Tube formation in the fruit fly (Drosophila melanogaster) egg chamber is an excellent model for vertebrate neural tube formation, as they closely resemble each other. During egg chamber development, a subset of cells, the roof cells and the floor cells, form two tubes that make eggshell structures called dorsal appendages (DAs). Roof cells, which express the protein Broad, constrict their apices (inner surfaces) to curve up and out of the epithelium. The floor cells elongate, dive under the roof cells, and zipper together to seal the floor; they are distinguished by expression of the protein Rhomboid. Defects in this process, especially in floor closure, create open tubes, similar to spina bifida. To observe cell movement in the developing egg chamber, we want to outline the shapes of cells with a fluorescent molecule. mCherry is a red fluorescent protein that has good fluorescence and a short maturation time (15 minutes), so we can observe the rapid process of tube formation. If one links the CD2 gene, which encodes a cell membrane adhesion protein, to the mCherry gene, red fluorescence will appear all around the cell surfaces. Such a fusion protein lets us observe changes in cell shape and cell movements during tube formation. When the CD2-mCherry segment is connected to the regulatory region for the rhomboid gene, fluorescence occurs in just the floor cells. I have created a rhomboid-CD2-mCherry construct that I will introduce into flies through injection with a recombinase that will integrate the construct into the fly genome. With this construct I can visualize DA tube development live by observing fluorescence in the floor cells. Analysis of these cells can give insight into conserved tube-forming processes.

Mast cells regulate both inflammatory responses and tissue repair in human diseases but there are conflicting reports on the role of these cells in the pathogenesis of various kidney diseases. Interleukin-6 (IL-6) is an interleukin that acts as both a pro-inflammatory and anti-inflammatory cytokine. From our preliminary studies we have demonstrated that IL-6 and its receptor gp130 are pro-fibrotic in renal fibrogenesis. Here we measure mast cell function in unilateral urethral obstruction, a well-characterized model of renal fibrosis, using mice in which Cre recombinase is expressed under the control of a segment of the carboxypeptidase A3 (Cpa3) promoter bred to IL-6 flox/flox mice. Mast cells from the C57BL/6 Cpa3-IL-6 (fl/fl) mice do not express IL-6. We have groups of 10 for each of the following strains: control1; Cpa3-Cre, control2; IL-6fl/fl and experimental mice; Cpa3-IL-6. Mice under general anesthesia had the ureter ligated at 2 sites distally from the kidney and sacrificed after 14d. We measured the level of fibrosis by measuring hydroxyproline colorimetrically and by measuring area of Picro Sirius red in stained kidney sections, and quantified by pixel analysis. Our preliminary results indicate that the absence of IL-6 in mast cells is important in renal fibrosis. When IL-6 was ablated from mast cells the level of fibrosis measured by collagen and the expansion of extracellular matrix was reduced. These preliminary results suggest that mast cell-derived IL-6 promotes renal fibrosis after unilateral urethral obstruction.

Quorum sensing is cell density-dependent communication that regulates many functions in bacteria. Signal synthases (I) produce acyl-homoserine lactones, which diffuse into the environment and activate signal receptors (R) when they reach a critical concentration. Synthases and receptors often form cognate pairs. The soil bacterium Burkholderia thailandensis encodes three I-R pairs. The I-R1 system controls cell-cell aggregation and activates many putative aggregation factors including the bce genes that likely encode an exopolysaccharide. The I-R2 system controls antibiotic production. The antibiotics are members of the bactobolin family and are effective against both Gram-positive and Gram-negative bacteria. Both aggregation and antibiotics are believed to promote competitiveness in the diverse soil communities where B. thailandensis is found. We have developed genetic screens to learn more about quorum-controlled antibiotics and aggregation in B. thailandensis. The first screen identified bactobolin-resistant isolates of the soil bacterium Bacillis subtilis, which is normally sensitive to bactobolin. We submitted these mutants for whole genome sequencing to investigate the mutations that confer resistance, and with our results we hope to gain insight into how bactobolin acts on target cells. The second screen was designed to isolate variants that overproduce aggregation factors. Quorum sensing mutants do not wrinkle but variants arise that produce aggregation factors by other means. We are isolating such variants and identifying the underlying mutations to understand how they compensate for the loss of quorum sensing regulation. This may reveal the elements involved in regulating aggregation. These variants will also be useful to learn about the advantages of aggregation in different contexts. Through our two screens we hope to better understand quorum-controlled aggregation and antibiotic phenotypes. Our findings will be useful in further studies of the importance of each of these in competition.

Methylobacterium extorquens AM1 is a methylotrophic bacteria ubiquitous in the environment and in particular on the undersides of leaves. This organism is able to metabolize both single carbon compounds like methanol and multi-carbon compounds such as succinate and pyruvate. M. extorquens has been studied for decades because of its potential for not only creating value added compounds from methanol, but important regulatory systems including the regulatory pathway of methanol metabolism, which has yet to be fully elaborated. Our laboratory focuses on single carbon compound metabolism and the regulation of the pathways required for growth. The first step in methylotrophic growth is the oxidation of methanol via methanol dehydrogenase encoded by the mxaFI genes. Promoter fusion data suggests a complex regulatory system involving two two-component systems MxbDM and MxcQE, two homologues of the main methanol dehydrogenase XoxF12, and an unknown transcriptional repressor. In order to elucidate this process, the direct binding of a transcriptional regulator to the mxaF promoter region must be demonstrated. Unfortunately all putative regulators have historically failed to show activity in vitro. In order to isolate direct regulators, whole cell extract will be washed over an abundance of the mxa promoter region that is bound to magnetic beads. Then using a magnet, the promoter region can be removed along with proteins that have bound from the cell extract, any non-specific binding proteins can be washed off using a low salt buffer, and specific binding proteins removed using a high salt buffer. The elutions containing the proteins of interest can be run out on an SDS PAGE gel, and the unique bands of interest excised and sent for mass spectrometry analysis for identification. Our work will define transcriptional regulators of the mxa operand, determining which specific proteins are binding the promoter region of that operand.

Bacterial conjugation is a form of horizontal gene transfer that serves as an important source of genetic plasticity and diversity in both Gram positive and Gram negative bacteria. It has also been proved to be advantageous for dissemination of antibiotic resistance. Conjugation is a plasmid-mediated mechanism that allows single stranded DNA to be transferred across cell membranes from a donor to a recipient cell. In Escherichia coli, the conjugative F (fertility) plasmid encodes all necessary proteins for assembling the transfer apparatus thus allowing transfer and propagation of DNA into a recipient bacterial cell. Previous conflicting studies of bacterial conjugation in E. coli have shown DNA transfer to occur between donor and recipient pairs in intimate cell-cell contact or between distant donor and recipient pairs, connected only by the pilus, a filamentous protein extended from the donor cell. Using various fluorescent protein constructs to differentially mark donor and recipient cells and F plasmid DNA itself, we visualize conjugation in real time with single-cell resolution by fluorescent microscopy. Here we show that conjugation occurs only when donor and recipient cells are in intimate and close physical contact with each other. In addition, there appears to be no preference for cellular orientation for transfer. Furthermore, we propose a simple rate equation to model DNA transfer between bacteria and measure the rate of DNA transfer per unit time per cell contact. This quantitative model is a tool to elucidate conjugation at a single-cell scale and will provide a deeper understanding into the mechanism of bacterial conjugation.

Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. Last summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. We constructed a strain of Escherichia coli that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. Using this method, we were able to produce on average 160 mg of alkanes per liter. Also, we identified a protease with gluten-degradation potential, and then reengineered it to have 100 times increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. Finally, to enable next-generation cloning of standard biological parts, BioBrick vectors optimized for Gibson assembly, an emerging DNA cloning technique, were constructed and used to construct the Magnetosome Toolkit: genes for biofabrication of magnetic particles.

Our goal is to use human adenovirus to investigate how human defensins interact with viral capsids to neutralize infection. Defensins are small antimicrobial peptides of the innate immune system that prevent bacteria and viruses from infecting their host, but the mechanisms by which defensins neutralize viruses are not yet well known. For human adenovirus (HAdV), we have shown that infection by serotype 5 (HAdV-5) is completely sensitive to defensins while that of HAdV-19c is resistant. Adenovirus is a non-enveloped DNA virus with an icosahedral capsid comprised primarily of the hexon protein. Fiber proteins attached to penton base proteins at the icosahedral vertices interact with host cells during cell entry. Previous studies showed that penton base and fiber proteins contain specific sequences important for conferring sensitivity to defensins. In order to identify these sequences in the HAdV-5 penton base protein, we constructed a number of chimeric viruses in which parts of the HAdV-5 penton base are replaced with the HAdV-19c penton base. Replacing the last half of HAdV-5 penton base, but not just the third or fourth quarters, with the corresponding sequence from the HAdV-19c penton base resulted in significant resistance to defensins. Based on these results, we created new chimeras by replacing the entire third quarter in combination with specific residues in the fourth quarter of the HAdV-5 penton base in order to determine exactly which residues confer resistance to defensins. After identifying these residues, we will create new chimeras by replacing the fourth quarter and mutating specific residues in the third quarter of the penton base. Together with quantitative binding studies to measure the impact of these changes on the defensin-adenovirus interaction, these studies will provide insight into the molecular mechanism of defensin-mediated neutralization of adenovirus that may be generalized to other non-enveloped viruses that are sensitive to defensins.

Congenital heart defects are the most common form of birth defect, affecting about one out of every 100 newborns in the US. Ten percent of these afflicted newborns will experience a narrowing of their aorta, formally called coarctation of the aorta. Coarctation can result in extreme hypoxia in tissues that do not receive sufficient blood supply, and can eventually lead to congestive heart failure; surgical repair is required for the infant to regain normal circulation. The etiology of coarctation has been linked in many cases to migration of cells from the ductus arteriosus into the aorta. The ductus arteriosus is a fetal vessel that must close at birth, and does so by constriction of its smooth muscle cells until its lumen is occluded. If these contractile cells migrate into the aorta, they can constrict, narrowing the aortic lumen. Preliminary data from experiments on the region where the ductus inserts into the aorta show a differential expression of chemorepulsive signaling proteins from the Slit and Robo families in ductal versus aortic tissue, suggesting that a distinct tissue boundary forms in this region. This is supported by previous studies in lineage mapping, which demonstrated a distinct somite-neural crest smooth muscle lineage boundary in the aortic arch. In order to elucidate the role that Slit and Robo proteins play in determining the ductus-aorta boundary, I use reverse transcription PCR analysis to examine the expression of Slit-Robo ligand and receptor proteins in perinatal ductal and aortic tissue. By comparing expression in normal samples with samples from patients with coarctation, more insight into the influence of Slit-Robo chemorepulsive signaling on boundary formation will be gained. Characterization of the ductus-aorta boundary by identification of differential ligand-receptor expression is critical to understanding the role that abnormal boundary formation plays in coarctation of the aorta.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis. Components of the outer and inner membrane, characteristic of Gram-negative bacteria, contribute to Salmonella’s mechanisms to avoid detection and killing by the host immune system. The anionic bacterial cell surface is disrupted by cationic antimicrobial proteins (CAMP) that increase outer membrane (OM) permeability and access to the inner membrane. To resist CAMP killing Salmonella activates the PhoPQ two-component system, which is essential for pathogenesis. Because many PhoPQ-regulated enzymes are dispensable during infection, we conducted a forward genetic screen that identified new components of the PhoPQ regulon involved in maintaining the OM permeability barrier. Sequencing revealed mutations in genes encoding a variety of membrane proteins including transporters, phospholipid and lipopolysaccharide biosynthetic enzymes and efflux pumps, as well as regulatory proteins. Amongst the results, yejM mutants expressed the highest levels of membrane damage. YejM is an inner membrane protein with a periplasmic enzymatic domain that is critically involved in maintaining the OM permeability barrier. Structural alignments suggest that YejM is a member of the alkaline phosphatase superfamily. Attempts at full length deletions of yejM were unsuccessful suggesting the transmembrane domain is essential for viability, whereas periplasmic deletion mutants were viable but expressed varied levels of OM damage. Complementation with periplasmic deletion mutants and plasmids carrying the periplasmic domain rescued the OM permeability defect. Using structural models generated by a Protein Homolog/analogy Recognition Engine (PHYRE), we located putative metal binding sites involved in catalysis. To investigate the enzymatic mechanism, specific metal-binding residues were mutated via Polymerase Chain Reaction. We aim to determine if these residues contribute to the OM permeability defect of the yejM mutant Salmonella.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction is one promising pro-longevity intervention, though like other aspects of longevity, its mechanism is also poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan and has been found to delay the onset of age-associated disease, not only in C. elegans, but in a wide range of species including yeast, flies, mice, and primates. We are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We will follow up on our collected data by measuring the full lifespans of all mutants that respond strongly in the screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

In any niche occupied by multiple species, competition is inevitable; whether through killing opponents, or depleting resources first. Ecological theory posits the existence of r- and K-selected competitors. r-selected organisms colonize well and reproduce quickly (often at the expense of individual survival). K-selected organisms succeed in stable, dense environments and are typically larger and live longer; typically investing in self defense mechanisms. This observed quantity vs quality competition is the foundation for the r/K selection theory. One limitation of existing r and K models is that factors causing organisms to be either r or K selected are not clearly defined. Release of toxic substances (i.e., interference competition) is an example of a factor that could allow for a species to be good at excluding others near the carrying capacity. To study this, we created a bacterial warfare model with two antibiotic producing bacteria, Chromobacterium violaceum (Cv) and Burkholderia thailandensis (Bt). These species were competed in at a 1:1 ratio and were disturbed (re-plated via coring) at varying intervals. Both bacteria produce their antibiotics when cell density is high. Cv behaves like an r-selected organism in that it grows quickly but has a weak antibiotic. Bt shows characteristics of K-selected organisms, growing slower, but creating antibiotics that rapidly kill Cv. Our initial observations show that Cv succeeds in frequently disturbed environments by not allowing Bt to effectively reach quorum, while Bt wins in more static environments where its ability to produce potent antibiotics to pay off. Also, controls indicate while Cv's antibiotic provides an edge, its growth rate appears to be the determining factor for success. These results provide evidence that disturbance frequency acts as a primary selective force in the antibiotic mediated competition, and may provide parallels and contrasts to other systems where r and K selected organisms compete.

The type VI secretion system (T6SS) is a large protein complex utilized by bacteria to transport proteins into other cells. Initially implicated in virulence, the T6SS also participates in interbacterial interactions. A T6SS found in the opportunistic pathogen Pseudomonas aeruginosa, the hemolysin coregulated protein secretion island I T6SS (H1-T6SS), has been found to deliver a set of toxins to other bacteria. This provides P. aeruginosa a fitness advantage over competing bacteria. The H1-T6SS can target neighboring P. aeruginosa, which protects itself with cognate immunity proteins. Deleting these immunity proteins leaves P. aeruginosa susceptible to self-attack. However, the H1-T6SS as well as other T6SSs can target other bacteria, specifically they only target certain species. One of the goals of this project is determine the nature of such specificity. These findings would implicate new functions for the H1-T6SS. To further this, I am seeking to extend the scope in which the T6SS is viewed, both mechanistically and genetically. In doing so I am searching for new genetic loci that participate in T6SS-dependent interactions. Taken together, this work provides insights into a new aspect of type VI secretion and may change the genetic composition of T6SSs.

Chronic infections with Pseudomonas aeruginosa are a significant cause of deterioration of lung function in patients with cystic fibrosis (CF), and have substantial clinical importance due to their marked resistance to treatment with antibiotics. Furthermore, existing laboratory methods for testing bacterial susceptibility to antibiotics are poor indicators of clinical response to therapy, which further increase the difficulty associated with treating these infections. Current research shows that phenotypically diverse P. aeruginosa subpopulations evolve within a single patient during prolonged infection, and that clinical antibiotic therapy causes some of these subpopulations to change significantly in relative abundance. Observed changes in abundance following antibiotic treatment signify varying degrees of antibiotic susceptibility within each patient’s P. aeruginosa population as a whole, with an increase in a particular subpopulation abundance indicating resistance, and a decrease in a particular subpopulation abundance indicating sensitivity. As previous studies have only evaluated P. aeruginosa antibiotic sensitivity prior to antibiotic treatment, we attempted to demonstrate the correlation between an increase subpopulation abundance following clinical treatment and the degree of resistance to antibiotics in laboratory testing. This was done by evaluating the antibiotic minimum inhibitory concentration (MIC) of a range of isolates collected from CF patients who had undergone antibiotic therapy, using antibiotics which had been given to each patient in clinical treatment. Our results demonstrate a marked lack of correlation between relative subpopulation abundance and antibiotic MIC, with clinically resistant subpopulations showing the same or lower MIC than subpopulations which were clinically sensitive treatment. We conclude that isolates resisting treatment do not have a different MIC than isolates responding to treatment, suggesting that mechanisms other than classic antibiotic resistance are responsible for treatment resistance in the CF lung. Further studies to evaluate these mechanisms, such as biofilm formation and formation of persister cells, are currently under way.

 Mycobacterium tuberculosis infection is a leading cause of death worldwide, but the full details of how the human immune system responds to these bacteria are not known. Our lab studies CD1 proteins which allow T-cells to respond to bacterial lipids. We discovered that low expression of CD1A on dendritic cells is associated with an increased susceptibility to tuberculosis; however, the molecular details of CD1A-deficiency are not known. I cloned and analyzed the CD1A promoter from four CD1A-deficient and five control individuals. I identified twelve genetic variations in the CD1A promoter. Of these, eight were single nucleotide polymorphisms (SNPs), three were insertions or deletions, and one was a variable tandem repeat. One SNP (-199 T/C) and the variable tandem repeat showed an association with CD1A-deficiency. A T/T genotype at position -199 from the transcription start site correctly identified five out of five control subjects while a C/C genotype identified three out of four CD1A-deficient subjects. The variable tandem repeat was a string of 20 to 60 thymine bases. The CD1A-deficient subjects showed uniform repeat lengths, while the repeat lengths among control subjects were variable. These data identify two loci within the CD1A promoter that will be the target of future studies aimed at understanding the molecular mechanism of CD1A-deficiency.

Different species within the bacterial genus Francisella are pathogenic in mammals. Recently, an emerging pathogen in fish, F. noatunensis, has been causing significant mortalities in tilapia and Atlantic cod. The Francisella Pathogenicity Island (FPI) is a gene dense region on the Francisella chromosome that encodes essential virulence factors for the mammalian pathogens F. tularensis and novicida. To determine if the functionality of virulence factors has been conserved for the genus, a gene knockout for the pathogenicity determinant protein A (pdpA) gene in the F. noatunensis FPI was generated as a potential vaccine candidate. PdpA has been shown to be essential for the ability of Francisella to cause disease for the human pathogen. The F. noatunensis knockout was generated via homologous recombination using a suicide vector containing an antibiotic cassette flanked by portions of pdpA. Since polarity can be an issue for knockouts in gene dense regions, two different pdpA mutants were generated in opposing polarity. It has recently been shown that F. noatunensis infection in zebrafish closely mimics Francisella infection in mammals implying conserved virulence strategies for the genus. Therefore, zebrafish were used to assess the level of attenuation for the F. noatunensis pdpA knockouts generated in our laboratory. In addition, iglC is another gene found in the FPI that results in attenuation after it has been inactivated in the human pathogen. Our laboratory has acquired an iglC mutant for F. noatunensis from a collaborator that has reduced virulence in tilapia and will serve as a reference for our pdpA knockout infections in zebrafish. The results from this study will show the physiological similarities within the genus relative to their phylogenetic relationships.

In developing countries nearly 80% of all diseases are linked to water and sanitation issues; globally, over two million people die annually from water-born diarrheal disease. It has long been known that some metals can exhibit powerful anti-bacterial properties. Copper has been used for hundreds of years as a means to purify drinking water but only recently have scientists begun to elucidate the mode of action for copper induced microbial death. In 2008 the U.S. Environmental Protection Agency officially registered copper alloys as biocidal agents. Now these alloys need to be studied to determine efficacy at different concentrations and with a variety of disease-causing microbes. This study investigates the powerful bactericidal effects of copper as a low cost accessible method to purify drinking water of pathogenic waterborne microorganisms. More specifically, E. coli was used as a model organism to test the efficacy of solid copper at killing microorganisms in drinking water over variable amounts of time. Efficacy of cell death was assessed with viable plate counts. Results show that ≥48cm2 of solid copper in 100 mL of water can kill E. coli (at an average concentration of 3.1x105 CFU/mL) within 24 hours. Further research is needed to determine the optimal copper to water volume ratio as well as the precise timing of cell death. It is also essential that the copper ion concentrations remain below EPA limits of 1.3 mg/L; preliminary findings produced inconsistent values. The results of this research could be beneficial to those living in locations where safe potable water is not readily available, or for those travelling to remote or foreign locations as well as hikers and campers.

The secretion of signals or toxins is a vital aspect of bacterial communication with its environment. We recently found that type VI secretion systems (T6SSs) in Gram-negative bacteria mediate interbacterial interactions by translocating protein effectors into neighboring bacterial cells, providing a large fitness advantage. One protein exported by the T6SS is a type VI-exported (Tse) cell wall amidase that degrades peptidoglycan, and secretion of this effector, Tse1, results in target cell lysis. Bacteria can also self-intoxicate by T6S, potentially posing a problem for the survival of a given species. However, bacteria encode cognate immunity (Tsi) proteins that bind and inactivate their Tse partners. As we identify more T6 effectors, it is becoming clear that Tse–Tsi pairs are part of a widespread protein–protein recognition network underlying bacterial diversity and community structure. This study sheds light on how this network influences interbacterial interactions by uncovering the mechanism of Tsi1-dependent inhibition of Tse1 and defining the molecular determinants of this recognition. I have reconstituted the complex in vitro by using recombinant proteins to probe for direct interaction. Then, by mapping conserved residues onto the recently solved structure of Tse1, I have identified and mutationally analyzed candidate surface residues that may be involved in Tsi1 recognition. Further understanding the determinants of this interaction may allow us to predict which bacterial species are vulnerable to Tse1 or provide a template for the design of novel effector-immunity complexes that escape recognition in naturally occurring bacteria as an antibacterial strategy.

E. coli are a species of bacteria that naturally inhabit the mammalian intestinal tract. They can thrive in environments with high fluid shear like the colon or the urinary tract due to their sticky hair-like projections called fimbriae which allow them to grip epithelial lining. These fimbriae are primarily composed of a coiled chain of a repeating protein unit called FimA. This helical coil unwinds in a gradual fashion when pulled and our goal is to see how mutating certain amino acids (all proteins are made of sequences of amino acids) inside FimA can affect the energy required to uncoil the helix. A microscopic cantilever is applied to different mutants for E. coli fimbria and an atomic force microscope records how much force the fimbria exerts back during a pull, indicating the helix uncoiling and how much force it requires. We hope to find mutants that uncoil more easily or not at all which would illuminate which amino acids in FimA are active in maintaining the helix and its uncoiling flexibility. The findings would have many applications practically and for expanding research. Rigid helices are a valuable test group for examining fimbria behavior independent of the effects uncoiling while the amino acids isolated as important can help design target drugs against E. coli infections in urinary tracts by weakening their binding. The protein structure knowledge earned is also of biomedical value as it can help design novel adhesives using bacterial fimbrial protein.