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.

Keywords: antibacterial agents; antibacterial drug discovery; antibacterial resistance; cell-penetrating peptides; design of antisense oligonucleotides; drug targets; gram-positive bacteria; riboswitch.

Figure 1

A chimeric antisense oligonucleotide pVEC-ASO-1 targets the SAM-I aptamer domain of the S-box polycistronic mRNA. (A) The chimeric antisense oligonucleotide binds to the complementary sequence of the SAM-I aptamer domain. (B) A double-stranded molecule is formed after binding the chimeric antisense oligonucleotide with the SAM-I aptamer sequence. (C) The recognized double-stranded molecule by the RNase H is enzymatically hydrolyzed. (D) The enzymatic hydrolysis of RNA leads to no gene expression of the S-box operon.

Figure 2

(A) Alignment of the sequences in pathogenic bacteria containing the SAM-I riboswitch sequence. The pathogenic bacteria S. aureus, S. sapropyticus, S. epidermidis, L. monocytogenes, C. tetani, C. perfingens, C. difficile, B. anthracis, and the non-pathogenic bacteria B. subtilis contains the SAM-I riboswitch sequence. The same color has the same nucleotide. (B) Gel electrophoresis of the SAM-I aptamer RNA from S. aureus. Total RNA is isolated and converted into cDNA. Two primers were used to amplify a SAM-I aptamer region targeted by the pVEC-AS0-1 (1) Determining the size of the DNA by 1000 bp. (2) The amplified cDNA from cells without treatment with pVEC-ASO-1. (3) The amplified cDNA treatment with pVEC-ASO-1.

Figure 3

Inhibition of bacterial growth by antisense oligonucleotide that targets SAM-I riboswitch mRNA. (A) pVEC-ASO-1, 2000 nM, binds with the mRNA for the SAM-I riboswitch and inhibits the bacterial growth of L. monocytogenes (the line with the upside-down triangles) and S. aureus (the line with the circles). In comparison, without pVEC-ASO-1, there is no inhibition of the bacterial growth of L. monocytogenes (the line with the triangles) and S. aureus (the line with the rectangles). (B) pVEC-ASO-1, 1000 nM, binds with the mRNA for the SAM-I riboswitch and inhibits the bacterial growth of L. monocytogenes (the line with the upside-down triangles) and S. aureus (the line with the circles). In comparison, without it, there is no inhibition of the bacterial growth of L. monocytogenes (the line with the triangles) and S. aureus (the line with the rectangles). (C) pVEC-ASO-1, 700 nM, also inhibits the bacterial growth of L. monocytogenes (the line with the upside-down triangles) and S. aureus (the line with the circles). Without pVEC-ASO-1, there is no inhibition of the bacterial growth of L. monocytogenes (the line with the triangles) and S. aureus (the line with the rectangles). (D) At a concentration of 350 nM of pVEC-ASO-1, there is almost no difference in the bacterial growth of L. monocytogenes (the line with the upside-down triangles) and S. aureus (the line with the circles) as in the presence as well in the absence of ASO-1 (the line with the triangles for L. monocytogenes; the line with the rectangles for S. aureus). (E) At a concentration of 150 nM of pVEC-ASO-1, no difference is observed in the bacterial growth of L. monocytogenes (the line with the upside-down triangles) and S. aureus (the line with the circles) with and without pVEC-ASO-1 (the line with the triangles for L. monocytogenes; the line with the rectangles for S. aureus). (F) At a concentration of 2000 nM of pVEC-ASO-1, it binds with the mRNA for the SAM-I riboswitch and inhibits the bacterial growth of S. aureus (the line with the circles). Without pVEC-ASO-1, there is no inhibition of the bacterial growth of S. aureus (the line with the rectangles). Compared to E. coli, there is no difference in its bacterial growth in the presence of 2000 nM pVEC-ASO-1 (the line with the upside-down triangles) as in the absence of pVEC-ASO-1(the line with the triangles).

Figure 4

Probing the pVEC-ASO’s toxicity level in a lung cancer human cell line. In a concentration of 2000 nM of pVEC-ASO-1, the survival of the cell line is 61%. In the next concentration of 1000 nM, the survival of the cell line is 94%. At a concentration of 700 nM, the survival of the cell line is 99%. In the absence of pVEC-ASO-1, the survival of the cell line is 100%.

Figure 5

Testing the antibacterial activity of pVEC-ASO-2 with five mismatches compared with pVEC-ASO-1. There was no inhibition of the growth of S. aureus even at 2000 nM.