Functional Analysis Of Peptide Deformylase Genes From Staphylococcus Epidermidis

Staphylococcus epidermidis is a kind of opportunistic pathogen that normally colonizes on the surface of human skins. In normal conditions, it does not cause diseases. But with the developments of usage of varies of artificial medical materials such as intravenous-detained tubes and urinary drainage tube, reports of staphylococcus epidermidis infections become more and more. Adding with the abuse of anti-biotics clinically in recent years, multi-resistant staphylococcus epidermidis has been reported and even super-resistant ones. It is urgent to develop new anti-staphylococcus epidermidis drugs and searching for the new drug target is the key step in developing new drugs. The goal of this research is to find new anti-staphylococal drug targets based on the published sequences of Staphylococcus epidermidis ATCC 35984 and 12228. The peptide deformylase is one of the new drug targets. There are two PDF coding genes in both Staphylococcus epidermidis ATCC 35984 and 12228-def and def2, coding SePDF1 and SePDF2, separately.Peptide deformylase is a key enzyme in prokaryotic protein synthesis. In normal prokaryotes, the synthesis of proteins starts with a formyl-methionine-tRNA (f-Met-tRNA). With the elongation of the peptide, the formyl group will be deleted by peptide deformylase. Then the MAP (methionine aminopeptidase) can excise the methionine in the N-terminal of the peptide and the peptide chain can fold correctly. While in eukaryotes, the methionine in the N-terminal which starts the peptide synthesis does not experience the formylation procedure. So the excision of formyl group exists only in prokaryotes. It has been reported that in prokaryotes, the gene encoding peptide deformylase could not be disrupted, which further proves the key role of peptide deformylase in bacterial metabolisms. So the peptide deformylase could be a new target for new anti-bacterial drugs. In order to develop new small leading compounds of anti-staphylococal drugs targeting peptide deformylase, the def and def2 were cloned form Staphylococcus epidermidis strain ATCC 35984 and the recombinant SePDF1 and SePDF2 were expressed, separately. Using FDH-coupled peptide deformylase activity measuring methods, SePDF1 was the active PDF in Staphylococcus epidermidis while SePDF2 was inactive. The small leading compounds targeting SePDF1 were designed and their anti-staphylococal and anti-PDF activities were measured, laying the foundation of new anti-Staphylococcus epidermidis drugs developments.Chapter 1 Function analysis of SePDFlIn order to investigate the function of SePDF1, the def gene, a 552 bp segment in staphylococcus epidermidis ATCC 35984 genome, was linked to the vector pET-22b after PCR amplification in vitro using the genome of Staphylococcus epidermidis 35984 as template. The recombinant vector was transformed into E. coli BL21 (DE3) to express recombinant SePDF1 with a 6×His tag through IPTG induction. Then using the high affinity of 6×His to Ni-NTA column, the recombinant SePDFl was purified from bacterial lysates. According to the instability of Fe2+ in vitro, Co2+, Ni2+ and Zn2+ were applied to substitute the metal cofactor of recombinant SePDFl in this research. Firstly Co2+, Ni2+ and Zn2+ were added directly into the LB medium using to culture E. coli expressing recombinant SePDF1. But the subsequent atom absorption spectrum indicated that the recombinant SePDF1 acquired using this method contains various kinds of metals, not only the one desired. In order to get the single-metal ion contained recombinant SePDF1, we applied the minimal medium of which every content can be manually controlled. By directly adding Co2+, Ni2+ and Zn2+ into the minimal medium followed by normal purifying procedures, a single-metal ion contained recombinant Co-SePDF1 or Zn-SePDF1 with high purity could be obtained. But for Ni-SePDF1, no Ni could be detected by atom absorption spectrum using the same protein purification procedures because of the low affinity of Ni to SePDF1 as we assumed. Applying FDH coupled assay, the activity of SePDF1 with different metal ion was measured using f-MAS (formyl-Methionine-Alanine-Serine) as the substrate. The result showed that Co-SePDF1 5 was the most active while that of Ni- and Zn-SePDF1 was much lower. It indicated the important role of metal ion binding domain in PDFs as the SaPDFB, with highly homogenous amino acid sequences to SePDFl, was most active when the metal cofactor was Ni. By analyzing the crystal structure of SePDFl which we previously obtained to that of SaPDFB, we found that there are no significant differences in the metal ion binding regions. The site-directed mutagenesis further proved the conclusion. By mutating the cysteine, one of the metal ion binding amino acid, the protein completely lost its activity. For the most active Co-SePDF1, the enzymatic parameters were measured. Its Km value was 2.227mM and kcat/Km was 6.3×104M-1*sec-1 using the Lineweaver-Burk methods. FPLC showed that SePDF1 was a monomer in solution. Combining the crystal structure we previously obtained, we use high throughput virtual screening by computer through small molecular leading compound pool and 88 molecules with potential SePDFl inhibition were found. Firstly we screened the small molecules for inhibiting Staphylococcus epidermidis strain 35984 use 128μg/ml as primary screening concentration and found that 6 had inhibiting effects. Further tests showed that two of these small molecules had a high bactericide but no peptide deformylase inhibiting abilities. These small molecules could not be used as potential drugs after the experiment that showed they were poisonous to eukaryotic cells.Chapter 2 The primary investigation of SePDF2 functionThe function of peptide deformylase is based on the formylation of Met-tRNA by formyl Met-tRNA transferase. The formyl Met-tRNA transferase coding gene fmt was located right besides the deft gene in Staphylococcus epidermidis 35984 like most Gram negative bacteria-their peptide deformylase coding gene was also located besides the fmt gene. Through bioinformatic analysis, the deft gene product SePDF2 belongs to Type I PDF family, not the same as SePDFl. The deft gene was constitutively transcripted through the life cycle of Staphylococcus epidermidis ATCC 35984. In order to investigate the function of SePDF2, the deft gene was amplified by PCR and cloned to pET-24+ vector for expressing 6xHis tagged recombinant proteins. After purified by Ni-NTA column, the enzymatic assays showed that SePDF2 had no peptide deformylase activities using the same methods measuring SePDF1. The amino acid sequence comparison indicated that there are several single point mutations in the three regions which was the activity center and highly conserved through all PDFs. Using the active BsPDF1 (Bacillus subtilis) which also belongs to Type I PDF family as template, the SePDF2 was changed by site-directed mutagenesis (A44G, A45V, A46G, I47L, S48A, S90C, I91L, L131Q, M133E) and the mutated SePDF2 was the same in the active center as the active BsPDF1. But it still got no peptide deformylase activity. The deft gene was consititutively transcripted but not translated in living bacteria. The function of def2 gene needs further investigation.