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

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 φE125 and Burkholderia pseudomallei phage φ1026b. 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.The Burkholderia cepacia complex (BCC) is a group of at least 17 Gram-negative species, the first identified strains of which were characterized as onion pathogens by W. H. Burkholder (9). Although these bacteria have a number of beneficial activities, including the promotion of crop growth and the degradation of organic pollutants, they have gained notoriety in the last two decades as serious opportunistic pathogens (19, 21, 25). BCC species, particularly B. multivorans and B. cenocepacia, cause serious respiratory infections in patients with cystic fibrosis and chronic granulomatous disease (42, 7). These infections are especially problematic due to symptom severity, the inherent antibiotic resistance of Bcc species, and the potential for rapid spread through susceptible patient populations (25, 23). Difficulties in treating these infections have led to the unfortunate practice of segregating patients, which has high economic, social, and psychological costs (18).Because of these clinical difficulties, interest in the isolation and characterization of Burkholderia-specific bacteriophages (or phages) has increased in recent years, with the apparent potential for using phages as therapeutic agents. Phage therapy is the clinical application of phages to prevent and/or to treat infections, which offers a promising alternative to antibiotic treatment for resistant bacteria such as those of the BCC (33, 39). A second benefit of these phage studies is that they may provide insight into the possible mechanisms of BCC virulence. For example, BcepMu, a transposable phage that specifically infects strains of B. cenocepacia, was found to carry genes similar to exeA, involved in toxin secretion, and mdmB and oafA, two acyltransferases (44). Finally, as Burkholderia phages tend to be underrepresented in comparative studies with respect to Escherichia coli and lactic acid bacteria phages, BCC-specific phage studies provide novel information about a relatively uncharacterized group of viruses.Although phage therapy using temperate virions can be effective (39), there are several reasons why lytic phages are generally considered the most appropriate candidates for use in phage therapy. One of the concerns is that phage integration can lead to lysogenic conversion and enhanced virulence (8). A second concern is that integration of temperate phages results in superinfection immunity due to expression of the phage repressor from the prophage. This protein binds to the operators of infecting phage DNA and represses gene expression, preventing both the initiation of the lytic cycle and the establishment of lysogeny (14). A third concern is that lysogeny affects the kinetics of infection. When a phage infects a cell and undergoes lysogeny instead of entering the lytic cycle, the cell survives, and no new phage particles are released (27). A final problem is that prophages can lead to specialized transduction after induction. Specialized transduction occurs after inexact excision of a prophage from the bacterial chromosome. Bacterial DNA flanking the prophage is packaged into the capsid, and this sequence, which can potentially encode virulence factors, can subsequently recombine into the chromosome of a new host (14).It has been estimated that more than half of tailed phages have evolved a temperate lifestyle, although some estimates have been greater than 90% (1, 22). This situation makes the isolation of naturally lytic phages extremely difficult, particularly when they must have a specific host range that includes clinically relevant bacterial species, such as B. cenocepacia (24). The use of classical genetics to produce lytic phage variants, for example, by plating temperate phages on lysogens and screening for clear plaque vir mutants, is complicated by the fact that such mutations are undefined.This report describes the characterization of KS9 (vB_BcenS_KS9), a prophage of Burkholderia pyrrocinia LMG 21824 (41), and its conversion to a lytic phage through specific molecular modification of gene 41 encoding its putative lytic phase repressor. Preliminary characterization of short sequences by Seed and Dennis (41) indicated that the genome of KS9, whose host range includes Bcc B. cenocepacia K56-2, shows similarity to the genomes of two non-BCC Burkholderia phages: φE125, a prophage of Burkholderia thailandensis E125 (47), and φ1026b, a prophage of Burkholderia pseudomallei 1026b (17). However, no phages closely related to KS9 have been functionally tested to demonstrate that proteins similar to gp41 function as true phage repressors. In the present study, we have used the BCC infection model of Galleria mellonella (40) to assess both the contribution of the KS9 prophage to BCC host virulence and the ability of a genetically modified KS9 to treat B. cenocepacia infections without stably integrating into the host bacterial chromosome as a prophage.