Evaluation of the metS and murB Loci for Antibiotic Discovery Using Targeted Antisense RNA Expression Analysis in Bacillus anthracis

Abstract

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-Nacetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter. Numerous such gene fragments that were overwhelmingly in the antisense orientation were identified for the metS1 and murB2 alleles, while no such orientation bias was seen for the metS2 and murB1 alleles. Gene replacement mutagenesis was used to confirm the essentiality of the metS1 and murB2 alleles, and the nonessentiality of the metS2 and murB1 alleles, for vegetative growth. Induced transcription of RNA from metS1 and murB2 antisense-oriented gene fragments resulted in specific reduction of mRNA of their cognate genes. Attenuation of MetS1 enzyme expression hypersensitized B. anthracis cells to a MetS-specific antimicrobial compound but not to other antibiotics that affect cell wall assembly, fatty acid biosynthesis, protein translation, or DNA replication. Antisense-dependent reduction of MurB2 enzyme expression caused hypersensitivity to beta-lactam antibiotics, a synergistic response that has also been noted for the MurA-specific antibiotic fosfomycin. These experiments form the basis of mode-ofaction detection assays that can be used in the discovery of novel MetS-or MurB-specific antibiotic drugs that are effective against B. anthracis or other gram-positive bacterial pathogens.

Physiological impact of transposable elements encoding DDE transposases in the environmental adaptation of Streptococcus agalactiae

“…Furthermore, IS1548 is oriented toward the murB gene, which might lead to an increased transcription of murB due to the presence of a putative promoter located at the 39 end of the IS element, as previously described for the IS1548 insertion site upstream of lmb (Al Safadi et al, 2010). In Bacillus anthracis, the downregulation of a murB homologue using targeted antisense RNA is responsible for hypersensitivity of the strain to b-lactam antibiotics (Kedar et al, 2007). This suggests that the potential upregulation of murB by IS1548 could cause a decrease in S. agalactiae susceptibility to b-lactams, although this class of antibiotics is currently still efficient in first-line chemoprophylaxis.…”

Regulation of mprF by Antisense RNA Restores Daptomycin Susceptibility to Daptomycin-Resistant Isolates of Staphylococcus aureus

“…Targeted antisense RNA technology is useful for identifying and regulating genes involved in antimicrobial resistance phenotypes (10,15,35). The purpose of this study was to demonstrate that modulation of mprF expression using inducible antisense RNA can restore DAP susceptibility in DAP r S. aureus strains.…”

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

“…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). Hitherto, limited but successful trials have extended to dominating resistant genes and bacterial species with highest incidence of resistance (Table 2), e.g., antisense targeting aac(6')-Ib (Sarno & et al, 2003;Soler Bistue & et al, 2007), act (Chen & et al, 1997;Gao & et al, 2005) in Escherichia coli, vanA in Enterococcus faecalis (Torres & et al, 2001), cmeA in Campylobacter jejun (Jeon & Zhang, 2009), mecA in Staphylococcus aureus (Meng & et al, 2006;Meng & et al, 2009), metS/murB in Bacillus anthracis (Kedar & et al, 2007) and oprM in Pseudomonas aeruginossa (Wang & et al, 2010).…”