Bioinformatics and genomic analysis for the potential of mRNAs as antibacterial drug targets in the genome of macrolide-resistant strains of Streptococcus pneumoniae infecting humans for novel antisense oligonucleotide design.

Introduction: The macrolide-resistant strains of Streptococcus pneumoniae are the latest additions to the WHO's List of Priority Antibiotic-Resistant Bacteria, responsible for over 3 million deaths annually, of which 300,000 are in children under 5 years of age. One of the most promising novel targets for antibacterial drug development is mRNA. The present study employs a rational framework based on four criteria related to mRNA distribution, function, and metabolism, and identifies them as suitable targets for antibacterial drug discovery. It focuses on seven of them, which are found in the S. pneumoniae genome: the RNase P, FMN, TPP, PreQ1-II, Purine, ykoK leader, and Glycine riboswitches.

Methods: The selection of targets and the subsequent rational design of ASOs targeting the macrolide-resistant S. pneumoniae are based on bioinformatics and genomic studies, including analyses of international databases, Clustal X multiple alignments, selection of appropriate motifs, BLAST searches, and biochemical pathway analyses.

Results: The mRNAs found in S. pneumoniae are grouped into four categories: most suitable, very suitable, suitable, and not suitable. Most suitable riboswitches (FMN, TPP, Purine, and PreQ₁₎ regulate essential metabolites, with no alternative biosynthetic pathways or transport. Very suitable riboswitches (ykoK leader) control the critical metabolite’s biosynthesis and transport. Suitable riboswitches (Glycine) have alternative biosynthetic pathways and do not control their transport. Five different antisense oligonucleotides (ASO) have been designed to target them.

Conclusions: Gene expression is regulated at the transcriptional or translational level. Using mRNA, we develop innovative strategies to design ASOs that inhibit S. pneumoniae growth. Our proprietary protocols for suitability and ASO design enable us to develop novel ASOs (with precise nucleotide sequences for specific binding, modifications that enhance stability and induce RNase H activity, and attached cell-penetrating peptides) targeted to the most suitable mRNAs, with potential as effective antimicrobial therapeutics with growth-inhibitory effects.