Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa

Pseudomonas aeruginosa causes life-threatening infections. Considering the current poor rate of release of novel antibiotics, let alone entirely novel classes of antibiotics, it is a worrying indication that we may soon run out of treatment options. Therefore, the development of innovative antibiotics targeting (not yet exploited) essential bacterial pathways will be crucial inthe near future. Strictly lytic bacteriophages, bacteria s natural enemies, rely completely on the bacterial metabolism for their propagation. Over a billion years of co-evolutionary struggle phages have evolved an incredible number of highly diverse proteins that either inhibit or adapt bacterialmetabolic processes to their own benefit. Many of them lead to cell-cycle arrest or even host lethality. As such, a novel source of Gram-negative antibacterials might originate from mining the thousands of available sequenced phage genomes. 158 early phage proteins encoded by nine different P. aeruginosa phages were selected as starting point of this work. We hypothesized that phage proteins, which are growth-inhibitory to their host when individually expressed, show the most promise in tackling crucial metabolic pathways. Consequently, the 158 selected proteins were first screened for their effect on P. aeruginosa growth. In total, nineteen unknown antibacterial phage proteins could be identified.To explore their possible mode of action and the molecular background of their toxicity, a systematic yeast two-hybrid (Y2H) against a random genomic fragment library of P. aeruginosa PAO1 was applied to identify their target(s) in Pseudomonas. This showed that bacteriophages influence the host metabolism using a variety of modes.A nice example is LUZ24 gp4. For this phage protein, one potential interaction partner in P. aeruginosa was identified, the PA4315-encoded transcriptional regulator MvaT, which was confirmed in vitro using coprecipitation assays. MvaT is a histone-like nucleoid structuring protein, which exerts a crucial role in compaction of the bacterial chromosome by the formation of oligomers. Moreover, the polymerization of the protein across AT-rich DNA strands, permits gene silencing of foreign DNA, thereby limiting any potentially adverse effects of such DNA. Recombinant MvaT-His and LUZ24 gp4-Strep were tested in gel shift assays, which proved the inhibitory effect of LUZ24 gp4 on MvaT DNA-binding activity. We therefore termed this gene product as Mip, the MvaT-inhibiting protein. A hypothesis on the biological role of Mip, one of the first proteins produced right after infection, can be made: Mip indeed prevents the AT-rich LUZ24 DNA from being physically blocked by MvaT oligomers right after its injection in the host cell. This strategy gives the phage a clear advantage since a physical blockage of its DNA rightafter injection, will not complete its infection cycle. Inhibition of MvaT by a phage-encoded protein will keep the phage DNA MvaT-free, thereby allowing phage transcription and thus completion of the phage infection cycle. Although microbial resistance is probably an unavoidable consequence of antibiotic therapy, a bacteriophage-based platform has a great potential with respect to identifying novel mechanisms and targets to treat bacterial infections. In fact, known phage-host interactions illustrate the potential for phage systems to be used for the identification of points in host metabolism that may be susceptible to small-molecule inhibitors. The most efficient and vulnerable targets are selected and validated through billions of years of co-evolution between phages and their hosts. As there is no dearth of bacteriophages in nature, the quest for lethal phage proteins as well as their cognate bacterial targets should be continued in order to expedite the research on antibacterial drug discovery.status: publishe