Iron limitation in M. tuberculosis has broad impact on central carbon metabolism.

Mycobacterium tuberculosis (Mtb), the cause of the human pulmonary disease tuberculosis (TB), contributes to approximately 1.5 million deaths every year. Prior work has established that lipids are actively catabolized by Mtb in vivo and fulfill major roles in Mtb physiology and pathogenesis. We conducted a high-throughput screen to identify inhibitors of Mtb survival in its host macrophage. One of the hit compounds identified in this screen, sAEL057, demonstrates highest activity on Mtb growth in conditions where cholesterol was the primary carbon source. Transcriptional and functional data indicate that sAEL057 limits Mtb's access to iron by acting as an iron chelator. Furthermore, pharmacological and genetic inhibition of iron acquisition results in dysregulation of cholesterol catabolism, revealing a previously unappreciated linkage between these pathways. Characterization of sAEL057's mode of action argues that Mtb's metabolic regulation reveals vulnerabilities in those pathways that impact central carbon metabolism.

CRISPR Interference Reveals That All-Trans-Retinoic Acid Promotes Macrophage Control of Mycobacterium tuberculosis by Limiting Bacterial Access to Cholesterol and Propionyl Coenzyme A.

Macrophages are a protective replicative niche for Mycobacterium tuberculosis (Mtb) but can kill the infecting bacterium when appropriately activated. To identify mechanisms of clearance, we compared levels of bacterial restriction by human macrophages after treatment with 26 compounds, including some currently in clinical trials for tuberculosis. All-trans-retinoic acid (ATRA), an active metabolite of vitamin A, drove the greatest increase in Mtb control. Bacterial clearance was transcriptionally and functionally associated with changes in macrophage cholesterol trafficking and lipid metabolism. To determine how these macrophage changes affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl coenzyme A (propionyl-CoA). Supplementation with sources of propionyl-CoA, including cholesterol, abrogated the restrictive effect of ATRA. This work demonstrates that targeting the coupled metabolism of Mtb and the macrophage improves control of infection and that it is possible to genetically map the mode of bacterial death using CRISPR interference. IMPORTANCE Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a leading cause of death due to infectious disease. Improving the immune response to tuberculosis holds promise for fighting the disease but is limited by our lack of knowledge as to how the immune system kills M. tuberculosis. Our research identifies a potent way to make relevant immune cells more effective at fighting M. tuberculosis and then uses paired human and bacterial genomic methods to determine the mechanism of that improved bacterial clearance.

Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response.

One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA, through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

Targeted transgene integration in plant cells using designed zinc finger nucleases.

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.