An M13 Phagemid Mediated Therapy to Silence Virulence Factors of Avian Pathogenic E. coli (APEC)

Abstract

Avian Pathogenic E. coli (APEC) causes various poultry infections, which leads to high rate of mortality and morbidity in poultry flocks. The current treatment strategies against APEC infections involve antibiotics and vaccines but they are becoming ineffective due to resistance and the emergence of new variants of APEC. Novel strategies have evolved to treat bacterial infection and to combat multidrug resistant bacteria. These novel strategies involve phage therapy, phagemid mediated therapy, probiotics, nanoparticles, quorum quenching and antimicrobial peptides. Phage therapy is one of the promising strategies to treat bacterial infections but a bit of modifications in phage genome via genetic engineering can broaden the host range, reverse antibiotic resistance and using phages to deliver sRNAs, antibiotics and antibodies into the target host. The phagemid-mediated therapy comprises of phagemid, which include sRNA expression cassette that contains pre-designed GUIDE sequences for the target gene. The M13 helper phages deliver the phagemids to the target host, achieved by the presence of f1 origin of replication. This study involved the designing of GUIDE sequence for pre-designed sRNA expression cassette, computationally analysis of sRNA binding energy, secondary structures and off target predication. Results showed that the designed sRNA has binding energy of -29.60kcal/mol with zero off targets. The real time RT-PCR showed that the csgA gene was repressed about 43% in phagemid treated APEC-O1 as compared to wild type APEC-O1. Similarly, the biofilm was reduced up to 34% in csgA silenced APEC-O1 compare to wild type APEC-O1. A little higher spreading was observed in csgA silenced APEC-O1 as compared to wild type APEC-O1, which showed that curli fimbria restrained the flagellar motility and tends to colonization. The study needs to be further investigated for the infection efficiency of M13 helper phages in vivo, using chick model. Furthermore, it is also important to search for other targets (genes) to eliminate or reduce the pathogenicity at higher level.