Targeting SAM-I Riboswitch Using Antisense Oligonucleotide Technology for Inhibiting the Growth of Staphylococcus aureus and Listeria monocytogenes

Abstract

With the discovery of antibiotics, a productive period of antibacterial drug innovation and application in healthcare systems and agriculture resulted in saving millions of lives. Unfortunately, the misusage of antibiotics led to the emergence of many resistant pathogenic strains. Some riboswitches have risen as promising targets for developing antibacterial drugs. Here, we describe the design and applications of the chimeric antisense oligonucleotide (ASO) as a novel antibacterial agent. The pVEC-ASO-1 consists of a cell-penetrating oligopeptide known as pVEC attached to an oligonucleotide part with modifications of the first and the second generations. This combination of modifications enables specific mRNA degradation under multiple turnover conditions via RNase H. The pVEC-ASO targets the S-adenosyl methionine (SAM)-I riboswitch found in the genome of many Gram-positive bacteria. The SAM-I riboswitch controls not only the biosynthesis but also the transport of SAM. We have established an antibiotic dosage of 700 nM (4.5 µg/mL) of pVEC-ASO that inhibits 80% of the growth of Staphylococcus aureus and Listeria monocytogenes. The pVEC-ASO-1 does not show any toxicity in the human cell line at MIC80’s concentration. We have proven that the SAM-I riboswitch is a suitable target for antibacterial drug development based on ASO. The approach is rational and easily adapted to other bacterial RNA targets.

Promising strategies employing nucleic acids as antimicrobial drugs

“…Recently, 2′OMe-PS/DNA-2′OMe gapmers were successfully delivered to S. aureus and L. monocytogenes by conjugating them with the CPP pVEC (amino acid sequence LLIILRRRIRKQAHAHSK, where L is leucine, I is isoleucine, R is arginine, K is lysine, Q is glutamine, A is alanine, H is histidine, and S is serine), inhibiting 80% of bacterial growth (MIC80) at 0.7 μM (4.5 μg/mL) of pVEC-ASO. 122 , 123 The conjugates showed good biocompatibility up to 1 μM tested in A549 human cells. Interestingly, the pVEC-ASO were designed to target two distinct riboswitches (TPP and S -adenosyl methionine I [SAM-I]) located in the 5′ UTR of mRNAs in Gram-positive bacteria.…”

“… 116 THI-box present in several operons TPP riboswitch L. monocytogenes Traykovska et al. 122 S-box operon SAM-I riboswitch L. monocytogenes ; S. aureus Traykovska and Penchovsky 123 MotA Virulence Cytoplasmic membrane protein; bacterial adhesion and biofilm formation P. aeruginosa Hu et al. 13 efaA Enterococcus faecalis antigen A; bacterial adhesion and biofilm formation E. faecalis Narenji et al.…”

“… 122 pVEC-2′OMe-PS/DNA-2′OMe gapmer MIC 80 : 0.7 μM N.A. L. monocytogenes ; S. aureus Traykovska and Penchovsky 123 2′OMe encapsulated in DPP decorated with PEG2000-DSPE Growth inhibition: 1 μM Sepsis mice model: 1.5 mg/kg (225 nmol/kg ASOs) ESBLs- E. coli Chen et al. 124 2′OMe/PGO Growth inhibition (54%): 20 μM N.A.…”

Antisense and Functional Nucleic Acids in Rational Drug Development

“…The design of therapeutic molecules, such as ASOs, which can bind in vivo to the aptamer domain, might be the answer to successfully creating entirely new classes of antibiotics [ 64 ]. Recent studies prove the inhibition effect of chimeric ASOs on the bacterial growth of S. aureus targeting SAM-I riboswitch [ 95 ]. The combined application of the first two ASOs, which target the glucosamine-6-phosphate ( glmS ) riboswitch and the nagA mRNA, block the synthesis of glucosamine-6-phosphate entirely and inhibits the bacterial growth of S. aureus [ 3 ].…”

Semaphorin 3C enhances putative cancer stemness and accelerates peritoneal dissemination in pancreatic cancer

“…In our preclinical study, SEMA3C knockdown enhanced the e cacy of GnP in reducing tumor growth and peritoneal carcinomatosis. In clinical applications, antisense oligonucleotides (ASOs), which in uence RNA processing and modulate protein expression, could potentially inhibit SEMA3C [22,23]. SEMA3C…”