Targeting Listeria Monocytogenes rpoA and rpoD Genes Using Peptide Nucleic Acids

Abstract

Treating intracellular pathogens remains a considerable medical challenge because of the inefficient intracellular delivery of antimicrobials and the frequent emergence of bacterial resistance to therapeutic agents deemed the drugs of last resort. We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF) 3 K cell penetrating peptide to target RNA polymerase a subunit (rpoA) and RNA polymerase sigma 70 (rpoD) in the intracellular pathogen Listeria monocytogenes. The PNAs tested displayed a concentration dependent inhibition of L. monocytogenes growth in pure culture at the micromolar level and significantly reduced intracellular L. monocytogenes in infected cell culture and Caenorhabditis elegans whole animal model. In vitro, the combined PNAs treatment was synergistic resulting in a clearance of L. monocytogenes at 0.5 · the individual PNA concentration. This study demonstrates the potential of anti-rpoA PNA as an antibacterial agent and will provide the basis for improving and developing these PNAs to better target intracellular pathogens like Listeria. This study also establishes C. elegans as a potential model for the screening of PNAs.

Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells

“…C. elegans AU37 (sek-1; glp-4) strain (glp-4(bn2) was used to investigate the antifungal efficacy of ebselen, as described elsewhere [20,38–41]. Briefly, L4-stage worms were infected either with Cryptococcus neoformans NR-41292 or Candida albicans ATCC 10232 for three hours at room temperature.…”

“…After infection, worms were washed with M9 buffer and treated for 24 hours either with DMSO or drugs (ebselen, amphotericin B, fluconazole, and flucytosine), at indicated concentrations. Post-treatment, worms were washed, disrupted using silicon carbide particles [20,39–41], and the resulting suspensions were serially diluted and transferred to YPD agar plates containing ampicillin (100 μg/ml), streptomycin (100 μg/ml) and kanamycin (45 μg/ml). Plates were incubated for 24–48 hours at 35°C before the colony forming unit (CFU) per worm was determined.…”

A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii

“…In order to assess the peptides’ ability to kill intracellular bacteria, murine macrophages (J774A.1) were infected, as described in previous studies 31 – 33 , 42 , 48 – 51 . Briefly, J774A.1 cells were seeded and incubated as described above for the toxicity assessment.…”

“…Infection and treatment of C. elegans was done, as described previously, with the following modifications 51 , 53 , 54 . Briefly, synchronized adult worms were transferred to modified NGM (0.35% peptone) agar plates seeded with a lawn of P. aeruginosa PAO1, colistin-resistant P. aeruginosa PAO1, or A. baumannii ATCC BAA-1605, for infection.…”

Chemical synthetic biology: a mini-review

“…Second, as for SB, it might have a tremendous impact on biotechnology, generating an entire set of tools for nanomedicine, diagnostic, drug/gene delivery, bioengineering, biosensoring, etc., based on artificial molecules. Examples of CSB approaches span from searching for nucleic acid alternatives, such as furanose-based DNA (Bolli et al, 1997), peptide-nucleic acids (PNAs; Egholm et al, 1993) and their conjugates (Alajlouni and Seleem, 2013), to the novel work on synthetic genetic polymers (XNA) capable of heredity and evolution (Pinheiro et al, 2012), and to synthetic genetic codes (Wong and Xue, 2011); and, on the other hand, to proteins composed by random amino acid sequences (Chiarabelli et al, 2006a, b), or from a subset of amino acids (Doi et al, 2005), or other unnatural building blocks, or by combinatorial approaches (Urvoas et al, 2012). In addition to these examples of CSB of “parts,” there is a flourishing research on CSB of “systems,” and in particular that one focused on the construction of synthetic cells (Luisi et al, 2006b), whose achievement represents the most ambitious goal of CSB.…”