Novel high throughput pooled shRNA screening identifies NQO1 as a potential drug target for host directed therapy for tuberculosis

Chemical regulation of macrophage function is one key strategy for developing host-directed adjuvant therapies for tuberculosis (TB). A critical step to develop these therapies is the identification and characterization of specific macrophage molecules and pathways with a high potential to serve as drug targets. Using a barcoded lentivirus-based pooled short-hairpin RNA (shRNA) library combined with next generation sequencing, we identified 205 silenced host genes highly enriched in mycobacteria-resistant macrophages. Twenty-one of these “hits” belonged to the oxidoreductase functional category. NAD(P)H:quinone oxidoreductase 1 (NQO1) was the top oxidoreductase “hit”. NQO1 expression was increased after mycobacterial infection, and NQO1 knockdown increased macrophage differentiation, NF-κB activation, and the secretion of pro-inflammatory cytokines TNF-α and IL-1β in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and anti-bacterial functions. The competitive inhibitor of NQO1 dicoumarol synergized with rifampin to promote intracellular killing of mycobacteria. Thus, NQO1 is a new host target in mycobacterial infection that could potentially be exploited to increase antibiotic efficacy in vivo. Our findings also suggest that pooled shRNA libraries could be valuable tools for genome-wide screening in the search for novel druggable host targets for adjunctive TB therapies.


Additional discussion points about the pooled lentiviral shRNA screening
Systematic genetic or chemical approaches for identifying host factors required for bacterial survival and replication have been limited [1][2][3][4][5] . Large-scale RNA interference (RNAi) screens to study bacterial infection were first performed in D. melanogaster cells infected with L. monocytogenes, Chlamydia spp. and Mycobacterium fortuitum 1,6,7 . A human kinase silencing RNA (siRNA) sub-library was screened in a human cell line to identify kinases required for intracellular growth of S. typhimurium 8 . A genome-wide RNAi screen in human cells have identified intracellular networks regulating M. tuberculosis survival 3 . Thus, high-throughput RNAi has emerged as a powerful molecular screening tool for studies of the host-pathogen interactions in different infection models 9 .
These previous large-scale screens used siRNA libraries constructed with chemically synthesized oligonucleotides and required massive parallel screening of thousands of individual siRNAs. This approach is expensive and time-consuming, and results are difficult to reproduce. Lentiviral-based libraries containing heterogeneous mixtures of short hairpin RNAs (shRNA) synthesized enzymatically from cDNAs do not require access to expensive HT equipment and can be performed in a single experiment.
Pooled shRNA library screens offer several advantages over siRNA: they are fast, efficient and inexpensive, result in deeper coverage and require minimal manipulation of cell cultures, resulting in more precise identification of targets; since shRNAs are transfected into cells via viral vectors (lentiviruses), they integrate into the cellular DNA resulting in stable knockdown of mRNAs, allowing for enrichment of the specific phenotype through multiple rounds of selection.
With one exception 1 , in all the RNAi screens reported before, host gene silencing was performed after mycobacterial uptake 2,3,8 . A major advantage of lentiviral-based shRNA targeting is the long-term silencing of host genes that allow for bacterial infection of cells in which gene expression down-regulation has already occurred. Thus, lentiviralbased shRNA allows for identification of host factors that play a role in the very early events leading to mycobacterial adaptation to the intracellular environment.
Since each RNAi screening is unique in its design, we reasoned different molecules could be identified using different approaches and this could widen the range of host targets for HDT. Although the lack of reproducibility of results of RNAi screens has been criticized, this may not be necessarily a weakness, since each screen may contribute new sets of validated targets to the pool of human molecules known to play a role in the mycobacteria-host interaction. Variability in results among different screens may depend on differences in both experimental set up (bacterial strain, infection protocol, transduction versus transfection, cell viability, etc.) and data analysis, and does not necessarily call into question the validity of the results but reveals the complexity of factors that play a role in the final outcome of mycobacterial infection. Careful validation of screening "hits" with different cell types, bacterial strains and different functional inhibition approaches (e.g. genetic vs. chemical) increases the reliability of screens.
The shRNA screening strategy used in this study was designed to identify host gene products that support mycobacterial uptake, replication and/or survival whose silencing or inhibition contributes to mycobacterial clearance in macrophages. However, another important application for this shRNA screening could be the identification of new host targets whose silencing or inhibition increase intracellular mycobacterial replication or survival. These host molecules are predicted to be involved in innate killing mechanisms or to negatively regulate other host factors required for mycobacterial survival. Drug agonists of these targets may improve mycobacterial control by macrophages. Since several rounds of selection are not feasible in heavily infected cells due to viability constrains, identification of shRNAs that increase mycobacterial growth in macrophages will require modifications of the current screening strategy. The use of lower MOIs, a first-step cell sorting immediately after infection, shorter culture times before selection and the use of more selective cell sorting gates (very high-, high-and low-CFP) may allow the isolation of cell clones carrying these type of shRNAs and their correspondent host genes.