Knockouts in Human Cells Point to Pathogenic Targets
News Nov 30, 2009
Whitehead researchers have developed a new type of genetic screen for human cells to pinpoint specific genes and proteins used by pathogens, according to their paper in Science.
In most human cell cultures genes are present in two copies: one inherited from the father and one from the mother. Gene inactivation by mutation is therefore inefficient because when one copy is inactivated, the second copy usually remains active and takes over.
In yeast, researchers have it easier: they use yeast cells in which all genes are present in only one copy (haploid yeast). Now Carette and co-workers have used a similar approach and used a human cell line, in which nearly all human chromosomes are present in a single copy.
“Having knockout cells for almost all human genes in our freezer opens up a wealth of biological questions that we can look at,” says Whitehead Fellow Thijn Brummelkamp. “In addition to many aspects of cell biology that can be studied, knockout screens could also be used to unravel molecular networks that are exploited by a battery of different viruses and bacteria.”
In this rare cell line, Carette and co-workers generated mutations in almost all human genes and used this collection to screen for the host genes used by pathogens. By exposing those cells to influenza or to various bacterial toxins, the authors isolated mutants that were resistant to them. Carette then identified the mutated genes in the surviving cells, which code for a transporter molecule and an enzyme that the influenza virus hijacks to take over cells.
Working with Carla Guimaraes from Whitehead Member Hidde Ploegh’s lab, Carette subjected knockout cells to several bacterial toxins to identify resistant cells and therefore the genes responsible.
The experiments identified a previously uncharacterized gene as essential for intoxication by diphtheria toxin and exotoxin A toxicity, and a cell surface protein needed for cytolethal distending toxin toxicity.
“We were surprised by the clarity of the results,” says Jan Carette, a postdoctoral researcher in the lab of Whitehead Fellow Thijn Brummelkamp lab and first author on the Science article.
“They allowed us to identify new genes and proteins involved in infectious processes that have been studied for decades, like diphtheria and the flu. In addition we found the first human genes essential for host-pathogen interactions where few details are known, as is the case for cytolethal distending toxin secreted by certain strains of E. coli. This could be important for rapidly responding to newly emerging pathogens or to study pathogen biology that has been difficult to study experimentally.”
Brummelkamp sees the work as only the beginning.
“Having knockout cells for almost all human genes in our freezer opens up a wealth of biological questions that we can look at,” he says. “In addition to many aspects of cell biology that can be studied, knockout screens could also be used to unravel molecular networks that are exploited by a battery of different viruses and bacteria.”
Previous work by the International Multiple Sclerosis Genetics Consortium (IMSGC) has identified 233 genetic risk variants. However, these only account for about 20% of overall disease risk, with the remaining genetic culprits proving elusive. A new study has tracked down four of these hard-to-find genes.READ MORE
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