Gymnotic Delivery of LNA/DNA Oligomers into Bacterial Pathogens

TRAGLIA G; DAVIES SALA CG; FUENTES B; SOLER BISTUE A

New Orleans

Congreso; American Society for Microbiology (ASM) General Meeting; 2015

American Society for Microbiology

Background: The need for new therapeutic agents due to the spread of antibiotic resistant bacteria led to development of different antisense technology strategies for drug design. However, the successful utilization of antisense compounds as antibacterials presents numerous challenges, among them their penetration into the cytosol. While delivery vehicles have been researched, relatively recent breakthroughs showed gene silencing in a variety of cells by delivery of naked oligonucleotides (gymnosis). Here we show gymnotic delivery of DNA oligomers containing locked nucleic acids (LNAs) (LNA/DNA oligomers) into bacterial pathogens.Materials and Methods: Gymnotic delivery of an LNA/DNA oligomer was tested on clinical Pseudomonas aeruginosa, Achromobacter xyloxosidans, Stenotrophomonas maltophilia, Staphylococcus aureus, Acinetobacter baumannii, A. pittii,Klebsiella pneumoniae, Escherichia coli, Serratia spp., Enterococcus spp. and Enterobacter cloacae isolates. Uptake of CAAGTACTGTTCCACCA LNA/DNA oligomer (LNA residues underlined) conjugated to Alexa Fluor 488 was quantified by flow cytometry. Uptake percentages are the average of three assays (20,000 cells/assay). Intracellular localization of LNA/DNA oligomers was confirmed by confocal laser scanning microscopy (CLSM).Results: Uptake of labeled LNA/DNA oligomer by cells in stationary phase was most efficient in A. baumannii with an average of 24.23% ± 8.9 of the cells internalizing the compound. Other bacteria like A. xyloxosidans, E. cloacae, and P. aeruginosa showed internalization levels between 9 and 16%. The rest of the bacteria assayed did not showed significantlevels of uptake in the conditions tested. CLSM analysis confirmed that the oligomers were internalized rather associated to the cells.Conclusions: Gymnotic delivery of LNA/DNA oligomers inside bacterial cells was successful for some bacteria. Although it occurred at a relatively low frequency, it is a starting point to look for strategies to achieve high frequency of gymnotic uptake.  The results suggest that utilization of LNA/DNA oligomers may contribute to overcoming the problem of uptake, a stumbling block in development of antisense compounds as antibacterials.