Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions

Abstract

The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3 end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage E125 and Burkholderia pseudomallei phage 1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo.

Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia

“…This situation may be analogous to the behavior of phage lambda int mutants, which are able to form abortive lysogens that give rise to sensitive cells upon subculture (19,21). In a study of a known temperate B. cenocepacia phage, KS9, only 18% of the phage-insensitive strains isolated following phage challenge were true lysogens, the remainder becoming insensitive presumably by some other mechanism (49). Phages Bcep22 and BcepIL02 may be a more extreme version of this behavior, in which lysogen formation, if it occurs, is below the rate of bacterial mutation to phage insensitivity.…”

Identification and characterization of ϕH111-1

“…13 ϕH111-1 was able to infect all 13 strains tested (Table S1), providing further evidence that this phage may be an appropriate candidate for therapeutic use (particularly if the lysogeny module were deleted). 8 Excluding B. cenocepacia, the ϕH111-1 host range was found to be relatively narrow as only B. multivorans ATCC 17616 and C5274 were susceptible to phage infection from a panel of 18 other Burkholderia strains tested (representing 8 additional BCC species) ( Table S1).…”

“…4 Although the therapeutic use of temperate phages is generally discouraged, the limited availability of obligately lytic BCC phages has necessitated the use of confirmed, putative, or modified temperate phages for several in vivo efficacy studies. [6][7][8] The use of such phages against Burkholderia is arguably safer than it is against many other pathogens because virulence factors in this genus have not been discovered to be encoded by temperate phages. 2 One of the challenges of prophage identification is the differentiation of inducible prophages from defective prophage remnants, a distinction with both evolutionary and practical implications.…”

Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity

“…In a type of coevolutionary arms race, phages can rapidly adapt to counter improvements in bacterial resistance, which is in direct contrast to the long development time required for new static chemical antibiotics (21). Recent studies exploring the treatment of Bcc infections via phage therapy have demonstrated promise (22)(23)(24)(25)(26), including our recent study showing Bcc phage efficacy in a mouse infection model (26).…”