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The adenovirus small e1a protein is a potent transcriptional modulator. Following infection of IMR90 cells, e1a causes a dramatic decrease in H3K18ac, a robust induction of genes involved in cell cycle progression, and a repression of genes involved in fibroblast homeostasis. Small e1a must interact with both RB and p300/CBP to transcriptionally reprogram IMR90 cells and to transform rodent cells. Our lab has conducted chromatin immunoprecipitation experiments followed by massive parallel sequencing (ChIP-seq) for Pol II following infection of IMR90 cells with adenoviruses that express wild-type e1a and e1a mutants that do not bind to p300/CBP (p300 - e1a) and RB (RB e1a). We have also conducted corresponding RNA-seq experiments following infection of IMR90 cells with our panel of e1a adenoviruses. Preliminary analysis of ChIP-seq studies for Pol II and RNA-seq data indicates a strong positive correlation between Pol II enrichment and RNA levels in uninfected cells and cells infected with an adenovirus that expresses wt e1a. Additional analysis will need to be carried out to determine the significance of Pol II localization and distribution. The genome-wide distributions of p300 and e1a remain unknown. Therefore, we propose to conduct ChIP-seq studies for p300 and e1a. We hypothesize that e1a relocalizes p300 genome wide and that e1a will colocalize with p300 and RB.
A BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF AZOC, THE AZOREDUCTASE ENZYME OF
CLOSTRIDIUM PERFRINGENSJessica Morrison, Shuo Dai, Jie Ren, Amanda Taylor, Mitchell Wilkerson, Cristee Wright, Aihua Xie, Gilbert John.
Oklahoma State University, Stillwater, OK.
Azo dyes are used widely across industries as colorants. Many microorganisms are able to reduce azo dyes by use of an azoreductase enzyme. It is through the reduction of the azo bonds of the dyes that carcinogenic metabolites are produced. The field of research on azoreductases is growing, but there is very little information available on azoreductases of strictly anaerobic bacteria. The azoreductase gene was identified in Clostridium perfringens (AzoC), a strict anaerobe that is found in human intestinal tracts. AzoC was biochemically characterized via UV-VIS spectroscopy and was found to have high activity, especially with Direct Blue 15. AzoC was found to work best at pH 9.0, 25 °C, and with NADH and FAD as cofactors. AzoC was biophysically characterized using mass spectroscopy,
FTIR, circular dichroism, and SDS PAGE. FAD was identified as the noncovalently bound cofactor of AzoC in a 1:1
ratio. By SDS-PAGE, AzoC was determined to be a trimer connected by disulfide bonds. The trimeric form does not seem to add to structural stability, as determined by thermal melt studies. Computational analysis showed that the secondary structure of AzoC is consistent with the structural characteristics of other azoreductases, suggesting that gut enzymes of similar function will have related structures.
THE BACTERICIDAL EFFECT OF SILVER NANOPARTICLES AND IONSDulce Romero-Urbina, J. Jesus Velazquez, Patrick Ketter, Joshua Davidson, Miguel Jose-Yacaman.
University of Texas at San Antonio, San Antonio, TX.
Nanomaterials have the potential to provide solutions to technological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, and water treatment. Recently, silver nanoparticles and their applications have attracted great interest due to the important antimicrobial activities of these nanomaterials, which allow their use in several industrial sectors. Silver nanoparticles can be used in various products to kill different types of bacteria and offer an alternative to antibiotics. The mechanism of how silver nanoparticles kill bacteria is not well understood.
It is known that free silver ions (Ag+) are highly toxic to a wide variety of organisms including bacteria. Several silver Graduate
amounts of free silver ions (Ag+) were most effective in killing bacteria. The preliminary results from inhibition curves show an increased antibacterial effect of Ag+ ions in the treatment of Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermis. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques were used to characterize the treated bacteria. Understanding the mechanism of how silver nanoparticles and ions attack bacterial cell is of great importance in order to most effectively design products that will kill bacteria.
341 ORAL ABSTRACTS
HOW DOES THE POXVIRUS K1 PROTEIN INHIBIT NUCLEAR FACTOR KAPPA B ACTIVATION?
Ariana Bravo Cruz, Joanna Shisler.
University of Illinois at Urbana-Champaign, Urbana, IL.
Nuclear factor kappa B (NF-κB) is a transcription factor that regulates the expression of multiple proinflammatory genes. Poxviruses have evolved multiple mechanisms to modulate the host inflammation response by encoding several proteins that inhibit NF-κB activation. One such protein is the K1 protein. The mechanism by which K1 inhibits NF-κB activation remains unclear. Recently, our lab demonstrated that the K1 protein inhibits virally induced PKR, an upstream activator of NF-κB. We initially hypothesized that the K1 protein prevented NF-κB activation solely by inhibiting PKR activation. However, recent evidence suggests that the K1 protein can inhibit NF-κB activation in a PKR-independent manner. First, ectopically expressed K1 inhibits the PKR-independent NF-κB activation pathway triggered by TNF and PMA via luciferase assay. Second, the K1 protein does not inhibit IκBα degradation, an event downstream of PKR activation. To confirm this PKR-independent mechanism, we used PKR-/- cells and observed that K1 still inhibited TNF and PMA-induced NF-κB activation. The K1 protein allows NF-κB nuclear translocation, representing the first report of a vaccinia virus protein that targets NF-κB while in the nucleus. In addition, we observed that the K1 protein coassociates with the p65 subunit of NF-κB. We hypothesize that the K1 protein binds to NF-κB to block NF-κB-DNA binding, an event required to drive the transcription of target genes. Increasing knowledge of how viruses modulate the host environment to promote infection is beneficial for the immunology field.
New therapies and viral vectors could be developed and applied to both basic research and recombinant vaccine development.
ENZYMATIC AND BIOPHYSICAL CHARACTERIZATION OF AZOM AND MUTANTSShelby Rice, John Cooper, Brian Couger, Gilbert John.
Oklahoma State University, Stillwater, OK.
Enterococcus faecium (E. faecium) is a microorganism naturally found in the human intestine, and it has been found to contain the gene azoef1, which encodes for azoreductase activity. Azoreductase reductively cleaves azo dyes commonly used as colorants for food, beverages, manufacturing of textiles, cosmetics, pharmaceuticals, and plastics.
The metabolic byproducts can be carcinogenic and mutagenic, thereby becoming detrimental to human health and the environment. Little information is known about the structure and function of AzoEf1. Therefore, the goal of this study was to identify important binding sites for azo substrates and cofactor interaction. Using in silico modeling methods, specific residues were determined to be functionally relevant for binding based on their spatial location within the enzyme’s active site. Therefore, 3 mutants were generated, and enzymatic and biophysical techniques were used to characterize the wild-type (AzoM) and mutant proteins. The results will provide a better understanding of the structure and function of azoreductase and contribute to the broader azoreductase field, thereby having an impact on human health, industry, and the environment.
GLYCOSYLATION OF PSR IS IMPORTANT FOR DISEASE IN STREPTOCOCCUS PNEUMONIAEAnel Lizcano, Ramya A.S. Badu, Cecilia A. Hinojosa, Carlos Orihuela.
University of Texas Health Science Center at San Antonio, San Antonio, TX.
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. PsrP (pneumococcal serine-rich repeat protein) is a virulence factor in S. pneumoniae that mediates lung-cell adhesion, facilitates biofilm formation, and is important for virulence. psrP-secY2A2, is the pathogenicity island which encodes PsrP; 10-glycosyltransferases (GTFs), glyA-glyG, nss, gtfA, and gtfB; and 7 components of an alternate secretion system. The accessory proteins in psrP-secY2A2 glycosylate transport PsrP to the bacterial surface. However, the importance of PsrP glycosylation during virulence in S. pneumoniae has not been investigated. Using western blots, we determined that glycosylation of PsrP is important for stability in 6 out of 10 GTF-deficient strains expressing recombinant PsrP. To investigate if deletion of each GTF affected virulence, we performed in vitro lung-cell adhesion assays. These resulted in alternate adhesion patterns: 4 out of 10 GTF deletion strains were attenuated for adhesion similar to the psrP deficient strain. One GTF-deletion strain had a 3-fold increase in adhesion pattern compared to the wild-type strain.
We also tested the ability to form in vitro biofilms in each GTF-deficient strain. They all showed a different biofilm phenotype as imaged by confocal microscopy. To test virulence in vivo, we infected mice intratracheally with each of the GTF-deficient strains. Five out of the 10 GTF-deletion strains were attenuated in vivo. Our findings indicate that
342 ORAL ABSTRACTS
glycosylation of PsrP in S. pneumoniae is important for protein stability, adhesion, biofilm formation, and virulence of PsrP. These studies provide evidence that protein glycosylation is an important post-translational event that can impact pathogenesis.
IDENTIFICATION OF CRITICAL RESIDUES IN THE FLA-A FLAGELLIN OF VIBRIO CHOLERAEStacey Stahl1, Karl Klose2.
University of Texas Health Science Center at San Antonio, San Antonio, TX, 2University of Texas at San Antonio, San 1 Antonio, TX.
Vibrio cholerae is the causative agent of the human diarrheal disease, cholera. The bacterium possesses a single polar flagellum that is required for motility and contributes to virulence. The V. cholerae genome encodes 5 distinct flagellins (FlaA, B, C, D, and E), but only FlaA is essential for flagellar synthesis and motility. The flagellins have been identified as causing reactogenicity in current vaccine strains; however, nonflagellated strains do not confer the same level of protective immunity. Thus, identifying the critical residues for motility can lead to targeted efforts for a motile vaccine strain lacking reactogenicity. Transcription of flaA is controlled by a class III (σ54-dependent) promoter, while transcription of the other 4 flagellins is controlled by class IV (σ28-dependent) promoters. Class III expression does not contribute to the critical function of FlaA because flaA expressed from a class IV promoter still allows flagellar synthesis. This suggests that specific residues in FlaA contribute to its critical role in flagellar synthesis. To identify important residues in FlaA, we constructed chimeric FlaA-FlaD proteins, and screened for their ability to provide motility in a flaA flaD V. cholerae strain. These studies have revealed that a critical residue lies in the N-terminus at aa 96, and in the C-terminus at aa 328, and these are predicted to be located in the D1 and D2 domains of the flagellin.
The D1 and D2 domains lie in close proximity to each other in the assembled flagellum and may interact. Further analysis should reveal how these specific FlaA residues contribute to flagellar synthesis.
A STICKY SITUATION: CELL-TO-CELL ADHERENCE BETWEEN C. ALBICANS AND ORAL BACTERIAJonathan Dornell, Michael Gustin.
Rice University, Houston, TX.
Cell-to-cell adhesion is the binding of one cell to another by means of adhesin molecules. This phenomenon enables Candida albicans to colonize host tissues and form biofilms. This mechanism also enables C. albicans to indirectly establish itself through C. albicans-bacteria adhesion. The ability to adhere to distinct cell types is an essential virulence factor for C. albicans infections such as oral candidiasis and denture stomatitis. Despite the importance of cell-to-cell adhesion for C. albicans colonization, there are many questions that remain unanswered. C. albicans and oral bacteria have been coisolated from varying microenvironments in the oral cavity, including the tongue, periodontal pockets, and dental plaque. Furthermore, the ability of C. albicans to establish itself in the oral cavity is influenced by the surrounding bacteria. In order to expand our understanding of pairings between C. albicans and oral bacteria, a coadhesion assay was developed using fluorescence measurements with a plate reader to quantitatively identify which bacterial species adhere with C. albicans. Additionally, this assay is flexible enough to further investigate C.
albicans-bacterial pairings with different techniques such as fluorescence microscopy. This coadhesion assay has demonstrated that C. albicans strongly adheres with the initial colonizer S. gordonii with the potential to identify novel pairings with pathogenic oral bacteria such as P. gingivalis. The results obtained from this project will contribute to our knowledge of C. albicans’s ability to colonize the oral cavity and which oral bacteria potentially facilitate C. albicans infections in the mouth.
OTHER BIOLOGICAL SCIENCESRoom 210A
IMPAIRED CHEMOTACTIC SIGNALING WITHIN PRIMARY CD4+ T CELLS RESULTS FROM
DOWNMODULATION OF CCR7 BY HIV-1 VPUPeter Ramirez1, Marylinda Famiglietti2, Alberto Bosque1, Vicente Planelles1.
University of Utah, Salt Lake City, UT, 2San Raffaele Scientific Institute, Milano, IT.