الفهرس 
INTRODUCTION
Listeria monocytogenesOnly 14 pages are availabe for public view
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Abstract
There is a pressing need for novel and innovative therapeutic strategies to combat infections caused by intracellular zoonotic pathogens such as Listeria monocytogenes thereby, limiting the spread of these pathogens between susceptible hosts. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers.
The ongoing work is focused on targeting of L monocytogenes rpoA gene which encodes alpha subunit of RNA polymerase; the key enzyme of gene expression. We first sequenced the rpoA gene in six different Listeria (4 human strains and 2 animal strains) and the blasting showed that they are nearly identical with each other as well as with the Listeria sequence data bases located at NCBI. Based on the obtained sequences we chose to target specific area (with 100% identity across Listeria species) including the start codon (ATG) and the upstream ribosomal binding site (RBS) with 12 sequence specific PNA that was covalently conjugated to five different CPPs.
We utilized three different models for testing the efficacy of the designed PNAs against L monocytogenes i.e. pure culture, infected macrophage cells and infected animal model (Caenorhabditis elegans (C. elegans)).
In general, neither the control PNA nor the free PNA had antilisterial effect up to 32 µM indicating PNA’s specificity for conveying its action as well as the impact of conjugating of the PNA with a cell penetrating peptide, respectively. Furthermore, changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against L. monocytogenes in the three test models.
At the pure culture, PRXR exhibited the most potent antisense effect and caused a significant inhibition in the bacterial growth as low as 1 µM and produced a 100% bacterial reduction at 4 µM and higher. The same PNA did not cause any antimicrobial effect when used against pure culture of MRSA up to 16 µM which further confirms the PNA’s specificity. The second most performing PNAs were PTAT and PRFR which had MICs of 1 and 2 µM and caused complete bacterial eradication at 4 µM and 8 µM, respectively. PKFF and PANT were the least effect PNAs used in this study with MICs of 16 µM and 32 µM, respectively. Additionally, a time-kill assay revealed three conjugated CPPs (PRXR, PTAT and PRFR) rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane.
The relative quantification using RT-q real time PCR confirmed that the anti-rpoA PNAs are working via specific inhibition of rpoA gene expression. Moreover, rpoA gene silencing resulted in inhibition of expression of important virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner indicating the potent indirect ability of the PNAs in inhibition of the production of the harmful toxins. Furthermore, the PNA’s safety profile was addressed and the PNA-inhibition of bacterial protein synthesis was found to be selective and it did not adversely affect mitochondrial protein synthesis.
The present study showed that the conjugated PNAs can retain their antisense effect against the intracellular Listeria harboring the macrophages and 8 µM PRXR was able to completely eradicate the intracellular L. monocytogenes after 4 hours treatment of the infected macrophage cells.
This study demonstrated that the mutant C. elegans strain AU37 as an appropriate and simple animal model for studying the pathogenicity of the intracellular Listeria and the killing kinetics was found to be strain specific.
However, all the used PNAs either at 16 µM or 32 µM led to a significant inhibition of the bacterial load inside the infected worms, higher concentration of the most potent PNA (PRXR) was required to attain complete bacterial eradication.
Finally, this study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.