Ana Castro and Joseph Giacalone present at Genetics Student Seminar on 3/20/17
The role of the anti-sigma factor RsiV in lysozyme sensing and stress response in Bacillus subtilis and Clostridium difficile
*Ana N Castro1, Lincoln T Lewerke1, Ben D Cortes2, Jessica L Hastie1, Craig D Ellermeier1
1Microbiology Department, College of Medicine, University of Iowa, Iowa City, IA; 2Biology Department, Benedictine College, Atchison, KS. *Anafirstname.lastname@example.org
ECF σ factors are alternative σ factors that allow many bacteria to sense and respond to changes in the environment. σV, an ECF σ factor, is found in low GC Gram-positive bacteria, induces resistance to lysozyme and is important for virulence in pathogens. In the absence of lysozyme, σV is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded by regulated intramembrane proteolysis (RIP). RIP is initiated by signal peptidase cleavage of RsiV at site-1 resulting in the release and activation of σV. We demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme. By altering the signal peptide, we find that the spacing between the cleavage site and the transmembrane is critical to RsiV avoiding signal peptidase cleavage in the absence of lysozyme. The lab previously determined the X-ray crystal structure of the extracellular domain of RsiV in complex with lysozyme. This structure revealed that RsiV does not bind near the signal peptidase cleavage site. This has led to our model in which binding of lysozyme to RsiV triggers a conformational change, which allows signal peptidase to recognize the cleavage site. Currently, we are determining the structure of full-length RsiV in both the absence and presence of lysozyme. Together this will provide information on the changes that occur to RsiV upon binding lysozyme that lead to σV activation. This will broaden our knowledge on a novel role for signal peptidase and could lead to novel drug targets.
Patient-specific iPSCs to investigate pathophysiology and develop treatments for RPGR-associated XLRP
Giacalone J.C. 1, Burnight E.R. 1,Sharma T.P., Wiley L.A.1, Ochoa D.1, Collins, M.M.1, Mullins R.F.1, Tucker B.A.1, and Stone E.M.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA
Purpose: Retinitis Pigmentosa (RP) is a heterogeneous disease that causes death of the light sensing photoreceptor cells of the outer neural retina and affects as many as 80,000 individuals in the US alone. X-linked RP (XLRP) is responsible for some of the most severe and earliest onset cases. The majority of XLRP cases are caused by mutations in the gene RPGR. The retinal-predominant isoform of RPGR, known as ORF15, contains a long repetitive sequence, which harbors the majority of the pathogenic mutations that cause RPGR-associated XLRP. The purpose of this study was to: 1) model RPGR-associated XLRP using patient-specific induced pluripotent stem cells (iPSCs) and 2) develop CRISPR/Cas9-based genome editing strategies for correction of disease-causing mutations.
Methods: Patient-specific iPSCs were generated from dermal fibroblasts ofpatients with molecularly confirmed RPGR-associated XLRP. Pluripotency was confirmed using the TaqMan Scorecard Assay. CRISPR/Cas9 constructs were generated to target patient-specific mutations. Gene targeting constructs and homology directed repair constructs were introduced to iPSCs via NEON transfection. Correction was confirmed via T7E1 assay and Sanger sequencing. Patient-specific iPSC-derived 3D retinal eyecups were generated and characterized via Western blot, immunocytochemistry and confocal microscopy.
Results: Seven iPSC lines were generated with varying mutations, disease severity, and disease phenotypes. Genome editing of patient-specific iPSCs was achieved with transfection efficiencies of 30 percent, and the resulting modified iPSC clones were isolated and expanded via reporter selection. Patient-specific iPSC-derived retinal eyecups were generated and displayed the retinal progenitor and photoreceptor-specific markers PAX6, OTX2, RCVRN, CRX and NRL.
Conclusions: With the advent of iPSC technology, we are now able to generate retinal cells from male patients with mutations in RPGR ORF15. We have shown that genome editing via the CRISPR/Cas9 system can successfully correct patient-specific mutations in iPSCs, which will serve as a valuable tool for characterizing the observed disease phenotype.