhibit any alginate lyase family-related enzyme activity nor does it have the ability to directly degrade hyaluronic acid,it promotes the degradation of hyaluronic acid by pneumococcal hyaluronidase SPD₀287.The results of quantitative proteomics revealed that the deletion of the spd0300 gene mainly affects the expression of regR.This effect is mainly achieved through cis-regulatory elements such as the second promoter and enhancer located at the 3’ end of the spd0300 gene.Through DNA pull-down and EMSA experiments,we found that the transcription factor ComE could interact with the cis-regulatory elements and regulate the expression of regR,thus affecting the utilization of hyaluronic acid by Streptococcus pneumoniae.