Infection caused by antibiotic-resistant bacteria has become a major global healthcare issue, and there is a rapidly growing need for the development of new antimicrobials. Antibiotic development based on incremental change that produces new iterations of old chemical structures is unlikely to yield innovative and effective new antimicrobials. A transformational change is urgently required, and there is increasing need for antimicrobials with a novel method of action (Overbye & Barrett, 2005).
One technology currently under investigation is the use of antisense oligonucleotides (AS-ODN) to target genes in a sequence-specific manner and inhibit gene function. Antisense sequences can be designed to bind complementary RNA gene sequences through Watson–Crick pairing to block translational initiation or elongation (Bennett & Swayze, 2010). It is thought the presence of an AS-ODN molecule sterically blocks the ability of the ribosome to bind to the mRNA, preventing translation (Good, 2003). Degradation of the targeted RNA gene may involve the recruitment and activation of RNase-H (Rassmussen et al., 2007) or RNase-P (Shen et al., 2009). Numerous clinical trials using AS-ODN technology are being conducted for the treatment of diseases, such as cancer (Iversen et al., 2003), HIV/AIDS and other viral infections (Tripathi et al., 2005) and various autoimmune disorders (Ricotta & Frishman, 2012), with many showing promising therapeutic potential. By contrast, the use of AS-ODN's to treat bacterial infections is less advanced.
It might be expected that microbial cell surface structures, such as lipopolysaccharide membranes, lipoteichoic acids, teichoic acids and peptidoglycan, would present formidable barriers to entry of AS-ODN's to the bacterial cell cytoplasm, but little evidence has been reported regarding their effect. AS-ODN's conjugated to membrane-penetrating peptides show improved bacterial penetration (Tilley et al., 2006) and coupling of a positively charged peptide (KFFKFFKFFK) to a peptide nucleic acid (PNA) construct greatly increased penetration of the PNA into Escherichia coli (Good et al., 2001). These delivery peptides probably enhance cell penetration by localization of the PNA to the negatively charged lipopolysaccharide of the outer membrane. The coupling of AS-ODN's to membrane-penetrating peptides (RFFRFFRFFXB) has been shown to increase their permeability into E. coli (Mellbye et al., 2010), S. enterica (Mitev et al., 2009), Klebsiella pneumoniae (Kurupati et al., 2007) and Burkholderia multivorans (Greenberg et al., 2010). Encapsulation of AS-ODN in liposomes has also opened up new possibilities for cell delivery. Liposomes may fuse with bacterial cell walls improving AS-ODN penetration through the bacterial cell wall, as well as facilitating the penetration of AS-ODN through the cytoplasmic membrane (Meng et al., 2009). Injection of anionic liposome encased PS-ODN targeted towards mec-A, into the tail vein of mice infected with methicillin-resistant Staphylococcus aureus (MRSA), has been shown to restore the MRSA to oxacillin susceptibility and to rescue these animals from lethal sepsis (Meng et al., 2009). Zoocin A, a peptidoglycan hydrolase, can facilitate the uptake of PS-ODN by S. mutans OMZ175 (Dufour et al., 2011), suggesting that at least in Gram-positive bacteria, the peptidoglycan layer is a major barrier to PS-ODN entry. Penicillin is a β-lactam antibiotic known for its ability to inhibit the synthesis of the peptidoglycan layer of bacterial cells leading to a loss of structural integrity (Tomasz, 1979). It may also stimulate autolysis of target bacteria by the deregulation of genes encoding autolytic enzymes (Penyige et al., 2002). Therefore, the use of such cell wall targeting antibiotics presents a possible method of inducing bacterial cell permeabilization that has already been extensively studied and is already approved for in vivo use (Moellering & Weinberg, 1971; Chung et al., 2009).