Treatment ofStreptococcus mutanswith antisense oligodeoxyribonucleotides togtfBmRNA inhibits GtfB expression and function

Abstract

We examined the effects of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODN) targeted to mRNA transcribed from gtfB, which encodes synthesis of water-insoluble glucans in Streptococcus mutans. Treatment of S. mutans with 10 muM antisense PS-ODNs inhibited gtfB mRNA transcription, GtfB expression and water-insoluble glucan synthesis. The architecture of biofilms formed by antisense PS-ODNs-treated S. mutans showed reduced biomass, more dispersed distribution with enlarged interspaces and fewer layers of attached cells. PS-ODN treatment had no effect on the growth of S. mutans. Our results indicated that it might be feasible to use antisense PS-ODN as a novel agent in caries prevention.

A New Small Molecule Specifically Inhibits the Cariogenic Bacterium Streptococcus mutans in Multispecies Biofilms

“…Our studies revealed that 2A4 altered expression of six biofilm-associated genes in planktonic cells of S. mutans. These genes included the adherence-associated gene pac (24), glucosyltransferase gene gtfB (10,42), glucan binding protein-encoding gene gbpB (36), acid-producing gene ftf (16,38), relA, and regulatory gene comDE. They have been linked to different steps of biofilm formation.…”

Streptococcus mutans biofilm inhibition using antisense oligonucleotide to glucosyltransferases B and C

“…On the basis of results reported by Guo et al (7), we selected 10 μM as the final concentration of PS-ODNs for treatment of streptococci, and also applied the similar transfection reagent (TurboFect™) consisting of a cationic polymer in order to facilitate penetration of the PS-ODNs to the bacterial cells. The cationic polymer forms complexes with PS-ODNs via electrostatic interaction between negatively charged oligonucleotide molecules and positively charged reagent molecules (7,15). Since such complexes maintain the positive charge, therefore they can bind to the negatively charged bacterial cell wall, and then can be taken up by the cells.…”

“…The currently available antisense technology provides the novel approach that can be applied for blocking the production of glucan polymers in S. mutans bacteria (7). It is based on the use of chemically modified single-stranded nucleic acid molecules (usually 20 nt in length), which can selectively hybridize to their target complementary mRNA through Watson-Crick base pairing, consequently leading to degradation of the bound mRNA by RNase H endonuclease, steric hindrance of ribosomal activity, inhibition of mRNA splicing or destabilization of pre-mRNA (8,9).…”

“…A large number of studies have demonstrated the efficacy of antisense oligonucleotides for silencing of various genes in bacteria, including S. mutans (10)(11)(12). Importantly, Guo et al (7) showed that phosphorothioate oligodeoxyribonucleotides (PS-ODNs) designed to antisense gtfB mRNA in S. mutans reduces the expression and activity of GtfB enzyme as well as water-insoluble glucan production and biofilm formation. The PS-ODNs represent DNA molecules containing a phosphorothioate-modified backbone, where one of the non-bridging oxygen atoms in the phosphodiester linkage is replaced by a sulphur atom in order to increase the resistance to endo-and exonucleases (13).…”

Antisense Antibacterials: From Proof-Of-Concept to Therapeutic Perspectives

“…Validated essential genes among different bacterial species include fbpA/ fbpB/fbpC (Harth & et al, 2002(Harth & et al, , 2007 and glnA1 (Harth & et al, 2000) in Mycobacterium tuberculosis, gyrA/ompA in Klebsiella pneumonia (Kurupati & et al, 2007), inhA in Mycobacterium smegmatis (Kulyte & et al, 2005), oxyR/ahpC in Mycobacterium avium (Shimizu & et al, 2003), NPT/EhErd2 in Entamoeba histolytica (Stock & et al, 2000(Stock & et al, , 2001, gtfB in Streptococcus mutans (Guo & et al, 2006), fmhB/ gyrA/hmrB (Nekhotiaeva & et al, 2004a) and fabI (Ji & et al, 2004) in Staphylococcus aureus, 23S rRNA (Xue-Wen & et al, 2007), 16S rRNA plus lacZ/bla (Good & Nielsen, 1998), and RNAse P (Gruegelsiepe & et al, 2006) in Escherichia coli, and acpP in Burkholderia cepacia (Greenberg & et al, 2010), Escherichia coli (Deere & et al, 2005b;Geller & et al, 2003aGeller & et al, , 2003bGeller & et al, , 2005Mellbye & et al, 2009Mellbye & et al, , 2010Tan & et al, 2005;Tilley & et al, 2007) as well as Salmonella enterica serovar Typhimurium (Mitev & et al, 2009;Tilley & et al, 2006). Mellbye et al, First proof-of-principle evidence was given by White et al in 1997 for successful increasing the bactericidal activity of norfloxacin by antisense inhibiting the marRAB operon in Escherichia coli (White & et al, 1997).…”