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Study Outlines Genetic Screening Strategy for Host-Pathogen Interaction Networks

NEW YORK (GenomeWeb News) – In a study appearing online today in Science, a Massachusetts-based research team describes a genetic screening strategy it is using to tease apart a set of complex interactions between bacterial pathogens and their host cells.

The approach — dubbed "insertional mutagenesis and depletion," or iMAD — couples mutagenesis of bacterial DNA with RNA interference, which is then used to knock down host cell genes.

"This strategy is broadly applicable, allowing the dissection of any interface between two organisms involving numerous interactions," Tufts University microbiology researcher Ralph Isberg, the study's senior author, and his colleagues wrote.

In the current study, for instance, Isberg and his collaborators from Tufts and Harvard Universities started by focusing on interactions between the pathogenic bacteria from the Legionella pneumophila species and cells from the fruit fly Drosophila melanogaster.

Using the iMAD screen, they tracked down dozens of proteins produced by L. pneumophila that seem to support the bug's growth within these fruit fly host cells — information that served as a jumping-off point for more detailed analyses of specific L. pneumophila mutants in cells from the fruit fly and from another host, amoeba.

"We used this technique to resolve the network of proteins secreted by the bacterium Legionella pneumophila to promote intracellular growth," the study's authors explained, "a critical determinant of pathogenicity of this organism."

L. pneumophila, best known for its role in Legionnaires' disease, is a species of gram-negative bacteria that can cause disease in humans who inhale aerosol particles from L. pneumophila-contaminated water. The bug infects other hosts, too, including free-living amoebas, which help it move from one host to the next.

Past studies have identified hundreds of L. pneumophila proteins that are apparently secreted by the bug as it sets up shop in host cell vacuoles. Even so, as with many host-pathogen relationships, relatively little is known about the full repertoire of pathogen and host proteins that interact with one another during infection.

In addition, attempts to get a clearer understanding of these complicated processes are often hindered by the fact that pathogens typically have multiple molecular methods for achieving the same sorts of interactions with host cells.

For their part, the researchers reasoned that they should be able to see some of these redundancies by mutating the bacterial genes that interact with one host pathway while simultaneously knocking down components from host pathways needed for other, parallel interactions.

To explore that possibility, the team started by screening L. pneumophila mutants generated with a transposon site hybridization, or TraSH, method, looking for genetic alterations that interfered with the pathogen's ability to grow in fruit fly cells that had lower-than-usual levels of their own specific proteins. In particular, researchers relied on RNAi to target components of membrane trafficking pathways contributing to processes in fruit fly cells that are known or expected to be exploited by L. pneumophila during infection and replication.

Starting with the 678 genes found in that screen, investigators narrowed in on 55 genes from a bacterial secretion system called Dot/Icm suspected of contributing to bacterial growth in host cells based on their localization and other features. From there, the team was able delineate 14 different functional groups for the bacterial genes based on the growth patterns of various L. pneumophila mutants in RNAi-treated fruit fly cells.

"We predicted that if a set of Dot/Icm [translocated substrates] targets a particular host pathway, mutations in individual members of that set should result in similar phenotypes," the team explained, noting that it tracked L. pneumophila mutant growth in Drosophila cells exposed to five different double-stranded RNA treatments.

Together with follow-up experiments aimed at further characterizing specific mutants in fruit fly or amoeba cells, the genetic interplay identified by iMAD helped the team create a network of interactions that highlighted some of the redundant pathways used by L. pneumophila as it interacts with its hosts.

"Our integrated approach can be applied to other bacterial pathogens, as well as other systems that have convergent biochemical pathways," Isberg and co-authors concluded, "and shows that it is now possible to define roles for individual proteins and their relative contributions to pathogenesis in different hosts."

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