Solving Pathogen Sudoku

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Science Translational Medicine  02 Jan 2013:
Vol. 5, Issue 166, pp. 166ec2
DOI: 10.1126/scitranslmed.3005530

When pathogenic organisms infect humans, they express a multitude of proteins, and similarly, host cells express numerous proteins that can either restrict or collaborate with the pathogen. Thus, in order to block only the protein (or proteins) required for virulence, one would need to know the specific pathogen-host interactions that are critical for infection. The problem can be equated to solving a Sudoku number puzzle with no hints: Although the milieu of proteins and multiple redundancies expressed during disease among the many for both pathogen and host can be determined, this information will not tell us the arrangement or key factor (or factors) required for infection. O'Connor and co-workers have now developed an approach to dissect and screen for factors important for pathogen-host interactions.

The new technique—called insertional mutagenesis and depletion (iMAD)—was used to probe for proteins of the bacterium Legionella pneumophila that are required for intracellular growth in cells from fruit flies (Drosophila). iMAD uses both bacterial mutagenesis and host RNA interference (RNAi) screening methods to identify double-stranded DNA from bacterial transposon mutants that altered growth of host cells in which selected genes were silenced by means of RNAi. The authors probed mutations that covered the entire L. pneumophila genome and were able to identify defects resulting from mutations in 55 genes that encode proteins secreted by the Dot/Icm type-IVb secretion system, which is required for L. pneumophila intracellular replication. L. pneumophila mutants were identified in the lidA and legA3 genes, which impaired growth in Drosophila cells in which the genes that encode membrane trafficking proteins Sec22 or Bet5. Similar defects were observed resulting from mutations in the bacterial wipB gene, when the fly cells were depleted for a separate membrane trafficking protein, Arf1. Further validation of these findings was provided by experiments that examined infection of L. pneumophila in natural host organisms. L. pneumophila lidA wipB double-mutant bacterial strains showed reduced growth in mouse macrophages, Acanthamoebae castellanii and Dictyostelium discoideum amoeba.

This dual-screening approach allows for determining specific interactions that previously have been difficult to assess. Certainly, this iMAD strategy is complex and can currently be used to examine only pathogens for which sufficient genetic techniques exist to construct mutant libraries for screening. Nonetheless, the potential to probe dually for specific pathogen-host factors important for virulence allows for much greater specificity in therapeutic development for both pharmaceutical-biochemical and genetic treatment strategies.

T. J. O'Connor et al., Aggravating genetic interactions allow a solution to redundancy in a bacterial pathogen. Science 338, 1440–1444 (2012). [Abstract]

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