Silencing of Curlin Protein via M13 Phagemid-Mediated Synthetic sRNA Expression Reduces Virulence in the Avian Pathogenic E. coli (APEC).

Abstract

Curli fimbriae, a virulent factor of the Avian Pathogenic Escherichia coli (APEC), is responsible for adhesion, biofilm formation, and colonization of pathogen. Major curli fimbriae protein is encoded by csgA gene. APEC is one of the leading causes of colibacillosis in poultry flocks and due to excessive use of antibiotics and vaccines in poultry, the emergence of various multi-drug resistant (MDR) bacterial strainsare is frequently reported. The growing concern of MDR bacterial strains necessitate novel antibacterial approaches to combat colibacillosis in poultry. RNA-based gene silencing is a very specific and robust strategy to target specific bacterial factors involved in pathogenicity and virulence. In this study, a phagemid-mediated sRNA expression system to target a vital gene, csgA, is employed. This comprises an M13 phagemid harboring a sRNA expression cassette and a pre-designed GUIDE sequences for the csgA target gene. To target the csgA gene at the mRNA level, a GUIDE sequence was computationally designed for pre-designed sRNA expression cassette. Online web tools were used to predict the binding energy, secondary structure, and off-target binding potential of the sRNA to optimize its expression. Results showed that the designed sRNA has a binding energy of -29.60kcal/mol with zero off-targets. After expression of the sRNA in the APEC cells, ̴ 45% reduction in the csgA level was observed via RT-PCR in the CS-APEC-O1 strains compared to the wt-APEC-O1. Similarly, the biofilm forming ability decreased by 40% in the CS-APEC-O1 strains. The swarming motility and hemagglutination efficiency were not affected by the sRNA expression. Future studies investigating the in vivo efficiency of M13 phagemid delivery are required to evaluate its candidacy in phage therapy.