| Streptococcus pneumoniae is Gram-positive pathogen that causes sinusitis, otitis media, pneumonia, sepsis, meningitis and other serious diseases. In the past, antibiotics are the primary therapeutic drugs for streptococcal infection. However, as the wide use of antibiotics, drug-resistant bacterial strains spread in the world, bringing a great deal of problems in the clinical treatment. Understanding the drug mechanism can provide useful information for the development of new antibacterial drugs and vaccines.Aim:To analyze the proteomic alterations of S. pneumoniae between control and the bacteria treated with vancomycin or ruthenium complex X-03 using 2-DE-based proteomics, and to investigate the differentially expressed proteins using follow-up bioinformatics tools and functional studies, aiming to understand the molecular action mechanism of vancomycin and X-03 against S. pneumoniae.Methods:2-DE was used to separate the total proteins and membrane proteins extracted from control and drug-trented bacteria. ImageMaster software was used to analyze the 2-DE images and identify differential expression proteins (DEPs). The DEPs were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and the peptide mass fingerprints (PMF) of these protein spots were obtained. Then the peptide mass spectra were matched against the NCBI database using the MASCOT search engine. Proteins over 95% confidence are considered acceptable. Bioinformatics tool was used to categorize the DEPs. Real-time PCR analysis was used to determine the differential expressions of the partial proteins in RNA level.Results:(1) A 2-DE map for the membrane proteins of S. pneumoniae was established. Around 60%(164 proteins) of the extracted proteins were identified to be membrane proteins with 2-DE coupled with MALDI-MS/MS and 2D-LC-ESI-MS/MS. (2) In the vancomycin treated bacteria, thirty-three DEPs were identified, including 29 up-regulated and 4 down-regulated proteins. The real-time PCR analysis showed that the changes in the mRNA level of genes galU,hprK,recA and spxB have the same tendency with their protein expression levels after vancomycin treatment. (3) In the X-03-treated bacteria,32 proteins were found to be down-regulated and 34 proteins were up-regulated. Bioinformatics analysis revealed that these altered proteins are mainly involved in protein translation, tRNA-aminoacylation, cell cycle regulation, fatty acid metabolism, oxidation, DNA replication or transcription, amino acid metabolism, carbohydrate and nucleic acid metabolism. With the real-time PCR analysis, the changes in the gene level of eno,accA and grpE had the similar pattern with their protein level, whereas the mRNA expression of secA was not consistent with its protein expression.Conclusions:(1) 2-DE proteomic maps of vancomycin-treated and-untreated S. pnemunoniae have been established. Proteins involved in various biological processes including translation, nucleotide and carbohydrate metabolism, cell cycle and regulation have been found to have expression alterations in response to vancomycin challenge. Further characterization of these protein alterations may provide useful information to help us understand the inhibitory mechanism of vancomycin against S. pnemunoniae. (2) Ruthenium complex X-03 could inhibit the growth of S. pneumoniae, with low toxicity to host cells. By comparing the protein expression differences between the X-03-treated and control bacteria, it can be suggested that X-03 may inhibit bacterial fatty acid synthesis, DNA replication, transcription and other biological processes, thereby retarding bacterial growth.
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