Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens

Abstract

There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. 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. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.

Figure 1. Time-kill analysis of PNAs against L. monocytogenes F4244.

(A) 32 μM of the five PNAs at 37 °C were incubated for 12 hours and samples were collected every two hours. (B) 8 μM of PTAT, PRXR, and PRFR at 37 °C were incubated for 2 hours and samples were collected every 20 minutes. The results are presented as means ± SD from two independent experiments (n = 3). (C) Permeabilization of the cytoplasmic membrane of L. monocytogenes F4244 indicated by percent of calcein leakage for 30 min exposure to PRXR and nisin. Samples were collected every 5 minutes. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).

Figure 2. Concentration-dependent reduction of rpoA, hly, plcA and plcB expression after treatment with PTAT.

Bacterial cultures were treated with 1, 2 and 4 μM PTAT for 3.5 hours at 37 °C. Total RNA was extracted from the treated and untreated cultures. The levels of mRNA were determined by RT-PCR. (A) The level of rpoA expression. (B) The level of hly expression. (C) The level of plcA expression and (D) the level of plcB expression. P value of (^*P ≤ 0.05) is considered as significant.

Figure 3. Effect of PNA, linezolid and ampicillin on mitobiogenesis.

In cell- ELISA was carried out in the presence and absence of these drugs, and the levels of mitochondrial (mt)-DNA encoded protein (COX-I) and nuclear-DNA encoded protein (SDH-A) in J774A.1 were quantified. Ratio of COX-I and SDH-A was calculated and the results were shown as percent inhibition of mitochondrial biogenesis.

Figure 4. Killing of C. elegans by different strains of L. monocytogenes.

Worms were grown on lawns of bacteria for 8 hours before washing and transferring to microtitre plate. (A) Worms fed E. coli OP50 or L. monocytogenes J016. (B) Worms fed E. coli OP50 or L. monocytogenes 19111. (C) Worms fed E. coli OP50 or L. monocytogenes F4244.

Figure 5

Evaluation of antimicrobial efficacy of PNAs in C. elegans model at 16 μM (A) and 32 μM (B). L. monocytogenes J0161 infected L4-stage worms were treated with PNAs and antibiotic for 18 h. Worms were lysed and the CFUs were counted and the percent bacterial reduction per worm in treated groups were calculated in relative to the untreated control groups. 10 worms in triplicates were used for each treatment. The results are presented as means ± SD from two independent experiments. Data without error bars indicate that the SD is too small to be seen. P values of (*≤ 0.05) are considered as significant.