Medical Microbiology Commons^™

H-Ns Binding And Repression Of The Ctx Promoter In Vibrio Cholerae, Emily A. Stonehouse, Robin R. Hulbert, Melinda B. Nye, Karen Skorupski, Ronald K. Taylor

Dartmouth Scholarship

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at …

Campylobacter Ureolyticus: An Emerging Gastrointestinal Pathogen?, Susan Bullman, Daniel Corcoran, James O'Leary, Brigid Lucey, Deirdre Byrne, Roy D. Sleator

Department of Biological Sciences Publications

A total of 7194 faecal samples collected over a 1-year period from patients presenting with diarrhoea were screened for Campylobacter spp. using EntericBios, a multiplex-PCR system. Of 349 Campylobacter-positive samples, 23.8% were shown to be Campylobacter ureolyticus, using a combination of 16S rRNA gene analysis and highly specific primers targeting the HSP60 gene of this organism. This is, to the best of our knowledge, the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis and may suggest a role for this organism as an emerging enteric pathogen.

Complement Factor H-Related Proteins Cfhr2 And Cfhr5 Represent Novel Ligands For The Infection-Associated Crasp Proteins Of Borrelia Burgdorferi, Corinna Siegel, Teresia Hallström, Christine Skerka, Hannes Eberhardt, Barbara Uzonyi, Tobias Beckhaus, Michael Karas, Reinhard Wallich, Brian Stevenson, Peter F. Zipfel, Peter Kraiczy

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.

METHODOLOGY/PRINCIPAL FINDINGS: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins …

Interferon Regulatory Factor 3-Dependent Pathways Are Critical For Control Of Herpes Simplex Virus Type 1 Central Nervous System Infection, Vineet D. Menachery, Tracy J. Pasieka, David A. Leib

Dartmouth Scholarship

The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via interferon (IFN) regulatory factor 3 (IRF-3)-dependent pathways. The absence of IRF-3 would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-3^−/− mice following intravenous HSV type 1 (HSV-1) challenge (K. Honda, H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi, Nature 434:772-777, 2005). In contrast, the …

Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication Via Interaction With Ddx3x, Chaomin Sun, Cara T. Pager, Guangxiang Luo, Peter Sarnow, Jamie H. D. Cate

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16-36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent …

Sialic Acid Transport And Catabolism Are Cooperatively Regulated By Siar And Crp In Nontypeable Haemophilus Influenzae, Jason W. Johnston, Haider Shamsulddin, Anne-Frances Miller, Michael A. Apicella

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: The transport and catabolism of sialic acid, a critical virulence factor for nontypeable Haemophilus influenzae, is regulated by two transcription factors, SiaR and CRP.

RESULTS: Using a mutagenesis approach, glucosamine-6-phosphate (GlcN-6P) was identified as a co-activator for SiaR. Evidence for the cooperative regulation of both the sialic acid catabolic and transport operons suggested that cooperativity between SiaR and CRP is required for regulation. cAMP was unable to influence the expression of the catabolic operon in the absence of SiaR but was able to induce catabolic operon expression when both SiaR and GlcN-6P were present. Alteration of helical phasing supported …

Association Between Chronic Liver And Colon Inflammation During The Development Of Murine Syngeneic Graft-Versus-Host Disease, Jason Anthony Brandon, Jacqueline Perez-Rodriguez, C. Darrell Jennings, Donald A. Cohen, Vishal J. Sindhava, Subbarao Bondada, Alan M. Kaplan, J. Scott Bryson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4⁺ T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals …

Bpab, A Novel Protein Encoded By The Lyme Disease Spirochete's Cp32 Prophages, Binds To Erp Operator 2 Dna, Logan H. Burns, Claire A. Adams, Sean P. Riley, Brandon L. Jutras, Amy Bowman, Alicia M. Chenail, Anne E. Cooley, Laura A. Haselhorst, Alisha M. Moore, Kelly Babb, Michael G. Fried, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5′ of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA …

Blood Kinetics Of Four Intraperitoneally Administered Therapeutic Candidate Bacteriophages In Healthy And Neutropenic Mice, Jumpei Uchiyama, Yoshihiro Maeda, Iyo Takemura, Russ Chess-Williams, Hiroshi Wakiguchi, Shigenobu Matsuzaki

Russ Chess-Williams

Due to multiple-drug resistant bacteria, phage therapy is being revisited. Although most animal experiments focus on therapeutic efficacy, the blood clearance kinetics of phages have not been well described. For further development of an efficient therapeutic strategy, information on phage blood kinetics is important. In this study, time-course concentration changes in peripheral blood of healthy and neutropenic mice were measured using four therapeutic phages (φMR11, KPP10, φEF24C, and KEP10). The results showed a two- to three-day rapid phage clearance, which fits a two-compartment model.

Blood Kinetics Of Four Intraperitoneally Administered Therapeutic Candidate Bacteriophages In Healthy And Neutropenic Mice, Jumpei Uchiyama, Yoshihiro Maeda, Iyo Takemura, Russ Chess-Williams, Hiroshi Wakiguchi, Shigenobu Matsuzaki

Russ Chess-Williams

Due to multiple-drug resistant bacteria, phage therapy is being revisited. Although most animal experiments focus on therapeutic efficacy, the blood clearance kinetics of phages have not been well described. For further development of an efficient therapeutic strategy, information on phage blood kinetics is important. In this study, time-course concentration changes in peripheral blood of healthy and neutropenic mice were measured using four therapeutic phages (φMR11, KPP10, φEF24C, and KEP10). The results showed a two- to three-day rapid phage clearance, which fits a two-compartment model.

Primary Human Mammary Epithelial Cells Endocytose Hiv-1 And Facilitate Viral Infection Of Cd4+ T Lymphocytes, Stephanie M. Dorosko, Ruth I. Connor

Dartmouth Scholarship

The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4(+) cells throughout the breast-feeding period, it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4(+) cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4(+) target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-, CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription …

Mononucleosis And Antigen-Driven T Cell Responses Have Different Requirements For Interleukin-2 Signaling In Murine Gammaherpesvirus Infection, Michael Molloy, Weijun Zhang, Edward Usherwood

Dartmouth Scholarship

Interleukin-2 (IL-2) has been implicated as being necessary for the optimal formation of primary CD8⁺ T cell responses against various pathogens. Here we have examined the role that IL-2 signaling plays in several aspects of a CD8⁺ T cell response against murine gammaherpesvirus 68 (MHV-68). Exposure to MHV-68 causes a persistent infection, along with infectious mononucleosis, providing a model for studying these processes in mice. Our study indicates that CD25 is necessary for optimal expansion of the antigen-specific CD8⁺ T cell response but not for the long-term memory response. Contrastingly, IL-2 signaling through CD25 is absolutely required …

Cross-Reactivity Of Antibodies Against Leptospiral Recurrent Uveitis-Associated Proteins A And B (Lrua And Lrub) With Eye Proteins, Ashutosh Verma, Pawan Kumar, Kelly Babb, John F. Timoney, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to …

Role Of Flgt In Anchoring The Flagellum Of Vibrio Cholerae, Raquel M. Martinez, Brooke A. Jude, Thomas J. Kirn, Karen Skorupski, Ronald K. Taylor

Dartmouth Scholarship

Flagellar motility has long been regarded as an important virulence factor. In Vibrio cholerae, the single polar flagellum is essential for motility as well as for proper attachment and colonization. In this study, we demonstrate that the novel flagellar protein FlgT is involved in anchoring the flagellum to the V. cholerae cell. A screen for novel colonization factors by use of TnphoA mutagenesis identified flgT. An in-frame deletion of flgT established that FlgT is required for attachment, colonization, and motility. Transmission electron microscopy revealed that while the flgT mutant is capable of assembling a phenotypically normal flagellum, …

Efficient Activation Of Reconstructed Rat Embryos By Cyclin-Dependent Kinase Inhibitors, Robin L. Webb, Kirk A. Findlay, Michael A. Green, Tina L. Beckett, M. Paul Murphy

Molecular and Cellular Biochemistry Faculty Publications

Background

Over the last decade a number of species, from farm animals to rodents, have been cloned using somatic cell nuclear transfer technology (SCNT). This technique has the potential to revolutionize the way that genetically modified animals are made. In its current state, the process of SCNT is very inefficient (<5% success rate), with several technical and biological hurdles hindering development. Yet, SCNT provides investigators with powerful advantages over other approaches, such as allowing for prescreening for the desired level of transgene expression and eliminating the excess production of undesirable wild-type animals. The rat plays a significant role in biomedical research, but SCNT has been problematic for this species. In this study, we address one aspect of the problem by evaluating methods of activation in artificially constructed rat embryos.

Principal Findings

We demonstrate that treatment with a calcium ionophore (ionomycin) combined with a variety of cyclin-dependent kinase inhibitors is an effective way to activate rat embryos. This is in contrast to methods developed for the mouse embryo, which tolerates much less specific chemical treatments. Methods developed to activate …

Antimicrobial And Antioxidant Activities Of Essential Oil And Methanol Extract Of Jasminum Sambac From Djibouti, Fatou Abdoul-Latif, Prosper Edou, François Eba, Nabil Mohamed, Adwa Ali, Samatar Djama, Louis-Clément Obame, Mamoudou Hama Dicko

Pr. Mamoudou H. DICKO, PhD

The essential oil of jasminum sambac from Djibouti was subjected to screening for their possible antioxidant activity by two complementary test systems, namely DPPH free radical scavenging and beta-carotene-linoleic acid assays. Butylated hydroxytoluene (BHT) was used as positive control in both test systems. In the DPPH test system, the IC50 value of essential oil and methanol extract were respectively 7.43 and 2.30 μg/ml. In the beta-carotene-linoleic acid system, oxidation was effectively inhibited by Jasminum sambac, the RAA value of essential oil and methanol extract were respectively 96.6 and 93.9%. When compared to BHT, the essential oil and methanol extract had …

Maternal Consumption Of Canola Oil Suppressed Mammary Gland Tumorigenesis In C3(1) Tag Mice Offspring, Gabriela Ion, Juliana A. Akinsete, W. Elaine Hardman

Biochemistry and Microbiology

Background: Maternal consumption of a diet high in omega 6 polyunsaturated fats (n-6 PUFA) has been shown to increase risk whereas a diet high in omega 3 polyunsaturated fats (n-3 PUFA) from fish oil has been shown to decrease risk for mammary gland cancer in female offspring of rats. The aim of this study was to determine whether increasing n-3 PUFA and reducing n-6 PUFA by using canola oil instead of corn oil in the maternal diet might reduce the risk for breast cancer in female offspring.

Methods: Female SV 129 mice were divided into two groups and placed on …

Proteomic Analysis Of Iron Acquisition, Metabolic And Regulatory Responses Of Yersinia Pestis To Iron Starvation, Rembert Pieper, Shih-Ting Huang, Prashanth P. Parmar, David J. Clark, Hamid Alami, Robert D. Fleischmann, Robert D. Perry, Scott N. Peterson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).

RESULTS: Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis …

Crystal Structure Of The Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features Of An Epoxide Hydrolase Virulence Factor, Christopher D. Bahl, Christophe Morisseau, Jennifer M. Bomberger, Bruce A. Stanton, Bruce D. Hammock, George A. O'Toole, Dean R. Madden

Dartmouth Scholarship

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other alpha/beta hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-A resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the …