Genetically Engineered Virulent Phage Banks in the Detection and Control of Emergent Pathogenic Bacteria

Abstract

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host.

Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing

“…Traditional phage engineering strategies, such as in vitro manipulation, allele-exchange within bacterial hosts, and phage crossing via co-infection of bacteria (Beier et al, 1977; Garcia et al, 2003; Lin et al, 2011) have been used to modulate phage host range (Pouillot et al, 2010; Tetart et al, 1998; Trojet et al, 2011; Yoichi et al, 2005), but these strategies are inefficient and unable to achieve multiple genetic modifications in a single step. Screening for a desired mutation after classical crossing or recombination experiments can require PCR, restriction digestion, or plaque hybridization on hundreds of individual plaques, which are all costly and time-consuming methods.…”

“…Conversely, our strategy rarely requires the screening of more than a few yeast clones, since we found that >25% of yeast clones contained properly assembled phage genomes (composed of up to 11 DNA fragments) that were bootable into functional phages after transformation into bacteria. Previously, a scheme for engineering phage T4 through electroporation of PCR products was devised (Pouillot et al, 2010), but it is based on a particular feature of the genetic regulation of T4 and cannot easily be applied to other phage families. Recently, the 5.4 kb filamentous coliphage ϕX174 was assembled in yeast in order to stably store the genome and aid in phage refactoring (Jaschke et al, 2012).…”

Engineering Phage Host-Range and Suppressing Bacterial Resistance Through Phage Tail Fiber Mutagenesis

“…We envision that the high-throughput mutagenesis and characterization of host-range determinants will be an important and useful tool to decipher host recognition by viruses. As more and more viral tail components are structurally resolved and sequenced (Montag et al, 1987; Pouillot et al, 2010; Tétart et al, 1996; Trojet et al, 2011), the breadth of viral models on which this strategy can be applied will also be expanded. Moreover, mapping phage-host interactions should enable the rational engineering of phage host-range.…”

Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1

“…Depending on needs, phage samples could either be obtained from reference stocks or may be engineered through molecular techniques (e.g. Pouillot et al 2010) or based on phage training (Morello et al 2011;Chan and Abedon 2012;Maura et al 2012). Regarding application of this latter method, an important challenge to trained phage use is the time elapsed between obtaining the bacterial sample for production and actually applying the trained phage to the site(s) of infection.…”