Abstract
The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2′-O-methylribonucleotides (2′-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2′-OMe PGO in mycobacteria. The results indicated that 2′-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2′-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2′-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2′-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2′-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.
Highlights
Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis, is responsible for nearly 2 million fatalities annually, and in 2017, 10 million new TB cases were documented (WHO report, 2018)
To determine whether ald 2′-OMe PGO (ald-PGO) treatment influences the transcription of the target ald gene in M. smegmatis-infected macrophages, we performed quantitative Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) analysis using total RNA isolated from infected macrophages. qPCR revealed a significant decrease of ald transcripts after ald-PGO treatment compared with untreated control, or scrambled 2′-OMe PGO (scr-PGO) treatment, indicating the intracellular effect of 2′-OMe PGO on mycobacteria (Figure 5B)
We provided evidence that an antisense 2′-OMe PGO could inhibit growth of mycobacteria, downregulate the expression of bacterial genes, and penetrate into intracellular mycobacteria through gymnosis
Summary
Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis, is responsible for nearly 2 million fatalities annually, and in 2017, 10 million new TB cases were documented (WHO report, 2018). The use of ASOs as antibacterial agents is limited, in particular, by their poor uptake into either extracellular or, especially, intracellular bacteria (Xue et al, 2018), and the majority of them have been designed to work in eukaryotic cell systems (Juliano, 2016), with the exception of peptide conjugates with phosphorodiamidate morpholino oligonucleotides (PMOs or morpholinos) (Daly et al, 2017) or, to a lesser extent, PNAs (Good et al, 2001). 2′-OMe PGO showed the ability to penetrate into intracellular macrophage-residing mycobacteria without the aid of transfection agents and suppress target gene expression, suggesting its potential as a novel and effective antimycobacterial agent
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