Abstract
Infectious disease accounts for the highest percentage of preventable deaths worldwide and today’s health care systems are predominately reliant upon antibiotics to treat bacterial infections bacterial strains. Serious 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. Antisense oligonucleotides (AS-ODN) target genes in a sequence specific manner and inhibit gene function. However, barriers such as peptidoglycan, cell surface proteins such as teichoic acids and lipopolysaccharide membranes are thought to currently prevent the use of AS-ODN from becoming an effective treatment option for microbial disease as effective antisense inhibition in bacteria requires the delivery of the antisense agent across these bacterial cell barriers. Other reasons such as antisense size, charge and hydrophobicity and the Gram classification of the bacterial strain are also implicated in contributing to uptake difficulty.
Streptococcus mutans is frequently implicated as the primary etiological agent in the development of dental caries - Severe dental disease can lead to a number of serious health problems, including cardiac disease and septicaemia. In a previous study done in 2008 the ability of the combined use of zoocin A, a bacteriolytic enzyme, and two targeted PS-ODN sequences (targeted towards fab-M and fba) to produce a synergistic inhibitory effect upon closely related streptococcal species was examined and showed that a combination of zoocin A and PS-ODN could be used to achieve a dose-dependent inhibitory response upon bacteria that were A) susceptible to zoocin A, and B) contained the PS-ODN target site, and it was concluded that the zoocin A was indeed causing damage to the susceptible bacterial cell walls and thus allowing the PS-ODN entrance to the bacterial cell interior. However the large size of zoocin A precludes its possible use in clinical settings.
The current study examines a further variety of lytic antimicrobial agents for their ability to deliver PS-ODN into S. mutans OMZ175 and produce a synergistic inhibitory effect upon growth, viability and target mRNA production. The overall hypothesis of this work was that the combined use of antisense and clinically relevant lytic agents would cause a target specific decrease in bacterial growth. This hypothesis was examined by three different experimental approaches that aimed to examine A) The down regulation of target mRNA, B) The measurement of intracellular PS-ODN and C) The effects of different lytic agents and PS-ODN’s on growth rates.
A RNA extraction and RT-qPCR method was developed to analyse gene expression levels in S. mutans OMZ175, including that of the fba target. Whilst a protoplasting protocol was unable to be successfully developed (in order to analyse membrane permeability), radiolabelled γ32 P-AP-ODN was used to determine which antimicrobial delivery mechanism allowed the greatest amount of PS-ODN delivery into S. mutans OMZ175 in different growth stages. Results indicated that of all delivery mechanisms tested only zoocin A and penicillin showed positive synergism together with targeted PS-ODN. A significant decrease in fba expression was shown for the first four hours post zoocin A + targeted PS-ODN treatment for both lag and exponential phase S. mutans OMZ175, which corresponded with the observation that γ 32P ATS2 molecules gained entry into lag and exponential phase S. mutans cells over the first 4 hours of combined γ 32P PS-ODN + zoocin A treatment. Penicillin was shown to be able to facilitate the entry and synergistic inhibition of fba in lag phase S. mutans OMZ175 only, resulting in prolonged suppression upon fba expression, corresponding with a greater amount of γ32 P-PS-ODN becoming cell associated compared with that observed for those cells treated with zoocin A. A comparison between zoocin A and penicillin highlights the fact that the different modes of action of each agent result in different amounts of PS-ODN cell association over time. This study suggests that the choice of delivery agent may influence the time point at which PS-ODN intracellular concentration will reach a critical threshold and allow gene suppression to occur and that different modes of action of delivery agents affect the rate at which the PS-ODN is delivered into the bacterial cell. This study also suggests that for S. mutans OMZ175, the peptidoglycan layer acts an effective barrier preventing PS-ODN penetration, but that this may vary between bacterial strains due to the variety of bacterial cell components capable of influencing AS-ODN, PNA or PMO penetration.