Silencing acpp and MEXA genes which take part in lipid metabolism and antibiotic resistance in pseudomonas aeruginosa isolates BY antisense oligonucleotids

The antisense treatment strategy is to use oligodeoxyribonucleotides for the purpose of silencing various genes before they start producing proteins. The antisense mechanism is established by binding oligonucleotide strands to the target mRNA by way of Watsons-Crick base pairing. After binding, two basic pathways prevent the relevant gene from producing protein. The first of these is the formation of RNase H-mediated degradation by the oligonucleotide at the site of binding. Production of protein is hindered because translation becomes impossible from the gene strand that was broken down by the RNase enzyme. The second occurs when the oligonucleotide binds and closes the relevant gene site. In this way, ribosome is sterically hindered from reading the gene strand, posttranscriptional procedures are hindered and the gene cannot make production. The objective of this project is to use appropriate antisense strands and formulation against Pseudomonas aeruginosa strains to silence the Acyl carrier protein (acpP) gene, which is essential for the bacterium, and the mexA gene of the mex group efflux pump, which increases the resistance of bacterium to antimicrobials. Since the lack of acpP, an essential protein for the lipid biosynthesis of the bacterium, will result in the death of the bacterium, proliferation of the bacterium will be prevented and its resistance will be eliminated through the inhibition of the pump gene. It is observed in the literature that methods such as liposome and peptide conjugation have been tried to enable these agents enter P. aeruginosa cells, but we have not encountered any use of niosome formulations. Use of niosomes for this purpose will be unique to our project. Studies exploring the effects of this method, which can be considered as a relatively new treatment approach, have not been encountered in our country. In this project, silencing the acpP and mexA genes was made in P. aeruginosa by using antisense oligonucleotides and in this way inhibiting bacterial growth and resistance mechanism. In this study, antibiotic resistance patterns were determined by VITEK MS in P. aeruginosa strains isolated from Ege University Hospital, Medical Microbiology Laboratory and the presence of the targeted genes were revealed through the polymerase chain reaction (PCR) method. In these methods, P. aeruginosa ATCC 27853 and positive controls which harbouring relevant genes were used. The strains that meet the above criteria were genotyped using PCR based Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). Ten P. aeruginosa strains that were found to belong to different genotypes were used. DNA sequence of targeted genes were determined by DNA sequencing for designing the antisense oligonucleotides. The results of this sequence analysis were assessed in line with gene banks and literature information and the oligonucleotides specific to P. aeruginosa bacterium were identified. Niosome formulations were prepared to enhance penetration of antisense oligonucleotides into bacterial cells. The Minimum Inhibitor Concentration (MIC) values of the formulations were determined using the liquid microdilution method in line with the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The expression levels of the silenced acpP and mexA genes were explored with real-time quantitative RT-PCR (qRT-PCR). The effectiveness of the efflux pump was investigated with the accumulation test and the MIC values were determined again in the presence of the antisense formulation to see the change in resistance level. Based on the results of VITEK MS, 41 MDR isolates from different clinics were selected for the first stages of study. 38 of these isolates were found to contain the acpP and mexA genes, and these isolates were found to belong to 11 different families, 10 major clones and one subtype according to ERIC-PCR results. Sequence analyzes were performed for the selected members from each family, and no mutation was found in the acpP gene. However, silent mutations were detected in the mexA gene. Nine niozom formulations were prepared to target the designed ASO sequences. Only F1 and F5 formulations were found to be complexed with ASO sequences. It was, however, found that the combined F5 formulation had a higher concentration of ASO sequence carrying capacity. Thus F5 formulation was used in the next stages of study. Significant gene inhibition was detected at the six, four and three isolates for ASO-1 ASO-2 and ASO-6, respectively, according to the RT-PCR results of these complexed sequences. No activity was seen in other series Keywords: Antisense, Pseudomonas aerguinosa, qPCR