A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility

Objectives A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi. Methods A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility. Results Approximately 88% of the S. Typhi strain’s 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR. Conclusions A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.

INCREASED LIFESPAN THROUGH ALTERED GCN4 / ATF-5 IN S. CEREVISIAE AND C. ELEGANS

In a whole-genome screen for deletions that increase lifespan in S. cerevisiae, we identified increased Gcn4 signaling as a mediator of increased lifespan. Gcn4 is a nutrient-responsive transcription factor whose entire pathway is functionally conserved from yeast through humans. Accumulation of uncharged tRNAs has been shown to upregulate Gcn4, and its mammalian ortholog, ATF4. Here we demonstrate that chemical inhibitors of tRNA synthetases significantly extend lifespan in both yeast and the nematode C. elegans, in a dose- and Gcn4-dependent manner.

An In Vivo Genome-Wide CRISPR Screen Identifies Novel Dependencies for Blast Crisis Chronic Myelogenous Leukemia

Poorly differentiated aggressive myeloid diseases such as Acute Myelogenous Leukemia (AML) and blast crisis Chronic Myelogenous Leukemia (bcCML) are often resistant to standard therapy and associated with significantly poor survival in both children and adults. There is thus a significant need for a better understanding of the mechanisms that drive disease progression and for finding novel therapeutic targets. Thus, to determine the molecular effectors of myeloid leukemia growth in vivo, we carried out a genome-wide CRISPR/Cas9 dropout screen using the lentiviral Brie gRNA library. This library targets 19,674 genes, and has on average 3 gRNAs for each gene, and 1000 control non-targeting gRNAs. We carried out this whole-genome screen in a mouse model of Cas9+ blast crisis CML (bcCML) driven by BCR-ABL/ NUP98-HOXA9 since this represents a very aggressive phase of myeloid cancer where 90% of the leukemic blasts are undifferentiated and cancer stem cell-like. This in vivo screen led to the identification of 3636 genes essential for leukemic growth and propagation in the bone marrow of recipient mice, constituting pathways such as metabolism, protein translation and DNA replication. The genes that were significantly depleted included known drivers of myeloid cancer progression and regulators of myeloid cancer stem cells (for example, Brd4, Kdm1a, Pafah1b1/Lis1, Rptor), indicating that our screening strategy can successfully identify functional drivers of cancer growth. While intrinsic signals that drive myeloid cancer progression are well described, little is known about how interactions with the surrounding microenvironment can control leukemic growth and propagation. Our whole-genome screen identified ~130 cell surface genes that are significantly depleted in the bcCML stem cells transplanted in vivo. Since environmental factors commonly signal through receptors on the surface of leukemic cells, this subset is likely to include most, if not all, genetic effectors of niche driven signals required for in vivo growth and propagation of aggressive myeloid leukemia cells. Of these 130 genes, several have earlier been shown by us and others to be essential for myeloid cancer progression including Itgb1, Cxcr4 and Cd44. We are currently testing the functional contribution of novel candidate cell surface molecules, which can integrate signals from the environment, on the in vivo growth and progression of myeloid malignancies. We anticipate that these studies will provide a basis for testing antibody-mediated therapeutic inhibition of specific microenvironmental signals on myeloid leukemia growth and propagation. Disclosures No relevant conflicts of interest to declare.