A functional genomics screen identifying blood cell development genes in Drosophila by undergraduates participating in a course-based research experience.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

RhlR-Regulated Acyl-Homoserine Lactone Quorum Sensing in a Cystic Fibrosis Isolate of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.IMPORTANCEPseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

Role of a Small Molecule in the Modulation of Cell Death Signal Transduction Pathways.

Inflammasomes activate caspase-1 in response to molecular signals from pathogens and other dangerous stimuli as a part of the innate immune response. A previous study discovered a small-molecule, 4-fluoro- N'-[1-(2-pyridinyl)ethylidene]benzohydrazide, which we named DN1, that reduces the cytotoxicity of anthrax lethal toxin (LT). We determined that DN1 protected cells irrespectively of LT concentration and reduced the pathogenicity of an additional bacterial exotoxin and several viruses. Using the LT cytotoxicity pathway, we show that DN1 does not prevent LT internalization and catalytic activity or caspase-1 activation. Moreover, DN1 does not affect the proteolytic activity of host cathepsin B, which facilitates the cytoplasmic entry of toxins. PubChem Bioactivities lists two G protein-coupled receptors (GPCR), type-1 angiotensin II receptor and apelin receptor, as targets of DN1. The inhibition of phosphatidylinositol 3-kinase, phospholipase C, and protein kinase B, which are downstream of GPCR signaling, synergized with DN1 in protecting cells from LT. We hypothesize that DN1-mediated antagonism of GPCRs modulates signal transduction pathways to induce a cellular state that reduces LT-induced pyroptosis downstream of caspase-1 activation. DN1 also reduced the susceptibility of Drosophila melanogaster to toxin-associated bacterial infections. Future experiments will aim to further characterize how DN1 modulates signal transduction pathways to inhibit pyroptotic cell death in LT-sensitive macrophages. DN1 represents a novel chemical probe to investigate host cellular mechanisms that mediate cell death in response to pathogenic agents.