Category Archives: Student Seminar
CRISPR-Cas9 Gene Editing Yields a Novel Rat Model of the Metabolic Syndrome
Karen C Clark BS, Janette M Pettus BS, Justin L Grobe, PhD Anne E Kwitek, PhD
Department of Pharmacology, University of Iowa Carver College of Medicine; Interdisciplinary Graduate Program in Genetics, University of Iowa Carver College of Medicine
Metabolic Syndrome (MetS) is the clinical presentation of three or more risk factors—central obesity, dyslipidemia (elevated triglycerides, low HDL), hyperglycemia and hypertension—each of which contributes to increased risk of heart disease, diabetes and stroke in more than 20% of U.S. adults. There is strong evidence that MetS and its symptoms are highly heritable, yet identification of causative genes remains elusive, likely due to the complexity of the syndrome. Genome-wide association studies in human populations have fallen short in determining the causative loci; therefore, we employ the genetically tractable inbred Lyon Hypertensive rat model to tease apart the complex etiology of MetS.
Using a genome-wide approach, we previously identified a completely novel gene on rat chromosome 17 (RNO17) using a combination of QTL and eQTL mapping and gene network analysis, and found that RGD1562963 (RGD) has genetic effects on components of MetS.
CRISPR-Cas9 gene editing was used to introduce insertion and deletion mutations (indels) in exon 2 of RGD, and we are currently studying the mutations’ effects in male and female LH-derived rats. Though experiments are ongoing, preliminary data indicates homozygous RGD mutant females have increased resting aerobic metabolic rate (RMR) compared to wild-type controls—as measured by respirometry and core body temperature—and are hypertensive, especially when challenged with a high salt diet.
Our studies suggest RGD exerts pleiotropic effects on various components of MetS, and inhibition of this gene at the whole body level is tolerated. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate a completely uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of components of the Metabolic Syndrome.
Missing the Marx: Groucho function in Wnt Signaled Asymmetric Cell Division
Kimberly N. Bekas; Bryan T. Phillips
Genetics Graduate Program, University of Iowa, Iowa City, IAAsymmetric cellular divisions (ACDs) are a fundamental component of developmental processes that result in two daughter cells with differential cell fate at birth. C. elegans uses a modified version of the conserved Wnt/beta-catenin signaling pathway to regulate its many ACDs in embryonic and larval development. The DNA binding protein TCF/POP-1 functions in the Wnt/beta-catenin asymmetry pathway to differentially regulate gene expression in the daughter cells resulting from an ACD. The ability of POP-1, to repress or activate gene expression relies on interactions with Groucho family corepressors or the coactivator beta-catenin/SYS-1, respectively. The Groucho corepressors function in fate determination and are expressed in asymmetrically dividing tissues, such as the seam cells, yet a role for these corepressors in ACD has not been demonstrated. For this reason, we investigated the function of Groucho in the seam cell lineage, which divides asymmetrically to produce a pluripotent seam cell and terminally differentiated hypodermal cell. Seam cell fate appears to heavily rely on repression rather than activation since knockdown of the POP-1 increases seam cell number while knockdown SYS-1 does not affect seam cell number. If Groucho were required for POP-1 repression in the unsignaled daughter after ACD, we expect to see duplication of the signaled fate following Groucho depletion, similar to the POP-1 RNAi phenotype. Surprisingly, no defects in seam cell fates following corepressor depletion were seen. However, we sensitized cells via POP-1 RNAi and determined that, at low levels of POP-1 knockdown, additional knockdown of Groucho results in an increase in seam cell number that resembles the full POP-1 knockdown phenotype. This enhancement provides evidence that Groucho functions in terminal differentiation of the dividing seam cells to confer hypodermal cell fate. Current efforts include testing the role of POP-1 domains that interact with SYS-1 and DNA, using specific pop-1 alleles in combination with Groucho loss-of-function. Preliminary evaluation of a transgenic strain encoding a POP-1 deficient in SYS-1 binding, pop-1(q645) coupled with a sys-1 hypomorph (q544), shows no significant defects in seam cell ACD. These data indicate that differential transcriptional repression between the two daughters provided by Grouchos, rather than activation SYS-1, may be the critical effect of Wnt signaling in the unsignaled daughter.
A Targeted Sequencing Study of Glutamatergic Candidate Genes in Attempted Suicide
Suicidal behavior has been shown to have a heritable component that is partly driven by psychiatric disorders. However, there is also an independent factor contributing to the heritability of suicidality. We previously conducted a whole exome sequencing study of bipolar suicide attempters and bipolar non-attempters to assess this independent factor. This whole exome study implicated glutamatergic neurotransmission in attempted suicide, as did our genome-wide association study (GWAS) of the attempted suicide phenotype. In the current study, we have conducted a targeted next-generation sequencing study of the glutamatergic N-methyl-D-aspartate (NMDA) receptor, neurexin, and neuroligin gene families in 476 bipolar suicide attempters and 473 bipolar non-attempters. The goal of this study was to gather sequence information from coding and regulatory regions of these glutamatergic genes to identify variants associated with attempted suicide. We identified 186 coding variants and 4,298 regulatory variants predicted to be functional in these genes. No individual variants were overrepresented in cases or controls to a degree that was statistically significant after correction for multiple testing. Additionally, none of the gene-level results were statistically significant following correction. While this study provides no direct support for a role of the examined glutamatergic candidate genes, further sequencing in expanded gene sets will be required to understand the role of glutamatergic signaling in the risk for suicidal behavior.
Soluble CR1 Gene therapy rescues renal phenotypes in a murine model of C3G
Xue Xiao1,2, Yuzhou Zhang1, Janice Staber3, Sanjeev Sethi5, Paul B. McCray, Jr.2,3,
Carla M. Nester1,3,4, Richard JH Smith1,2,3,4
1Molecular Otolaryngology and Renal Research Laboratories, Caver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 2Interdepartmental PhD Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 3, 4Departments of Pediatrics and Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 5Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
C3 glomerulopathy (C3G) encompasses two prototypical diseases, dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Both diseases are characterized by fluid-phase dysregulation of the alternative pathway (AP) of complement that leads to C3 deposition in the renal glomerulus. Unknown disease triggers and lacks of specific treatments lead to end-stage renal failure in 50% of patients. Soluble complement receptor 1 (sCR1) is a soluble form of a membrane bound regulator of complement. Short-term studies show that sCR1 is capable of restoring complement control in a murine model of C3G, the Cfh-/-/huCR1-Tg mouse. However, within days of terminating treatment, complement dysregulation is again evident. In this study, we sought to determine whether continuous presence of sCR1 could provide long-term complement control in the C3G murine model. Using the piggyBac transposon system coupled with hydrodynamic tail vein injection, we delivered a construct of sCR1 (LHR A-C) to the C3G murine model to provide constitutive sCR1 expression in mouse circulation. Animals were followed for 6 months. sCR1 expression was detected by real time PCR and ELISA in mouse liver and circulation respectively in 6 months. C3 levels approximately doubled and clearance of glomerular C3 and C3 fragments deposition was documented by immunofluorescence. Electronic microscopy showed a reduction in dense deposits in injected as compared to control animals. There were no changes by light microscopy. Renal function improvements had been revealed from stabilized 24-hr urine albumin creatinine ratio. In this study, we proved that long-term expression of sCR1 could rescue the renal phenotype in C3G mice and may be a viable treatment for patients with this disease.
THE ROLE OF ENDOGENOUS MICROBIOTA IN SEIZURE-LIKE BEHAVIOR OF SHUDDERER, A DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL MUTANT
It is widely recognized that mutations in genes encoding voltage-gated sodium (Nav) channels contribute to the etiology underlying various seizure disorders. Shudderer (Shu), a gain-of-function mutant for the Drosophila Nav channel gene, exhibits neuronal hyperexcitability and seizure-like behavioral defects, including spontaneous leg jerking, twitching, and heat-induced convulsion. Intriguingly, we have recently discovered that food supplemented with milk whey acts as a nutritional therapy, drastically suppressing these behavioral phenotypes. Microarray analysis revealed high levels of insulin receptor (InR) expression in Shu mutants relative to wild-type (WT) flies, indicating Shu has reduced insulin signaling. Following milk whey treatment, InR expression in Shu mutants returned to wild-type levels, suggesting milk whey increases insulin signaling. Because the endogenous gut microbiota are known to impact metabolic and developmental homeostasis through insulin signaling, we hypothesized that the microbiome plays a role in Shu phenotypes and their diet-dependent modification. Raising Shu mutants and WT flies in either antibiotic-containing or sterile food was sufficient to eliminate the gut microbiota. Further, both treatments were found to significantly suppress Shu behavioral phenotypes while having no obvious effect on WT behavior. Culturing extracts of homogenized flies on LB agar plates revealed drastic differences in the number and possibly the species of bacteria found in Shu and WT flies raised on conventional or milk whey-supplemented food. To confirm these results, we plan to perform high-throughput sequencing of the bacterial 16S ribosomal gene to identify differences in the gut microbiome composition of Shu and WT flies in the context of both conventional and milk whey-containing diets. This and future experiments are expected to provide us with a better understanding of the interplay between dietary therapy and the microbiome in the context of seizure disorders.
Homophilic protocadherin cell-cell interactions drive dendrite complexity
Growth of a properly complex dendrite arbor is a key step in neuronal differentiation and a prerequisite for neural circuit formation. Diverse cell surface molecules, such as the clustered protocadherins (Pcdhs), have long been proposed to regulate circuit formation through specific cell-cell interactions. Here, using transgenic and conditional knockout mice to manipulate g-Pcdh repertoire in the cerebral cortex, we show that the complexity of a neuron’s dendritic arbor is determined by homophilic interactions with other cells. Neurons expressing only one of the 22 g-Pcdhs can exhibit either exuberant, or minimal, dendrite complexity depending only on whether surrounding cells express the same isoform. Furthermore, loss of astrocytic g-Pcdhs, or disruption of astrocyte-neuron homophilic matching, reduces dendrite complexity cell non-autonomously. Our data indicate that g-Pcdhs act locally to promote dendrite arborization via homophilic matching and confirm that connectivity in vivo depends on molecular interactions between neurons, and between neurons and astrocytes.
The role of copy number variation in cleft lip and palate.
L. A. Harney1,2,3, B. W. Darbro1,3, A. Long2, J. Standley1, A.M. Hulstrand2,3, H. Liu4, R.A Cornell3,4, D.W. Houston2,3, J. C. Murray1,3, J. R. Manak1,2,3
1) Department of Pediatrics, University of Iowa, Iowa City, IA; 2) Department of Biology, University of Iowa, Iowa City, IA; 3) Interdisciplinary Genetics Program, University of Iowa, Iowa City, IA; 4) Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA
Clefts of the lip and/or palate (CL/P) occur in about 1 in 700 live births. Categorized as non-syndromic (NSCL/P) or syndromic (SCL/P), individuals with NSCL/P have isolated clefts and account for about 70% of clefting cases whereas syndromic occurrences include additional cognitive or structural anomalies. Although genome-wide association, candidate gene, and animal model studies have been used to study CL/P, a largescale analysis to determine the contribution of copy number variation (CNV) to CL/P has yet to be performed. We performed the largest high resolution array-based comparative genomic hybridization study to date to identify copy number variants associated with NSCL/P in a cohort of 868 cases from the Philippines and 212 individuals with SCL/P of mixed ethnicities. A preliminary analysis is underway which prioritizes likely causative CN events for follow-up in zebrafish and frogs. Focusing on rare copy number losses, we identified 196 genes that were deleted in greater than one individual while 735 genes were deleted in a single case; collectively, the majority of genes were not previously implicated in clefting. After comparing the list of deleted genes to OMIM, DECIPHER, NCBI, and MGI databases, four were selected for functional follow-up in zebrafish. These genes, ISM1, PKP2, MYO5C and ULK4, are all novel clefting candidates, are overlapped by a CNV loss in greater than one individual, and appear in less than 1% of the cohort. Six additional genes identified have been previously implicated in clefting through association studies (NTN1, PCYT1A), variant analyses (ZNF750, CDH1, OFD1), or chromosomal microarrays (IMMP2L).Together, these studies will define the contribution of copy number variants to disease incidence of CL/P.
TFAP2A drives melanocyte gene expression in parallel with MITF
H E Seberg1, E Van Otterloo2, S K Loftus3, J P Lambert4, G Bonde2, R Sompallae5, J F Santana1, J R Manak1, A C Gingras4, W J Pavan3, R A Cornell1,2
1 Interdisciplinary Graduate Program in Genetics, University of Iowa
2 Department of Anatomy and Cell Biology, University of Iowa
3 Genetic Disease Research Branch, National Human Genome Research Institute, Bethesda, MD
4 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
5 Bioinformatics Division, Iowa Institute of Human Genetics, University of Iowa
Disruption of the transcription factor network governing melanocyte development contributes to the pathogenesis of pigmentation disorders and melanoma. The activity levels of an important member of this network, MITF, have been proposed to control melanoma phenotype. Mid- to high-level MITF activity drives growth and differentiation, while lower levels confer a stem cell-like, invasive quality. Transcription Factor Activator Protein 2 alpha (TFAP2A) expression is reduced in advanced stage melanoma tumors, and mutations in TFAP2A cause pigmentation phenotypes in humans, mice, and zebrafish. Because TFAP2A is widely expressed in the neural crest and skin, its specific role in melanocytes and relationship to MITF has been unclear. To determine the position of TFAP2A in the melanocyte gene regulatory network, we first used microarray analysis of wildtype and tfap2a null zebrafish to profile genes that are downregulated in the absence of TFAP2A. We then conducted anti-TFAP2A ChIP-seq to create a profile of TFAP2A-bound loci in melanocytes. Genes at the intersection of these profiles are likely direct targets of TFAP2A. These include melanin synthesis genes, such as dct, tyrp1, and trpm1, most of which are also thought to be direct targets of MITF. Comparison with published MITF ChIP-seq showed that TFAP2A peaks overlap MITF peaks more often than expected by chance. In reporter assays, deletion of TFAP2A binding sites in a minimal TRPM1 promoter decreased its activity, similar to published results for deletion of MITF binding sites from this element. Furthermore, we found that tfap2a and mitfa interact both physically in vitro and genetically in zebrafish. These results provide evidence that TFAP2A and MITF work in parallel to promote melanocyte differentiation, and show that widely-expressed TFAP2A can directly regulate expression of lineage-specific melanocyte genes. In addition, TFAP2A expression may be able to influence levels of MITF, driving cells toward differentiation and away from an invasive state.
Eric’s Talk Title
Investigating the Human Exome in Suicidal Behavior
|9/17/2015||Eric Monson||12:00-12:50 PM||ML B111|
|10/15/2015||Lisa Harney||12:00-12:50 PM||ML B111|
|11/12/2015||Michael Molumby||12:00-12:50 PM||ML B111|
|12/10/2015||Sophia Gaynor||12:00-12:50 PM||ML B111|
DEFICIENCY SCREENING; IDENTIFICATION OF GLUTATHIONE S-TRANSFERASE 1 AS A GENETIC MODIFIER OF THE DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL GENE, PARALYTIC
Hung-Lin Chen, Toshihiro Kitamoto
Voltage-gated sodium (Nav) channels are essential for generation and propagation of action potentials in neurons. Dysfunction of Nav channels often causes neuronal hypo- or hyper-excitability, resulting in a variety of neurological disorders, such as epilepsy. Epilepsy is one of the most common neurological disorders in the world. As estimated 50 million people worldwide suffer from this devastating pathological condition and a significant portion of patients (~30%) do not respond to anti-epileptic drugs (AEDs). Thus, identifying new target genes for AEDs is of emergent clinical need. In this study, I take advantage of Drosophila genetics to identify modifier genes that can reduce neurological phenotypes caused by mutations in the Nav channel gene, paralytic (para). Shudderer (Shu) is a mutant allele of para, displaying neuronal hyper excitability as well as behavioral and morphological abnormalities. These include seizure-like behaviors, down-turned wings, and indented thorax. Here we carried out a deficiency screen to identify modifiers of Shu. Our working hypothesis is that the Shu mutant phenotypes are enhanced or suppressed when the activity of genes functionally interacting with Shu is reduced by 50%. We systematically introduced molecularly defined deficiencies into the Shu mutant background and tested if it results in a modification of the mutant neurological phenotypes. After morphological and behavioral analyses, we identified a short genomic deletion in the second chromosome that can ameliorate Shu’s phenotypes. This deletion covers six genes. By using RNA interference and P-insertion mutants, we narrow down the modifier of Shu to glutathione s-transferase 1 (GstS1). GstS1 mutations reduced frequency of seizures in another Drosophila Nav channel mutant that carries a mutation causing genetic epilepsy with febrile seizures plus (GEFS) in humans. Immunochemistry showed a GstS1 mutation increases GABA levels in the Shu mutant brain. It indicates that deletion of GstS1 may reduce neurological phenotype of Shu by enhancing GABAergic inhibition. Since Drosophila and human share fundamental biological pathways, my study may provide a new direction for AED development.
THE ROLE OF MATERNAL WNTS AND FRIZZLEDS IN DORSO-VENTRAL AXIS SPECIFICATION
Melissa Marchal1 and Douglas Houston1
1The Interdisciplinary Program in Genetics and The Department of Biology, University of Iowa, Iowa City, IA, 52242
The proper localization of maternal mRNAs and proteins in the egg is required for many developmental processes, including dorsoventral (DV) axis determination. In Xenopus laevis in particular, maternal factors involved in patterning the DV body axis are localized during oogenesis to the oocyte vegetal cortex. Upon sperm entry, these determinants are translocated to the future dorsal side of the embryo, in a microtubule dependent process called cortical rotation. The major outcome of cortical rotation is the asymmetric activation of the Wnt/β-Catenin signaling pathway in dorsal nuclei, where β-Catenin acts to transcriptionally activate dorsal-specific genes at the midblastula transition. However, the upstream mechanisms initiating β-catenin stabilization have remained elusive. Recent evidence has suggested that secreted Wnt ligands Wnt11 and Wnt5a may act together in axis formation. However, the function of other maternally expressed Wnts and their cognate receptors (frizzleds) remains uncharacterized. We have examined the expression of wnts and frizzleds (fzds) in the oocyte and throughout early development. Through this analysis, we have found that fzds 1, 4, 6, and 7 are expressed abundantly in the oocyte, with fzd7 maintaining high expression levels until the tailbud stages. Using a maternal depletion approach we have begun to identify the roles of these maternal factors in DV axis patterning. We present evidence that maternal fzd1-depleted embryos show a ventralization phenotype and partial defects in dorsal-specific gene expression, while fzd4-depleted embryos show a dorsalization phenotype and the expansion of dorsal-specific markers. Additionally, we show that oocytes depleted of fzds 1, 4, and 7, have significant microtubule defects, suggesting that Frizzled-dependent signaling may play a role in cortical rotation.
Genome-wide Expression Profiles in Drosophila yakuba and D. santomea.
Population genetic models predict that the X chromosome will evolve at a faster rate than autosomes (i.e., faster-X evolution). The basis for faster-X evolution is that genes are under the highest level of selection in males. Evaluations of protein-coding sequences have indeed shown an excess of divergence on the X chromosome compared to autosomes, particularly in genes expressed higher in males than in females (i.e., male-biased genes). In addition, whole-genome analyses of gene expression in Drosophila yakuba and D. santomea males indicate that X-linked genes are more differentially expressed between species (i.e., faster-X evolution of gene expression) than autosomal genes. This trend is once again strongest for male-biased genes. However, these studies utilized only males and were therefore limited in their expression profiles. Here, we will investigate the whole-genome profiles of D. yakuba and D. santomea males and females to determine the relative rates of evolution for all gene classes. We also assess the way that divergent genes are inherited in hybrid and the regulatory factors that influence their expression patterns.
Whole Exome Analysis of Individuals and Families with CRMO
For my primary research project, I am working to determine the genes and pathways involved in the development of chronic recurrent multifocal osteomyelitis (CRMO), a rare autoinflammatory bone disease presenting in infancy and childhood. We currently have whole exome sequence data from 35 individuals with CRMO. For six of the isolated cases, we have data for one or two relatives with inflammatory disease, and for three pairs of sisters and three individuals with CRMO, we have exome data for both unaffected parents, for a total of 53 exomes. Nearly all of the probands have close relatives with psoriasis or Crohn’s disease. For all of the exome data, I have processed the data from fastq to vcf format using the Burrows-Wheeler Alignment (BWA) software, SAMtools, Picard, and the Genome Analysis Toolkit (GATK). Preliminary analysis of the data suggests that variants in genes involved in IL-17 and RANK signaling are enriched in our CRMO cohort, and I am currently working on developing and analyzing a control dataset for comparison using the publicly available 1000 genomes, EVS and ExAC databases. Additionally, our laboratory will send an additional 26 samples for exome sequencing this summer – the samples are 6 individuals with CRMO and their unaffected siblings and parents. I am also performing an experiment this summer to determine the effect of a putative enhancer mutation in the first intron of a candidate gene. The variant is enriched in our CRMO cohort and likely disrupts an NR4A2 binding site.
Characterization of the NPHP10/AIMP2 interaction
Nephronophthisis (NPHP) is a recessive kidney disorder that is the leading cause of early onset, end-stage renal failure. Many proteins mutated in cystic kidney disease have been shown to localize to the primary cilia and centrosomes, providing a coalescing mechanism for NPHP-related ciliopathies (NPHP-RC). Aside from renal failure and kidney cysts, retinal degeneration and dysplasia or degeneration of the cerebellum are also seen in many NPHP-RCs. SDCCAG8 is a nephronophthisis gene (NPHP10), with patients exhibiting retinal and renal abnormalities, obesity, and learning disabilities. Mutations in SDCCAG8 were also found in several BBS patients, making SDCCAG8 the 16th BBS gene (BBS16). However, little is known about the molecular functions of NPHP10 and how loss of NPHP10 function leads to the observed phenotypes. Our research has shown that NPHP10 interacts with components of the multi-aminoacyl tRNA synthetase complex (MSC), including 8 out of 9 aminoacyl tRNA synthetases (ARS) as well as aminoacyl-tRNA-synthetase-complex interacting multifunctional protein 2 (AIMP2). Further work determined that a direct interaction likely occurs between NPHP10 and AIMP2. Our current work focuses on determining the biological significance of this interaction.
Consequences of Recombination rate variation among Drosophila Melanogaster populations
Recombination is a crucial biological process that shapes evolutionary change within and between species. At the same time, accurate estimates of recombination rates are essential for correct inferences of selection and demographic events. In this study, we use the most accurate population genetic method, LDhelmet, to estimate and compare recombination rates in three Drosophila melanogaster populations. Recombination rates not only change in total magnitude but also in their relative distribution within chromosomes (landscapes). We show that differences in recombination landscapes between populations do not accumulate at the same rate than nucleotide differences. We also show that population-specific differences in recombination landscapes play a significant role explaining population-specific differences in nucleotide diversity. Our results suggest that inter-population differences in local recombination rates and the corresponding differences in local Background Selection (BGS) need to be considered as a possible explanation for population-specific differences in nucleotide diversity at specific genomic regions.
A NATURALLY OCCURRING MUTATION IN THE FERROUS IRON UPTAKE GENE FEOB CONFERS ENHANCED RESISTANCE TO OXIDATIVE STRESS IN A FRANCISELLA TULARENSIS VACCINE STRAIN
J Fletcher1, C Bosio2, B Jones1,3
1 The Interdisciplinary Graduate Program in Genetics, University of Iowa
2 Laboratory of Intracellular Parasites, Rocky Mountain Labs, National Institute of Allergy and Infectious Diseases Department of Microbiology, Carver College of Medicine, University of Iowa
3 Department of Microbiology, Carver College of Medicine, University of Iowa
Francisella tularensis is a highly virulent bacterial pathogen with an extremely low infectious dose (~10 CFU) and high rates of mortality if left untreated (30-60%). F. tularensis has an extensive history as a bioweapon, and there is no vaccine currently licensed. For these reasons the CDC considers F. tularensis a Tier 1 Select Agent. The unlicensed Live Vaccine Strain (LVS) provides moderate protection against virulent strains; however, we have recently discovered that various lab stocks differ in their virulence and ability to confer immunity. Genome sequencing of high virulence (RML, LD50 ~200 CFU) and low virulence (ATCC, LD50 ~9,000 CFU) strains has identified nine differences, of which four are non-synonymous substitutions. One such mutation occurs in the ferrous iron uptake gene feoB in RML. While iron is required for cellular function, ferrous iron can participate in the Fenton reaction with H2O2, leading to inactivation of essential iron-sulfur cluster enzymes, and DNA damage. Part of the innate immune response involves the oxidative burst in the phagosome and mitochondria-derived ROS in the cytosol. Fully virulent strains of F. tularensis are known to be highly resistant to such host defences, and have low levels of intracellular iron. Accordingly, the RML strain was highly resistant to exogenous H2O2 in vitro relative to the ATCC strain. Overexpression of the ATCC feoB allele, but not the RML allele, leads to significantly increased sensitivity to H2O2. Furthermore, the RML strain grows poorly under conditions of iron starvation, and an iron-responsive lacZ reporter had ~3-fold higher activity in the RML strain relative to ATCC under these conditions. Overexpression of the iron-responsive transcriptional repressor fur leads to reduced growth in the RML strain, but not ATCC. These results are consistent with the hypothesis that RML has less intracellular iron, and that this may lead to increased resistance to host-mediated oxidative stress.