Scientists Use Knockout Cells to Discover New Genes Involved in Viral and Bacterial Resistance

Researchers from Whitehead Institute for Biomedical Research say they’ve developed a new type of genetic screen for human cells to pinpoint specific genes and proteins used by pathogens. The identification of such factors could aid the future development of new therapeutics to combat infectious disease. Their paper, titled “Haploid genetic screens in human cells identify host factors used by pathogens,” appears in the November 27 issue of Science.

In most human cell cultures, genes are present in two copies (paternal and maternal). Gene inactivation by mutation is therefore inefficient because when one copy is inactivated, the second copy usually remains active and takes over. With yeast, however, researchers use haploid cells, in which all genes are present in only one copy. Now researchers have used a similar approach with a human cell line, in which nearly all human chromosomes are present in a single copy.

In this rare cell line, the scientists 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. The researchers 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.

The Whitehead researchers 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 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.”

This work was done in Thijn Brummelkamp’s lab. He is a fellow at Whitehead who 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.”