KEEPING UP INFECTIVITY:
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With inquisitive minds and tools as simple as a Waring blender, the work of early phage researchers such as Max Delbruck, Seymour Benzer, and Alfred Hershey generated much of the knowledge underlying contemporary molecular biology. But in a decision that echoes current debates about focusing too narrowly on canonical model organisms,1 Delbruck insisted that research be done only on the T-phages that infect...
Like all phages,
In response, the phage has developed the ability to switch tail-fiber protein tropisms, resulting in multiple states. When Miller and his collaborators compared the viral sequences, they discovered that all the tropisms contained a 134-basepair region of variability (VR1) and an almost 90% identical template repeat (TR). Adjacent to the TR was the unexpected
Experiments in which
To understand the mechanism, Miller's group is attempting to knockout the repair enzyme MutY. "If you line up an incorrect base across from an [adenine], MutY will take out the A at different efficiencies depending on which base it is mispaired with. Other enzymes then affect excision and repair synthesis to result in the complementary base being inserted across from the base that was mispaired with A," says Miller's nearly identically named UCLA colleague, Jeffrey H. Miller, who discovered the
© 2004 Nature Publishing Group
Putative DGRs are shown alongside
J.F. Miller has dubbed the actors in this process diversity-generating retroelements (DGRs). His UCLA colleague compares this mechanism to the mammalian adaptive immune system, pointing out that phage are not alone in cleverly achieving targeted genomic diversity. "It's an additional example of a mechanism for mutagenizing a portion of a gene or only one gene instead of mutagenizing everything," says J.H. Miller.
In a recent
The mechanism appears in the genomes of everything from mammalian commensal bacteria to photosynthetic marine cyanobacteria. "This variability mechanism has found its way into other organisms to do other things," says J.F. Miller. "Now the question is: What [is] the universe of these bacterial elements doing for their bacterial hosts?"
Luis Villarreal, professor of molecular biology and biochemistry at UC, Irvine, finds the evolutionary implications particularly exciting, "It's a biological peculiarity, but to me it still makes a big point," says Villarreal. "If you open up a book on phage biology, this should be prominent. It's the link towards a way to solve the problem of creating genetic diversity in the prokaryotic world and creating diversity via a similar solution in eukaryotes, such as in our adaptive immune system." Incidentally, Villarreal is working on a book about viral evolution.
THE BOTTOM LINE
J.F. Miller, who partnered in a biotech startup called Avidbiotics in Delaware, sees two developmental pathways: "We're simultaneously pursuing two major applications. The first involves antimicrobials/phage therapy, and the other is focused on diversifying proteins of interest as a means to find new drugs."
Avidbiotics intends to create an effective means of phage therapy that would overcome the fears that phage treatment could lead to the unintended transfer of genomic material. Rather than using whole phage, they would create dozens, if not hundreds, of tail fibers, each with a different protein, and combine them to use against bacteria. Tail fibers alone are enough to kill bacteria by piercing and depolarizing the cell membrane, but since they do not contain genomic material, they would not replicate, solving not just the transfer issue, but enabling accurate dosing. "The FDA is adamant about understanding the dosing of therapeutics," says David Martin, J.F. Miller's partner and a former head of R&D at Genentech, "If you've got a therapeutic that replicates exponentially, you cannot control that dose."
No matter how fast the target bacteria might mutate, this "cocktail of tails" would likely have enough different proteins to find a receptor to bind. Eventually, of course, the bacteria would mutate beyond whatever was in the mix, so the plan is to survey isolates and keep pace by developing new cocktails – still a faster process than traditional antibiotic development, according to J.F. Miller.
Martin tempers optimism with a realistic assessment of the challenges involved to receive federal approval in the United States. "There's a lot of naïveté among companies that are trying to develop bacteriophage therapy ... about what it's going to take to even start a clinical trial in the United States, much less wind up with an approved product," says Martin.
The DGR itself could also become a bench tool, says J.F. Miller. "If a virus can use this to diversify a receptor, maybe if we understand the mechanism, we can transplant into other proteins to diversify them," he says. If it works, phage research will have come a long way from Waring blenders.