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

UNLABELLED: 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-1ß 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.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Rv2468c, a novel Mycobacterium tuberculosis protein that costimulates human CD4+ T cells through VLA-5.

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5ß1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.