Biology subjects

Sassetti, C. M.

Publications and source records attributed to Sassetti, C. M..

4 recordsLinked to original sources

bioRxiv2018

New inhibitors of Mycobacterium tuberculosis identified using systems chemical biology

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified >10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECTs ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

microbiology↗

bioRxiv2018

CNBP controls c-Rel dependent IL-12 β gene transcription and Th1 immunity

An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Herein, we identified Cellular nucleic acid binding protein (Cnbp) as a specific regulator of interleukin-12{beta} gene transcription and Th1 immunity. Cnbp resides in the cytosol of macrophages and translocates to the nucleus in response to a broad range of microbial ligands. Cnbp-deficient macrophages had a selective impairment in their ability to induce IL12{beta} gene transcription. Cnbp interacted with c-Rel, an NF{kappa}B/Rel family member that controls IL12{beta} gene transcription. c-Rel nuclear translocation and DNA binding activity were dependent on Cnbp. Furthermore, Cnbp itself bound the IL12{beta} promoter. Lastly, Cnbp-deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL12{beta}, as well as a reduced Th1 cell IFN{gamma} response essential to control parasite replication. Collectively, these findings identify Cnbp as a key regulator of c-Rel dependent IL12{beta} gene transcription and Th1 immunity.

immunology↗

bioRxiv2018

ORBIT: a new paradigm for geneticengineering of mycobacterial chromosomes

Current methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic \"targeting oligonucleotide\" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a \"payload plasmid\" that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes in M. tuberculosis and M. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target- or modification-specific double-stranded DNA recombination substrates.

genetics↗

bioRxiv2017

The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis infection.

Protection from infectious disease relies on two distinct mechanisms. \"Antimicrobial resistance\" directly inhibits pathogen growth, whereas \"infection tolerance\" controls tissue damage. A single immune-mediator can differentially contribute to these mechanisms in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase complex (Phox) produces anti-microbial superoxides and protects from tuberculosis in humans. However, Phox-deficient mice do not display the expected defect in resistance to M. tuberculosis leaving the role of this complex unclear. We re-examined the mechanisms by which Phox contributes to protection from TB and found that mice lacking the Cybb subunit of Phox suffered from a specific defect in tolerance, which was due to unregulated Caspase1 activation, IL-1{beta} production, and neutrophil influx into the lung. These studies demonstrate that Phox-derived superoxide protect against TB by promoting tolerance to persistent infection, and highlight a central role for Caspase1 in regulating TB disease progression.

immunology↗