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Oral Presentations |
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Poster Presentations |
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*Best Oral or Poster Presentation, Graduate
# Best Oral or Poster Presentation, Undergraduate |
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The Effects of Farming, Construction and Weather on the Water Quality of the Squaconning Creek
Kelly Arebalo* Delta College, Essexville, MI. 48732
The effects of farming, construction and weather on the water quality of the Squaconning Creek was evaluated by testing the Dissolved Oxygen, pH, Temperature Difference, Total Phosphates, Nitrates, Turbidity, BOD, Total Solids, and Fecal Coliform. The Fecal Coliform testing included quantity and identification of specific types of bacteria. The three areas tested included the West Stream which is upstream from construction and surrounded by farmland, North Stream which is north of construction and surrounded by woodlands and fields and a downstream site south farmland and construction. Sites were tested in April, June and August 2008. |
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Generation of Branched Chain Fatty Acids through Lipoate Dependent Metabolism Facilitates Intracellular Growth of Listeria monocytogenes
Kristie Keeney*, Lisa Colosi, Walter Weber, and Mary O’Riordan, University of Michigan, Ann Arbor, MI 48103
The Gram-positive bacterial pathogen Listeria monocytogenes has evolved mechanisms to rapidly replicate in the host cytosol, implying efficient utilization of host-derived nutrients. However, the contribution of host nutrient scavenging versus bacterial biosynthesis toward rapid intracellular growth remains unclear. Nutrients that contribute to growth of L. monocytogenes include branched chain fatty acids, amino acids, and other metabolic intermediates generated from acyl-CoAs, which are synthesized using lipoylated metabolic enzyme complexes. To characterize which biosynthetic pathways support replication of L. monocytogenes inside the host cytosol, we impaired lipoate dependent metabolism (LDM) by disrupting two lipoate ligase genes that are responsible for bacterial protein lipoylation. Interrupting LDM modestly impaired replication in rich broth medium, but strongly inhibited growth in minimal medium and host cells, and impaired the generation of amino acids and branched chain fatty acids. Addition of short branched chain fatty acids (BCFA) and amino acids restored growth of the A1A2- mutant in minimal medium, and BCFA alone rescued intracellular growth and spread in L2 fibroblasts. LDM was also required in vivo, as a wild-type strain robustly out-competed the lipoylation deficient mutant in a murine model of listeriosis. Thus, LDM influences branched chain fatty acid composition, which is critical for intracellular growth and virulence of L. monocytogenes.
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Tropism of Tanapoxvirus in Human Cancer Cells
Hui Lin Lee* and Karim Essani
Laboratory of Virology, Western Michigan University, Kalamazoo, MI 49008
Oncolytic viruses are infectious agents that preferentially target and destroy cancerous cells without harming normal cells. This is an ideal situation that has not been successfully achieved for a great number of anti-tumor chemotherapeutic agents. For the past century, many different types of viruses have been investigated for their anti-cancer properties with a handful currently undergoing clinical trials. However, there is no magic bullet to target every single type of cancer, so a vast library of oncolytic viruses should be assembled to best serve as a means for cancer treatment. We are exploring the potential of Tanapoxvirus (TPV) as an oncolytic virus. We examined the tropism of a green fluorescent protein (GFP) labeled TPV (TPV-GFP) in various human cancer cell lines. TPV-GFP replicates efficiently in some human tumor cells, and is restricted in others as determined by viral titer at 7 days post infection. We discovered that replication in one glioblastoma cell line, and one colorectal cancer cell line is over a log fold higher than in the control Owl Monkey Kidney (OMK) cell line. Replication in two renal cancer cell lines is also increased. Curiously, replication of TPV-GFP in another two glioblastoma cell lines revealed a marked reduction in viral titer, as with a breast cancer cell line, an osteosarcoma cell line, and particularly, a melanoma cell line. Our findings provide valuable groundwork for further studies on the mechanisms responsible for greater replication of TPV-GFP in cancerous cells, and harness that function in the development of a superior oncolytic virus.
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Investigating the Role of CpsA in Regulation of Capsule Production in the Systemic Pathogen Streptococcus iniae
Brett R Hanson*, Lowe, Beth A, Neely, Melody N., Wayne State University School of Medicine, Detroit MI, 48201.
Streptococcal systemic pathogens are a significant cause of morbidity and mortality worldwide and are able to cause severe disease in people of all ages. The large repertoire of specialized virulence factors accumulated by streptococcal pathogens has allowed them to become remarkably efficient at causing disease. The polysaccharide capsule is a particularly important virulence factor allowing for systemic dissemination of infection. Previous work has demonstrated that capsule is not constitutively produced during streptococcal infection, indicating the existence of a regulatory system involved in control of capsule production. The CpsA protein is highly conserved in several streptococcal systemic pathogens and has been implicated as the most likely candidate for transcriptional control over the capsule operon. The aim of this project is to characterize CpsA protein topology, functional domains, cis DNA elements, protein interactions, and possible regulatory mechanisms in the systemic pathogen Streptococcus iniae. CpsA protein topology will be assessed with reporter protein fusions constructed using SOE PÇR. Functional domains will be identified using targeted deletions and mutations. Potential upstream elements involved in control of CpsA will be discerned through random mutagenesis and assaying with the PhoZ reporter gene under control of the CpsA promoter. EMSA and DNase footprinting will enable identification of DNA sequences bound by CpsA while immunoprecipitation experiments will be used to confirm any interactions between CpsA and other proteins. Analysis of this poorly characterized transcriptional activator will help provide insight into a prominent virulence determinant within many streptococcal systemic pathogens and may lead to new therapeutic targets.
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Elucidation of a Mechanism by which Lactobacillus sp. Inhibit Gonococcal Adherence to Epithelial Cells.
Rachel R. Spurbeck* and Cindy G. Arvidson, Michigan State University, East Lansing, MI 48824
Lactobacilli are the most common microbes of the indigenous microbiota of the healthy human vaginal tract. High levels of vaginal lactobacilli have been correlated with a decreased risk of sexually transmitted infections, including gonorrhea. We recently have shown that L. jensenii and L. gasseri, two Lactobacillus species prevalent in the vagina of healthy women, inhibit gonococcal adherence to and invasion of epithelial cells (Spurbeck and Arvidson, 2008). Here, we report the results of our efforts to determine the mechanism(s) by which this inhibition occurs.
Other researchers have shown that lactobacilli inhibit adherence of uropathogens by several mechanisms, including modulation of the host immune response, co-aggregation with the pathogen, and production of bacteriocins, hydrogen peroxide, and biosurfactants. We have determined that lactobacilli do not co-aggregate with gonococci, or secrete any molecules that inhibit gonococcal growth. We have also determined that lactobacilli inhibit gonococcal adherence to epithelial cells independently of an epithelial cell response, and Lactobacillus-mediated inhibition of gonococcal adherence is not targeting the adherence mediated by the gonococcal Type-IV pilus. Furthermore, we demonstrate that a heat stable component of L. jensenii contributes to the inhibition of gonococcal adherence to host cells, and that lactobacilli can release a compound that, by itself, inhibits gonococcal adherence to epithelial cells. This work was presented at the Midwest Microbial Pathogenesis Conference in Madison, WI, September 26-28, 2008.
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Investigating virulence mechanisms of the systemic pathogen Streptococcus iniae
Jonathan Allen*, Beth Lowe, and Melody Neely, Department of Immunology and Microbiology, Wayne State University, MI
Systemic streptococcal pathogens are a major cause of severe invasive infections. We have developed an infectious disease model using the systemic pathogen, Streptococcus iniae, and a natural host, the zebrafish (Danio rerio). Following an intramuscular injection, S. iniae rapidly disseminates throughout the body and can be found surviving intracellularly in macrophages. We have shown that S. iniae-infected macrophages do not result in macrophage cell death in tissue culture, but rather promote survival even in the presence of staurosporine, a potent inducer of apoptosis. Results show that S. iniae infection of macrophages results in the upregulation of anti-apoptotic factors such as BclXL and FLIP. We have also identified a streptococcal transcriptional regulator, CpsY, that is required for systemic survival and invasion to the brain. The cpsY gene is located adjacent to the capsule synthesis operon, but has been shown to have little effect on capsule biosynthesis, suggesting that it is involved in the regulation of some other important virulence factors. An in-frame deletion mutant of cpsY (delta cpsY) can invade and survive in macrophages like wild type; however, delta cpsY is killed by neutrophils in the presence of serum. With increasing incidence of invasive streptococcal infections and emerging resistance to commonly used antibiotics, these pathogens are a significant challenge for the medical community. Identification of the pathogenic mechanisms used by systemic streptococcal pathogens and the virulence factors involved is a key step in the development of new therapeutic strategies to combat invasive streptococcal infection.
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Role of a Two-component Signal Transduction System of the Streptococcus pyogenes Salivaricin Locus in Virulence
Phanramphoei Namprachan* and Melody N. Neely. Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI
Streptococcus pyogenes can cause severe invasive diseases, in part, due to its ability to evade the host immune response. Host immune evasion is facilitated by the ability of S. pyogenes to survive phagocytosis and killing by neutrophils. A previous mutagenesis screen identified several mutants highly attenuated for virulence in a zebrafish infectious disease model. One strain, containing a transposon insertion in the salK gene, also shows a high degree of attenuation for survival in an in-vitro human neutrophil assay. SalK of S. pyogenes is 87% homologous to a histidine kinase of the S. salivarius lantibiotic locus, and is located upstream of a response regulator, SalR, constituting a putative two-component signal transduction system. Complete deletion of salK or salKR genes also confers attenuation in both the zebrafish model and the neutrophil assay. To investigate transcriptional regulation of the S. pyogenes sal locus, we have identified two possible promoters, one upstream of salA and one located within the salY gene, upstream of salKR. Transcriptional analysis showed that the two promoters have independent regulation. Particularly, the salY promoter expression increases two-fold in the salKR deletion strain or when 500 bp upstream of the putative promoter is deleted suggesting a possible repression site for the response regulator, SalR. Furthermore, expression of salK from a constitutive promoter does not restore virulence to the salK deletion strain in the neutrophil assay, suggesting that regulated differential expression of salK is essential for virulence. Further analysis aims to elucidate the role of the sal operon in virulence.
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Investigation of Bile Resistance Mechanisms in Probiotic Lactobacillus reuteri ATCC 55730
KRISTI J WHITEHEAD* 1, S. Roos 2, J. Versalovic 3, R. Britton 1
1Michigan State University, East Lansing, MI, 2Swedish University of Agricultural Sciences, Uppsala, Sweden, 3Baylor College of Medicine, Houston, TX
Passage through the gastrointestinal tract exposes bacteria to a multitude of stresses, including the acidic environment of the stomach and bile in the small intestine. The ability to survive exposure to these stresses and colonize the gastrointestinal tract of the host is generally regarded to be an important characteristic for probiotic bacteria. We are investigating mechanisms through which Lactobacillus reuteri ATCC 55730, a human probiotic, is able to survive exposure to bile in the small intestine. Growth studies revealed that L. reuteri could survive and grow in physiologically relevant concentrations of bile after a 1-2 hour adaptation period. Two sets of microarray experiments were performed using DNA microarrays containing 1879 open-reading frames from the L. reuteri genome to compare the gene expression profiles of cells that had not been exposed to bile to those that had been exposed to bile for 15 minutes (bile shock) and cells that had not been exposed to bile to those that had begun growing in the presence of bile (bile adaptation). Based on the microarray data, nine genes were chosen for mutational analysis; the mutant strains were then tested to determine their levels of bile resistance. Three of the mutants, a putative ClpL chaperone, a putative esterase, and a gene of unknown function, had significantly lower levels of survival than wild-type cells when exposed to physiologically relevant bile concentrations. A fourth mutant, a multi-drug resistance protein, had a final culture density roughly half that of a wild-type culture after exposure to bile, indicating a role for this protein in bile adaptation. Analysis of the upstream regions revealed a conserved 13 base pair element present for 25% of the genes over-expressed during bile shock. Current work in the lab is focused on determining the distribution of this element throughout the L. reuteri genome, as well as developing tools for use in determining the bile-inducibility of this putative transcriptional element. These investigations may elucidate mechanisms through which L. reuteri responds to bile exposure in the small intestine of the host.
A portion of this work has been presented at the ASM General Meeting.
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Mutation and Characterization of the Recombinant Streptococcal Protein IdeZ
KYLE FLIPPO* and Jennifer Hess, Aquinas College, Grand Rapids, MI.
The enzyme IdeZ is a cysteine proteinase which enhances the virulence of Streptococcus zooepidemicus. IdeZ is so specific in its cleavage of substrates, that it only has one known substrate, immunoglobulin G (IgG). In this project, plasmid DNA containing recombinant IdeZ obtained from E.coli, was mutated using site specific primers. These primers were designed to target amino acids that were thought to influence the cleavage of IgG. It was found that mutations involving the histidine and aspartic acid residues in the active site of the enzyme and a leucine residue that is structurally important to the active site of the enzyme all prevented IgG cleavage from occurring. Attempted mutation of the cysteine residue in the active site was unsuccessful, leaving the IdeZ DNA sequence as wild-type and leaving the protein functionally normal. In contrast, the successful mutation of a glutamine residue also near the active site did not alter the enzymatic functions or, consequently, typical IgG cleavage patterns. The characterization of IdeZ function and effects of causative mutations could have far-ranging medical and veterinary applications, such as cleavage of IgG to treat auto-immune arthritis.
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Evaluation of a New Approach for Delivering Disinfection: Efficacy of Chlorine and Bromine Halogenated Contact Disinfection Units for Reduction of Bacteriophages
LAUREN PETERSON*1,3,4, JOSH MOSBERG1, DR. JOAN ROSE1,2,3
1Michigan State University Department of Fisheries and Wildlife, East Lansing, Michigan, 2CAMRA: Center for Microbial Risk Assessment, 3Michigan State University Department of Microbiology, East Lansing, Michigan, 4Michigan State University Lyman Briggs College, East Lansing, Michigan.
Diarrheal diseases caused by contaminated drinking water kills more than 2.5 million people a year (Kosek, 2003). Children under five years of age and individuals in developing countries are typically the victims (Kosek, 2003). A new approach for delivering chlorine or bromine via contact disinfectants has been evaluated in a gravity fed household drinking water design. Three different halogenating canisters (both bromine and chlorine) were challenged with viruses to determine the efficiency of these systems for producing safe water. Bacteriophage levels were determined using a double overlay assay with the host Escherichia coli (ATCC #15597). Results indicated that both systems were effective against viruses, however the bromine canister produced an average reduction of 5.41 ± 1.39 log10 and the chlorine canister gave an average reduction of 3.81 ± 2.08 log10. Only the bromine units consistently produced water below the 4 log10 EPA level for contact disinfectants (USEPA, 2001). Contact disinfectants offer advantages over current disinfection approaches, including cost. Residuals, flow rates and dwell time as well as future configurations to deliver disinfection with and without filtration are important considerations.
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