Identification and differential expression analysis of Hfq-bound novel small RNAs in Serratia marcescens by nitrocellulose-based partitioning.

BACKGROUND: The rising prevalence of multidrug-resistant bacteria poses a looming threat to healthcare and necessitates novel therapeutic strategies beyond conventional antibiotics. One such organism is Serratia marcescens, which possesses a broad range of virulence factors and has been listed as a high-priority pathogen by the WHO due to its increasing antibiotic resistance. Small RNAs are promising targets against antibiotic-resistant bacteria, as they are key regulators of bacterial gene expression. This study aimed to identify novel sRNA and elucidate their roles in regulating virulence-associated genes in S. marcescens.

METHODS AND RESULTS: Hfq-associated sRNA were isolated using nitrocellulose membrane-based partitioning following Hfq overexpression and purification, and identified through high-throughput sequencing. A total of 159 novel sRNA candidates were identified. Two candidates, SM_Hfq_npc119 and SM_Hfq_npc07, exhibited growth phase-dependent differential expression in Northern blot analysis. SM_Hfq_npc119 was highly expressed during the lag phase and under biofilm conditions but downregulated under oxidative stress, suggesting a role in early adaptation and virulence. An inverse correlation between SM_Hfq_npc119 and its predicted target, Type IV pilus PilW mRNA, suggests negative post-transcriptional regulation. SM_Hfq_npc07 expression peaked during log phase and was predicted to target the tssB gene of the Type VI secretion system, implicating a role in virulence regulation.

CONCLUSIONS: This study expands the current understanding of sRNA-mediated post-transcriptional regulation in S. marcescens by identifying novel Hfq-associated sRNA with potential roles in virulence. This findings provides a foundation for future functional investigations and highlight sRNAs as promising targets for the development of novel diagnostics and therapeutic strategies.