CAMBRIDGE, Mass. (November 26, 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.
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 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."
SOURCE