Engineering of the temperate Burkholderia phage Milagro for use as a therapeutic

Abstract

The Burkholderia cepacia complex (Bcc) causes life-threatening respiratory tract infections in persons with cystic fibrosis (CF). In CF patients, end-stage pulmonary disease often requires lung transplantation. However, pre-transplant colonization with antibiotic-resistant strains of Burkholderia is predictive of poor post-transplant outcomes. To address this issue, phage therapy has been proposed as a treatment of multiple drug resistant bacterial infections. However, the majority of characterized Bcc phages are temperate and are therefore unsuitable for phage therapeutics. Moreover, the few virulent (obligately lytic) phages that have been isolated have extremely limited host ranges, usually restricted to the original host used for isolation. To overcome this limitation, we have engineered a virulent, broad-host range derivative of the temperate phage Milagro, designated Milagro vir gp20:Milo Δcl. The engineered phage has a mutation in the predicted lysogenic repressor binding site, a deletion of the lysogenic repressor gene, and a fusion of the phage tail fiber with the receptor-binding domain of the broad-host range tailocin (bacteriocin) BceTMilo. Phage Milagro vir gp20:Milo Δcl has a substantially expanded host range and is able to infect multiple Bcc species including B. cenocepacia, B. multivorans, B. gladioli and B. vietnamensis. To establish a platform for phage engineering, a CRISPR/Cas9 system was developed in Burkholderia that system allows for direct gene editing of virulent phage.