Abstracts

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When: Monday, September 25, 2006, 4:00-5:00 PM

Abstract: StcE is a large zinc metalloprotease produced by Escherichia coli O157:H7 during human infection. The stcE gene is encoded by the pO157 virulence plasmid and positively co-regulated with other virulencefactors. StcE is secreted by the plasmid-encoded type II secretionsystem, and purified StcE specifically cleaves O-glycosylated proteinsincluding C1 esterase inhibitor, a negative regulator of the complementcascade, and two salivary mucins. In addition, StcE binds to multipletypes of immune cells in culture and aggregates two T cell lines, Jurkatand MOLT-4. Leukocyte surface glycoproteins play important roles incell adhesion and signalling. StcE binds to and cleaves two largesurface glycoproteins, CD43 and CD45, on Jurkat T cells, primaryneutrophils, and HL-60s, a neutrophil-like cell line. Cleavage of CD43by StcE results in a loss of the extracellular domain from the surfaceas demonstrated by Western blot and immunofluorescence microscopy, whilethe CD43 intracellular domain remains intact. StcE also cleaves within theextracellular domain of CD45. The interaction between StcE and leukocyteglycoproteins suggest that this protease may play an immunomodulatoryrole during infection with enterohemorrhagic E. coli (EHEC). CD43 andCD45 provide an anti-adhesive force in cell-cell interactions and areimportant in regulation of leukocyte activation. StcE-mediated cleavageof CD43 and CD45 may cause leukocytes to become hyperadhesive andsensitive to stimulation, resulting in an increase in inflammation atsites of infection. Identifying the outcomes of this interaction willshed further light on potential modulation of the inflammatory immuneresponse to EHEC by the StcE protein.

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When: Monday, June 26, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Wilmara Salgado Pabon, Dillard Lab

Title: The role of the putative relaxase TraI in DNA processing via the Neisseria gonorrhoeae Type IV secretion system.

Abstract: N. gonorrhoeae is a strictly-human pathogen and the causative agent of gonorrhea. A great majority of N. gonorrhoeae strains, and a few N. meningitidis strains, carry a genomic island that appears to have been horizontally acquired and may contribute to the virulence of these bacteria. The gonococcal genetic island encodes a type IV secretion system (T4SS) utilized by N. gonorrhoeae to secrete chromosomal DNA into the extracellular milieu for natural transformation. T4SSs in general are major driving forces of bacterial evolution and adaptation through the horizontal transfer of genes. Also, they are often virulence factors of a diverse set of extracellular and intracellular pathogens. However, despite the fact that T4SS where first described more than half a century ago, it was not until recently that DNA processing became a focus of investigation. T4SSs can inject DNA into virtually any eukaryotic cell type. Therefore, there is a possibility that they could be exploited as tools for the delivery of DNA of interest into a large number of target cells. Relaxases are the key element for DNA processing for type IV secretion. Hence, my project focuses on the characterization of the putative relaxase TraI in N. gonorrhoeae. This research will shed light into the characteristics of the DNA substrate, type IV secretion, and natural transformation of the very unique T4SS of N. gonorrhoeae.

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When: Monday, June 12, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Sharon Altman, Brandt Lab

Title: Stopping the enemy at the gate: characterizing an inhibitor of poxvirus entry.

Abstract: Recent concerns about the possible use of smallpox as a bioweapon have led to an increased call for agents active against poxviruses.  We previously identified a peptide, designated EB, which inhibits Herpes simplex virus entry. We now report that the EB peptide is active against vaccinia virus (VV).  Using a yield reduction assay the IC50 of EB was determined to be 15 uM while the IC50 value of scrambled version EBX was >200 uM, indicating that the effect is sequence-specific.  Dialysis of EB-treated VV resulted in the complete recovery of infectivity, indicating that EB is not viricidal.  Cells pretreated with EB were not resistant to infection, suggesting that interactions between EB and cells do not contribute to the anti-VV activity.  To determine if VV attachment was inhibited, recombinant VV containing beta-galactosidase was pretreated with EB and added to cells at 4 degrees celcius and the amount of attached beta-galactosidase activity was measured.  There was no decrease in beta-galactosidase activity in EB-treated samples, indicating that VV attachment is not inhibited by EB treatment.  To investigate the possibility that EB may inhibit VV entry, cells were infected for three hours with recombinant VV expressing lacZ under the control of an early promoter.  Samples infected with EB-treated VV exhibited significantly less beta-galactosidase activity than untreated samples in a dose-dependent manner, indicating that EB is most likely blocking VV entry.

We have identified a novel peptide inhibitor of VV infection active at low micromolar concentrations.  The inhibition is sequence-specific and reversible and blocks VV entry in vitro.  These results raise the possibility that the EB peptide could be a useful prophylactic or therapeutic agent against poxvirus infection.

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When: Monday, May 8, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Lisa Fox, Gumperz Lab

Title: Factors that influence NKT autoreactivity.

Abstract: NKT cells are a unique population of T cells that recognize lipids and glycolipids presented by CD1d antigen presenting molecules. An unusual feature of NKT cells is their autoreactivity: they respond functionally to CD1d-mediated presentation of self-lipids. Myeloid dendritic cells (DCs) constitutively express CD1d, and are efficient antigen presenting cells (APCs) for NKT cells. The regulation of self-lipid presentation by APCs, however, is not well understood. PPARγ (peroxisome proliferator-activated receptor γ) is a ligand-activated transcription factor expressed in myeloid cells that regulates genes involved in lipid uptake and storage. Activation of PPARγ in DCs results in upregulated expression of proteins that can mediate lipid uptake and may alter lipid metabolism within the cell. Therefore, we hypothesized that PPARg activation may affect the self-lipids presented by CD1d. To test this hypothesis, we analyzed responses of human NKT cell clones to myeloid DCs that were treated to activate or inhibit PPARg. NKT cell autoreactive responses were enhanced when DCs were treated to activate PPARg, and diminished after DC treatment with a PPARg inhibitor. In contrast, presentation of the exogenous lipid α-galactosylceramide (α-GalCer) was not affected by treatment of the DCs. Taken together, this suggests that PPARg activation is affecting the self-lipids that are presented by the DCs.

Speaker: Xiaohua Wang, Gumperz Lab

Title: Dynamics of NKT cell interactions with CD1d+ APCs.

Abstract: Recognition of foreign peptide antigens by conventional T cells initiates a dynamic activation process involving T cell adhesion and polarization to the APC, signal transduction and formation of a supra-molecular clustering called the immunological synapse. NKT cells differ from conventional T cells in that they recognize lipid antigens presented by CD1d molecules and respond functionally to both self and foreign lipids. Little is known about the dynamics of NKT cell interactions with APCs. In particular, it is not clear whether the recognition process of self-antigens differs from that of foreign glycolipid that is more potent in inducing T cell activation. Here we investigate how a series of human NKT cell clones interact with APCs in the presence of the high affinity foreign lipid antigen a-Galcer or of self-antigen alone. Stable T cell:APC conjugates were formed between NKT cells and CD1d transfected APCs, but not with untransfected cells. All of the clones attained a similar level of stable adhesion and showed clear CD3 polarization to CD1d+ APCs that were pre-pulsed with a-GalCer. The clones also formed stable conjgates with un-pulsed CD1d+ APCs presenting self-antigen alone and the kinetics of conjugate formation appeared similar, but there was little evidence of T cell CD3 polarization. The level of stable T cell adhesion to un-pulsed APCs varied substantially from clone to clone, but correlated with the self-antigen induced cytokine secretion response of NKT cells. Antibody blocking experiments suggested that the adhesion molecule LFA-1 is involved in both cases, but self-antigen response showed a greater dependence on LFA-1 co-stimulation than a-GalCer induced response did. These results show that recognition of self-antigens can lead to stable conjugate formation between NKT cell and APCs in a CD1d dependent manner, but may differ qualitatively from that induced by a-GalCer as to their super-molecular structure and downstream signaling.

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When: Monday, April 24, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Carlene Chun, McFall-Ngai Lab

Title: Microarray analyses of the squid-vibrio symbiosis illuminates conserved host responses to bacterial colonization.

Abstract: On Monday April 24, Carlene Chun from the McFall-Ngai lab will present on “Microarray analyses of the squid-vibrio symbiosis illuminates conserved host responses to bacterial colonization.” Her abstract is included below.
Biologists are becoming increasingly aware that beneficial interactions of bacteria with animals are more prevalent than pathogenic interactions. This new-found recognition begs the question: how are animal-bacterial interactions regulated to produce the alternate outcomes of health and disease? In response to Vibrio fischeri colonization, the light organ of the squid undergoes a program of rapid morphogenesis, involving many host responses common to other types of bacterial colonization, i.e. pathogenic and gut microbiota. Examples of this shared response are apoptosis, hemocyte trafficking and increases in microvillar density1-4. However, it is unknown if similarities in host response exist on a transcriptional level. In this study, we used spotted microarrays to identify genes differentially expressed in the squid light organ in response to wild-type V. fischeri colonization. Here we show that the expression of 171 transcripts changed at least 2-fold in response to the presence and absence of the squid symbiont. We found that symbiosis induces transcripts involved in DNA binding/transcription, protein metabolism, general metabolism and innate immunity such as those in the NF-kB and oxidative burst pathways. Symbiosis down-regulates cytoskeleton-related transcripts. To determine if our results were similar to gene expression changes reported in vertebrate hosts, we compared our findings to the results of previously published microarray experiments done in zebrafish and mouse systems. We identified 18 genes or genes in a similar gene family differentially regulated in all three systems, squid, mouse and zebrafish. Our results demonstrate that common transcriptional responses in response to bacterial colonization are shared among vertebrate and invertebrate hosts. To further analyze the role of one of these common pathways, the oxidative burst pathway, in the squid-vibrio symbiosis, we are sequencing 7 genes implicated in the pathway using rapid amplification of cDNA ends (RACE), performing quantitative real-time PCR and localizing gene products via immunocytochemistry. Here we show transcriptional down-regulation of NADPH oxidase gp91phox and glutathione peroxidase, in addition to localization of a halide peroxidase in the epithelial cells lining the pores, ducts, antechambers and deep crypts of the light organ. The modulation of genes, such as those involved in the oxidative burst pathway, may represent a common host response to bacterial colonization and the direction of modulation may determine whether pathogenesis or a healthy partnership will ensue.

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When: Monday, April 10, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Carolyn Lipke, Goodrich-Blair Lab

Title: The two-component regulator, PhoPQ, is required for resistance of Xenorhabdus nematophila to antimicrobial peptides.

Abstract: phoPQ mutant strains of X. nematophila show decreased resistance to antimicrobial peptides. However, these strains do not show other phenotypes of the VMO strains.

Speaker: Youngjin Park, Goodrich-Blair Lab

Title: Virulence modulation (VMO) of entomopathogenic bacterium, Xenorhabdus nematophila, on host insect Manduca sexta.

Abstract: Characterization of spontaneously derived strains of X. nematophila that no longer kill insects. These strains colonize nematodes normally, but show reduced mortality in insects, reduced suppression of antimicrobial peptide expression, and reduced resistance to antimicrobial peptides.

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When: Monday, March 27, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Rose Szabady, Welch Lab

Title: E. coli O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome in humans.

Abstract: Enterohemorrhagic E. coli (EHEC) intimately adhere to the colonic epithelium, forming attaching and effacing lesions and secreting a Shiga-like toxin that causes epithelial destruction. StcE is a zinc metalloprotease secreted by EHEC that cleaves specific heavily O-glycosylated proteins. One of these substrates is CD43, a large, mucin-like cell surface protein found on immune cells that has been implicated in adhesive and signalling processes of T lymphocytes. StcE cleaves CD43 at the T cell surface and promotes aggregation of Jurkat cells, a T cell-like line. We hypothesize that StcE cleavage of CD43 from the T cell surface causes lymphocytes to become hyperactive and adherent, increasing the pathologic inflammatory response during EHEC infection.

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When: Monday, February 13, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Bailey Freeman, Donna Paulnock’s Lab

Title: Macrophage-mediated Immunosuppression in African Trypanosomiasis.

Abstract: Infection of mammalian hosts with the extracellular protozoan parasite Trypanosoma brucei rhodesiense results in a chronic infection characterized by recurring waves of parasite growth followed by clearance by host antibody responses.  Work from our laboratory and others has demonstrated that both relatively resistant C57BL/6 mice (surviving ~60 days) and susceptible BALB/c mice (surviving ~25 days) mount a readily detectable immune response to the first wave of parasites, consisting of pro-inflammatory cytokine production, antibody production, and Th1 polarization.  This Th1 response, characterized by substantial IFN-g production, has been functionally and genetically linked to resistance to the parasite.  However, responses to subsequent waves of parasites are much less robust, and the reasons for this are largely unknown.  We are currently testing the hypothesis that macrophages are the key cells responsible for this dramatic alteration in host immunity over the course of infection.  Initial studies have shown that there is a dramatic increase in the number of macrophages in the spleen and peritoneal cavity of both resistant and susceptible mice, and preliminary data suggests that these cells are functionally altered.  Assessment of the response of resident spleen and peritoneal macrophages to a variety of microbial ligands at various times post-infection revealed that macrophages isolated early in infection produce the pro-inflammatory cytokines TNF-a and IL-12 upon stimulation. However, as infection progresses, production of these molecules declines, and instead IL-10 is produced in abundance.  Additionally, flow cytometric studies of splenic and peritoneal cell populations at various stages of infection have revealed a subset of cells that have the surface phenotype CD11b⁺Gr-1⁺CD31^high, which has been associated with immature macrophages.  Such cells are known mediators of immunosuppression in other systems, but their role in trypanosomiasis has never been examined.  We are currently exploring the potential relationship between maturation state and response capability, as well as the impact these cells have on generation of an effective Th1 response, in order to determine the potential implications of these alterations in macrophage responses for host resistance to trypanosomes.

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When: Monday, January 23, 2006, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Josh Troll, Margaret McFall-Ngai Lab

Title: Characterization of putative Vibrio fischeri-signal receptors in Euprymna scolopes.

Abstract: The Hawaiian bobtail squid, Euprymna scolopes, forms a mutuality association with the luminous Gram-negative marine bacterium Vibrio fischeri. Each generation of E. scolopes must re-establish the association with V. fischeri by recruiting the bacterial symbiont from the environment to live in the specialized ‘light organ’. Light organ structures that facilitate colonization by V. fischeri, such as a ciliated epithelial field are eliminated following the successful entry into the light organ crypt spaces by V. fischeri. A peptidoglycan (PGN) subunit identical to tracheal cytotoxin (TCT) from Bordetella pertussis and Neisseria gonnorhea is secreted by V. fischeri in culture. Pharmacological application of TCT to E. scolopes was sufficient to induce light organ morphogenesis. I have identified four transcripts from the E. scolopes light organ that code for distinct homologs of the peptidoglycan recognition proteins (PGRP) and have named them EsPGRP1-4 accordingly. All four EsPGRPs formed a consistent group which excluded PGRPs from any other organism in neighbor-joining tree analysis. Key residues known to be required for binding and catalytic breakdown of PGN (similar to T7 lysozyme) are conserved in all four EsPGRPs, with the exception of one serine substitution for a cysteine. Consistent with PGRP similarity to T7 lysozyme, EsPGRP2 lyses E. coli cells when expressed for protein purification. To evaluate EsPGRP catalytic activity against V. fischeri PGN, each EsPGRP was cloned into an V. fischeri expression vector and conjugated into either wt or auto-inducer mutants. No lysis of V. fischeri cells was observed upon induction, but protein expression could not be confirmed. EsPGRPs 1, 2, & 4, were also cloned into drosophila S2 cell expression vectors for protein purification. EsPGRP2 expressed well in S2 cells and EsPGRP4 expressed weakly. No expression was seen for EsPGRP1. Purification of EsPGRPs 2 & 4 is under way and further biochemical characterization will be performed.

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When: Monday, November 28, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Chris Kuckleburg

Title: Expression of phosphorylcholine on Haemophilus somnus LOS contributes to bovine platelet activation.

Abstract: Haemophilus somnus is a gram-negative bacillus that causes respiratory and reproductive disease in cattle. During systemic Haemophilus infection, vascular inflammation, thrombosis and an acute form of vasculitis known as thrombotic meningoencephalitis can develop. We hypothesized that platelet interactions with H. somnus and the endothelium may contribute to the vascular inflammation and thrombus formation that is frequently observed. Previously we demonstrated that H. somnus and its lipooligosaccharide (LOS) can activate bovine platelets. In addition, H. somnus but not its LOS, can induce platelet aggregation. Platelet aggregation by H. somnus was significantly inhibited in the presence of the platelet activating factor (PAF) receptor antagonist, WEB 2170. The platelet response to H. somnus could not be attributed to contaminating leukocytes, a potential source of PAF. Nor did inhibitors of phospholipase A2 (AACOCF3 and cPLA), an enzyme necessary for PAF production, affect platelet aggregation by H. somnus. It has been previously demonstrated that phosphorylcholine expression on the LOS of Haemophilus influenzae can lead to activation of human epithelial cells through the PAF receptor. H. somnus has also been shown to incorporate phosphorylcholine into its LOS. To further understand the potential role of phosphorylcholine in platelet activation, two isogenic strains of H. somnus were selected and characterized for the presence or absence of LOS phosphorylcholine expression by antibody specific staining. It was found that an H. somnus strain expressing phosphorylcholine induced platelet aggregation, while the phosphorylcholine deficient strain did not. Surprisingly, both strains induced platelet P-selectin expression, as measured by flow cytometry. This finding suggests that phosphorylcholine expression may be necessary for inducing platelet aggregation, but is not required for platelet granule secretion (i.e. P-selectin expression). The regulation of phosphorylcholine expression by H. somnus may represent a possible virulence mechanism which contributes to platelet activation and thrombus formation during systemic H. somnus infection.

Speaker: Dave McClenahan

Title: Altered Mannheimia haemolytica adherence to bovine lung epithelial cells infected with bovine herpes virus.

Abstract: Infection of cattle with bovine herpes virus (BHV-1) often leads toco-infection with the bacterial respiratory pathogen Mannheimia haemolytica.The mechanism by which viral infection synergizes with M. haemolytica is notwell understood, but may involve alterations in bacterial adhesion to thehost cell. The purpose of this study was to determine if infection of bovinelung epithelial cells with BHV-1 would result in increased adherence of M.haemolytica. Bovine lung epithelial cells were exposed to BHV-1 virus forvarious time points prior to incubation with M. haemolytica. After theincubation period, bacteria were recovered and colony counts were performed.Bacterial adherence to epithelial cells infected with BHV-1 for 3 hours wasdecreased as compared to uninfected epithelial cells. In contrast,epithelial cells infected with BHV-1 virus for 12 hours displayed increasedbacterial adherence as compared to uninfected epithelial cells.Extracellular matrix macromolecules such as heparan sulfate may be involvedin this adherence, as concurrent incubation of M. haemolytica and epithelialcells with soluble heparin or heparinase decreased bacterial adhesion. Inconclusion, it appears that prior BHV-1 infection of epithelial cellsresults in altered adherence of M. haemolytica to epithelial cells, and thisadherence is partially dependent on heparan sulfate on the extra cellularmatrix of the cell.

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When: Monday, October 24, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Torsten Wurm, Ph.D. (Kalejta’s lab), Institute For Molecular Virology

Title: Interaction of the coronavirus N protein with the host cell.

Abstract: The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. The subcellular localization of the avian infectious bronchitis virus (IBV) and the murine mouse hepatitis virus (MHV) N proteins (group I and group III coronaviruses, respectively) was examined by indirect immunofluorescence and confocal microscopy both in the absence and –if available- in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments. Both the N proteins of IBV and MHV were shown to localize either to the cytoplasm alone or to the cytoplasm and to a subnuclear structure, subsequently identified as the nucleolus, indicating that nucleolar localization of the N protein is a common feature of the coronavirus family. Pull-down assays demonstrated that MHV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. In cells infected with IBV or transfected with either IBV or MHV N the distribution of fibrillarin is reorganized into a globular distribution pattern. Both N protein and a fibrillarin-EGFP fusion protein colocalized to the perinuclear region and the nucleolus. In addition, results obtained by confocal microscopy and flow cytometry suggest that the N protein might function to disrupt cell division independent of apoptosis.

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When: Monday, September 26, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Dana Mordue (Knoll lab), Medical Microbiology & Immunology

Title: A functional genomics approach to isolate genes critical for parasite pathogenesis.

Abstract: A functional genomics approach was used to identify genes in the obligate intracellular parasite Toxoplasma gondii that are important for the establishment of chronic infection in mice but are not required for growth in cultured fibroblasts.  To achieve this aim, a library of over 6000 insertional mutants was constructed and evaluated in a murine model of chronic infection using a modification of signature-tagged mutagenesis.  Mutants were selected that grew normally in fibroblasts in vitro but were altered in their ability to colonize the brain 22 days following intraperitoneal challenge with the tachyzoite form of the parasite.  Over 50 mutants were ultimately identified that were defective in some aspect of in vivo pathogenesis.  The genes and proteins in a subset of these mutants have been identified and will be discussed.  An additional screen was performed to identify mutations that affect the ability of the parasite to survive in activated macrophages but do not affect survival in naïve macrophages.  Studies are underway to identify the parasite genes that contribute to parasite survival in activated macrophages.

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When: Monday, June 27, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Rose Szabady (Welch lab, MPath trainee), Medical Microbiology & Immunology

Title: Characterization of Vibrio cholerae TagA, a StcE-like metalloprotease.

Abstract: We have identified by sequence similarity a family of related bacterial metalloproteases, called the SLiMe (StcE-Like Metalloprotease) family after E. coli O157:H7 StcE, the prototype member of this family. StcE (Secreted protease of C1 esterase inhibitor from EHEC), has been purified and characterized as a protease of specific heavily glycosylated substrates. StcE has also been shown to promote the intimate adherence of enterohemorrhagic E. coli in vitro and to aggregate cultured T cells and erythrocytes.

The Vibrio cholerae genome encodes two SLiMe family members, TagA (ToxR-activated gene A) and a TagA-related protein. TagA is a ~112 kDa putative secreted lipoprotein. TagA aggregates cultured T cells and cleaves C1 esterase inhibitor to the same size product as StcE, but we have been unable to demonstrate StcE-like cleavage of mucin substrates. Members of the SLiMe family may share proteolytic activity against certain types of substrates but may also recognize unique substrates relevant to the pathogenic mechanisms of their host bacteria.

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When: Monday, June 13, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Xiuxu Chen (Gumperz lab), Medical Microbiology & Immunology

Title: Role of CD4-mediated co-stimulation in activation of CD1d-restricted T cells.

Abstract: CD1d-restricted T cells (or "NKT" cells) recognize glycolipids presented as antigens by CD1d antigen presenting molecules, and have been shown to have potent effects on a wide variety of immune responses in vivo. In some cases CD1d-restricted T cells contribute to Th1-type immunostimulatory responses, whereas in others they appear to promote tolerogenic or immunosuppressive responses.  Insight into how CD1d-restricted T cells mediate such diverse effects has come from the observation that CD4+ and CD4- CD1d-restricted T cells appear to be functionally distinct subsets, suggesting that they may contribute to different processes in vivo.  However, the factors that regulate these T cells remain poorly understood.  Classical MHC-restricted T cells generally require co-stimulation from CD4 or CD8 co-receptors for optimal activation, and this is accomplished by co-receptor binding to the MHC molecule.  However, CD4 appears to be unable to bind CD1d, and therefore it is unclear whether this molecule contributes to the distinct functional properties of CD4+ CD1d-restricted T cells, or is simply a lineage marker.  Our data indicate that CD4 can potently enhance the TCR signaling of CD1d-restricted T cells, and that it may play a constitutive role in the activation of these cells.  Co-ligation of CD4 with the CD3 complex markedly increases CD1d-restricted T cell intracellular Ca2+ flux, while sequestration of CD4 down-regulates CD3-mediated Ca2+ flux.  The effect of CD4 is translated to the functional outcome, as blockade of CD4 using a mAb reduces CD1d-restricted cytokine responses to CD1d+ APCs.  We are currently investigating how CD4 alters the functions of CD1d-restricted cells, and whether MHC class II molecules on certain CD1d+ APCs function as a CD4 ligand.

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When: Monday, May 9, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Sharon Altmann (Brandt lab; MPath trainee), MMI and Ophthalmology

Title: A Peptide with Broad Spectrum Antiviral Activity Inhibits Vaccinia Virus Infection.

Abstract: The recent increase in acts of international terrorism has led to a renewed discussion of the possibility that terrorists could use the orthopoxvirus variola major, the causative agent of smallpox, as a weapon for bioterrorism. Although vaccination has been shown to provide long term immunity against smallpox, the severity of side effects associated with vaccination makes this strategy of control less than ideal in the event of a smallpox outbreak.  To date, there is no FDA approved drug for the treatment of smallpox or other orthopoxvirus infections, and investigations of new treatments are hampered by a lack of understanding of the basic biology of the virus itself.  Our laboratory has identified a twenty amino acid peptide that inhibits in vitro infection by vaccinia virus, the prototypic orthopoxvirus. Initial tests have shown that this peptide, EB, has an IC50 value of 15 uM, does not inactivate vaccinia virus, and may block viral attachment.  By further characterizing how EB inhibits vaccinia virus infection, we hope to develop a better understanding of the life cycle of this and other orthopoxviruses.

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When: Monday, April 25, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Ron Peck (Bangs lab), MMI

Title: Stage-specific requirement for the lysosomal membrane glycoprotein, p67, in Trypanosoma brucei.

Abstract: p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei.   The processing, turnover, and targeting of p67 have been characterized, but its function remains unknown.  There are 8 copies of the p67 gene in the T. brucei genome, making deletion analysis technically challenging. Therefore, we have used a tetracycline-inducible RNA-interference (RNAi) system to explore the role of p67.  Induction of p67 RNAi for 24 hours results in an 80-95% reduction in p67 protein production in both bloodstream and procyclic stage trypanosomes.  The reduction of p67 results in a severe growth defect in the bloodstream stage but has very little effect on growth in the procyclic stage. We have focused on the effects of p67 RNAi in bloodstream cells. Gross cellular morphology is clearly affected within 24 hours post-induction and is very pronounced after 48 hours. Surprisingly, p67 ablation has no discernable effect on processing or localization of the major lysosomal protease, trypanopain, as demonstrated by pulse-chase labeling and immunofluorescence microscopy.  Receptor-mediated endocytosis is also unaffected as shown by uptake of a fluorescently-labeled tomato lectin. Immunofluorescence microscopy reveals no effects on subcellular architecture, but closer examination by electron microscopy suggests that lysosomal integrity may be compromised by the loss of p67. Interestingly, RNAi of p67 appears to protect cells from lysis by the trypanosome lytic factor found in human serum.  Collectively these data are most consistent with a structural/protective role in the intensely hydrolytic lysosome of bloodstream trypanosomes.

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When: Monday, April 11, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Erica Behling-Kelly (Czuprynski lab), Pathobiological Sciences

Title: Haemophilus somnus adheres to the glycocalyx of bovine brain endothelial cells and promotes alterations in the permeability of the cell monolayer.

Abstract: Haemophilus somnus is a pleomorphic, gram negative coccobaccillus capable of contributing to the development of respiratory disease, reproductive failure, arthritis and septicemia in the bovine.  The syndrome of neurological complications that can result from systemic infection with H. somnus, currently coined thromboticmeningioencephalitis (TME), is characterized by a fibrinopurulent meningitis, haemorrhage with abscessation and a thrombotic vasculitis of the central nervous system (CNS). Haemophilus somnus induced septicemia in the bovine is remarkably similar, from both a clinical and microbiological standing, to meningococcal sepsis in humans.  Marked inflammatory cell activation, disseminated intravascular coagulation, and vascular compromise are among the hallmarks of both these inflammatory conditions.  We are currently utilizing an in-vitro model system to investigate the interactions between bovine brain endothelial cells and H.  somnus, in an effort to elucidate mechanisms utilized by extracellular pathogens to trigger intravascular coagulation and break down of the blood-brain barrier.  The ability of H. somnus to adhere to or invade the endothelial cells that comprise the blood-brain barrier is a critical factor in the pathogenesis of TME.  In the current study, we demonstrate that H. somnus adheres to bovine brain endothelial cells (BBEC) in-vitro in a time dependent manner.  Activation of the BBEC by tumor necrosis factor-alpha, a cytokine that is present in the bloodstream during septicemia, enhances the adherence of H. somnus.  Our data indicate that adhesion of H. somnus to the bovine blood-brain barrier likely involves the heparan sulfate proteoglycans in the glyocalyx of the endothelial cell. The lack of direct cellular invasion by H. somnus led us to hypothesize that the bacteria gains entry to the CNS by a paracellular mechanism. We have initiated in-vitro studies to investigate the ability of H. somnus to alter endothelial cell monolayer permeability and transendothelial electrical resistance.  Thus far, we have demonstrated that H. somnus increases monolayer permeability, as indicated by albumin flux, and decreases electrical resistance.   We have also demonstrated the ability of H. somnus to induce procoagulative changes in BBEC.  Future studies are planned to investigate the role these procoagulative changes play in altering adhesion of H. somnus to the endothelial cell monolayer as well as permeability of the endothelium.

Speaker: Dhammika Attapatu (Czuprynski lab), Pathobiological Sciences

Title: Cellular trafficking of M. haemolytica leukotoxin.

Abstract: The bovine respiratory pathogen Mannheimia haemolytica produces a potent exotoxin (LKT) which binds to ruminant leukocytes. Recent data shows that the LKT binds to the beta 2 integrin, LFA-1 (CD18/CD11a) on the leukocyte surface. In this study we examined intracellular signaling events that follow LKT binding to BL-3 (bovine lymphoma) cells. We found evidence that LKT is associated with cell surface lipid rafts independent of LFA-1, and transported into cells in a cytoskeleton dependent manner. Scanning immuno-electron microscopy of LKT treated cells revaled the presence of LKT on the mitochondrial outer membrane. This observation suggested the possibility that LKT exerts its effects via mitochondria mediated cell death pathway. We also observed Caspase 9 activation, up regulation of pro-apoptotic BAD and BAX proteins and down regulation of the anti-apoptotic BCl-2 and Akt-1.  These findings suggest a direct action of LKT on mitochondia which may play a significant role in LKT mediated bovine cell death.

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When: Monday, March 14, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Subramanya Hegde, MMI (Gumperz lab)

Title: CD1d-restricted NKT cells induce monocyte differentiation.

Abstract: Dendritic cells (DCs) are professional antigen presenting cells that play a crucial role in sensing microbial compounds and initiating and modulating the subsequent adaptive immune response. Monocytes can function as precursor cells for DCs, although DCs may also mature from other bone marrow precursors.  It is not clear what stimulates monocytes to differentiate into dendritic cells in physiologic conditions. Monocytes constitutively express CD1d antigen presenting molecules, and this suggests they may interact with CD1d-restricted T cells (also known as "NKT" cells). However, little is known about how this interaction influences the monocytes.  Here we show that CD1d-restricted T cells can induce monocyte differentiation into cells that resemble immature DC. Monocytes exposed to NKT cells acquired immature DC cell surface markers. Confocal immuno-fluorescence analysis showed that MHC class II was intracellularly co-localized with LAMP-1, as occurs in immature DCs. Moreover, the NKT-stimulated monocytes could mature normally upon exposure to LPS, and then had a phenotype characteristic of mature DCs. The monocyte differentiation effect was mediated by secreted factor(s) produced by activated NKT cells, since activated NKT cells induced monocyte differentiation in transwell experiments, whereas rested NKT cells did not.  However, rested NKT cells could become activated to produce the factor(s) by interacting with CD1d molecules on fresh monocytes in the absence of added antigens.  Hence, NKT cell recognition of self antigens may be sufficient to induce secretion of monocyte differentiation factors, and therefore stimulation of differentiation by NKT cells could function as a rapid pathway of DC differentiation in vivo that is not dependent on foreign antigens.

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When: Monday, February 28, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Carolyn Lipke, Bacteriology (Goodrich-Blair lab)

Title: Regulation of a secreted protease, PrtA, in Xenorhabdus nematophila.

Abstract: The Gram-negative bacterium Xenorhabdus nematophila is a potent insect pathogen, and is known to actively suppress aspects of the insect immune system.  Additionally, the bacterium must be able to survive within this host environment, where additional stresses may be present. We have identified a gene in X. nematophila (mgtB) that is necessary for transcription of a gene (prtA) encoding a secreted protease, both genes being required for full virulence in Manduca sexta insects. MgtB is predicted to encode a Magnesium (Mg2+) transporter, but how this protein may play a role in virulence factor regulation is not known.  In other pathogens, PhoPQ acts as a two-component regulator of virulence factors (including mgtB) by sensing entry into the host environment and altering transcription of genes necessary for virulence and survival in the host environment.  We hypothesize that PhoPQ and MgtB regulate prtA and other genes encoding virulence factors involved in suppressing the insect immune system.  This work will provide insights into X. nematophila pathogenesis, which in turn could lead to new methods for controlling agricultural pests.  Additionally, parallels between insect and mammalian innate immunity make the elucidation of X. nematophila virulence mechanisms relevant to our understanding of mammalian pathogens.

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When: Monday, February 14, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Fang Li, Chemical & Biological Engineering (Palecek lab)

Title: Identification and characterization of Candida albicans genes involved in cell adhesion, morphogenesis and virulence.

Abstract: Candida albicans is the most common fungal pathogen of humans. Among the factors involved in C. albicans pathogenesis are adhesion to host cells, biofilm formation and the dimorphic transition. The gene EAP1 was isolated using a parallel plate flow chamber to screen a flo8 Saccharomyces cerevisiae strain expressing a C. albicans genomic library. Eap1p belongs to a large family of GPI-anchored glucan-cross-linked cell wall protein. Disruption of EAP1 in C. albicans reduced adhesion to polystyrene and HEK293 kidney epithelial cells and suppressed filamentous growth. EAP1 was also required for in vivo C. albicans biofilm formation.

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When: Monday, January 24, 2005, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Lindsey Moser, MMI (Schultz-Cherry lab)

Title: Insights into Potential Mechanisms of Astrovirus-Induced Diarrhea.

Abstract: Astroviruses are the second leading cause of virally induced diarrhea in children under the age of two.  Despite this prevalence, very little is known about the pathogenesis of astrovirus infection or the mechanism by which astroviruses induce diarrhea. Using a turkey model for astrovirus infection, our laboratory has shown that infection results in only mild histological changes without a dramatic inflammatory response.  We have therefore pursued other possible  routes for diarrhea induction, most specifically by examining the  effect of astrovirus infection on intestinal barrier permeability.

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When: Monday, December 6, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Jeremy Glasner, Animal Health & Biomedical Sciences (Perna lab)

Title: Organizing and Understanding the Genomes of Pathogenic Enterobacteria.

Abstract: We have been sequencing, annotating and comparing the complete genome sequences from enterobacteria including pathogens of both plants and animals. These analyses reveal many of the features that distinguish these phenotypically diverse organisms as well as some surprising similarities. We are developing an ambitious bioinformatics resource that aims to integrate and curate the annotations and comparisons of these sequences and link them to genetic and biochemical characterizations where possible. To facilitate querying of this massive database of information we are working towards the creation of a controlled vocabulary of genome descriptions with a focus on gene products involved with host-pathogen interactions.

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When: Monday, November 22, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Becky Montgomery, Biochemistry & Molecular Virology

Title: Cellular Determinants of HSV Entry.

Abstract: In the current entry model proposed for herpes simplex virus type 1 (HSV-1) entry into mammalian cells, glycoprotein gC and/or gB interact with cell surface glycosaminoglycans (GAGs) to promote attachment of HSV-1 to cells. Next, gD interacts with one of several cellular co-receptors (HVEM, nectin-1, or 3-O-sulfated heparan sulfate proteoglycans) to promote stable docking of virus to cells followed by envelope-cellular membrane fusion involving gB and gH-gL.  The goal of these studies was to further characterize the interactions of GAGs and identified co-receptors with regards to productive HSV-1entry. We found that HSV-1(KOS) entered CHO-K1 (GAG-replete) or CHO-745 (GAG-deficient) cell lines in a co-receptor dependent manner (either HVEM or nectin-1).   Overall cellular attachment was greatly reduced in GAG-deficient cells as compared to GAG-replete cells. However, attachment was not dependent on co-receptor expression in GAG-deficient cell lines.  Thus, HSV-1 binds to a currently unidentified receptor in GAG-deficient CHO cells. Additional studies used soluble GAGs. Studies using soluble GAGS determined a novel GAG inhibitor, CS-E potently inhibited HSV-1 and HSV-2 infection of cells, but did not inhibit infection by other alphaherpesvirues.  This inhibitor, and heparin, also blocked HSV-1 infection of GAG-deficient 745-HVEM and 745-nectin expressing cells.  Further detailed chondroitin-sulfate (CS-E) inhibition studies of HSV-1 infection in K1-nectin-1 cells revealed a biphasic inhibition profile (IC50(1) ~0.003 mg/mL and IC50(2)~0.8 mg/mL), suggesting that CS-E inhibited 2 distinct HSV-1 entry steps in cells expressing GAGs and nectin-1. IC50(1) was comparable to the CS-E IC50 determined for HSV-1 entry in 745-nectin-1 or 745-HVEM cells (GAG-deficient).   IC50(2) mimicked the heparin IC50 observed in K1-nectin-1 or K1-HVEM cells (GAG-replete) for preventing HSV attachment to cells.  We conclude that HSV-1 uses two different entry pathways in co-receptor expressing CHO cells.   These pathways (1) require co-receptor, and either HVEM or nectin-1 can fulfill this requirement; (2) exhibit different cellular requirements for virus attachment; and (3) differ in their post-attachment requirements.  These studies report the first evidence for virus entry in which the same cellular receptor participates in two biochemically distinct entry pathways within one cell type.

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When: Monday, October 25, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Gireesh Rajashekara, Animal Health & Biomedical Sciences (Splitter lab)

Title: Brucella melitensis: Determining virulence determinants.

Abstract: Disease caused by Brucella is an enormous problem worldwide in animals and humans and is considered to be a potential bioterrorist agent in the USA. Well-characterized virulence factors of many pathogenic bacteria such as cytolysins, capsules, exotoxins, secreted proteases, pili/fimbriae, flagella, phage-encoded toxins, and virulence plasmids are absent in Brucella. Thus identification of virulence factors has remained elusive.  We have taken the comparative genomics approach and in vivo real-time genetic screening to define factors responsible for host preference and virulence.  First, we have used the whole genome microarray of B. melitensis to compare the genomes of five other Brucella spp.  Secondly, we are using the bioluminescent B. melitensis in the IRF-1-/- mouse model of infection to define the genetic requirements of Brucella for virulence.

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When: Monday, October 18, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Peter Redford, Medical Microbiology & Immunology.

Title: How Does Uropathogenic Escherichia coli Know When It's in the Bladder?

Abstract: Pathogenic E. coli strains carry an array of virulence factors that permit them to survive and propagate in areas outside their niche in the bowel lumen. In animal infection studies, we found that DegS, an activator of the alternative sigma factor Sigma-E, is essential for infection. In nonpathogenic E. coli the Sigma-E regulon functions in repair and maintenance of the outer membrane. We hypothesize that in uropathogenic E. coli the Sigma-E system also serves as a sensor of the host environment and as an activator of virulence factors.

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When: Monday, May 10, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Tom Grys, Medical Microbiology & Immunology (Welch lab)

Title: E. coli O157:H7 shedding by bovine hosts: the "ins and outs" of large animal studies.

Abstract: Animal studies are routine in the microbial pathogenesis field. However, large animal studies are less common, and present a different set of issues for the experimenter, such as sample collection, statistics, and cost.  Enterohemorrhagic strains of E. coli are carried asymptomatically by a number of ruminants. Typically, the animals will shed the bacteria for a few weeks and clear the "infection."  Contamination of meat and other animal products can occur during processing of infected animals, posing a public health risk.  StcE is a secreted protein from E. coli O157:H7 that has mucinase activity and may play a role in colonization of human and/or ruminant hosts.  The presentation will focus on a recent experiment to measure shedding of a wild-type strain and a stcE knockout strain after a simulated natural infection of bovine hosts.

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When: Monday, April 26, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Meg Bohse, Medical Microbiology & Immunology (Woods lab)

Title: The YPS3 locus of Histoplasma capsulatum.

Abstract: Histoplasma capsulatum is a thermally dimorphic, pathogenic fungus that is the causative agent of histoplasmosis, an infection of particular danger to the immunocompromised.  The YPS3 gene of H. capsulatum encodes a protein that produced only during the pathogenic yeast phase of infection and is also expressed differentially in H. capsulatum strains of differing virulence and thermotolerance.  Our research focuses on determining the role this protein plays in H. capsulatum virulence and the mechanism of Yps3p secretion and surface localization.

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When: Monday, April 5, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Nancy Keller, Plant Pathology

Title: Aspergillus fumigatus virulence factors: lessons from the genetic model Aspergillus nidulans.

Abstract: Aspergillus fumigatus is an increasingly significant medical menace in this era of HIV and immunosuppressed patients.   Although a number of putative virulence factors have been postulated for this fungus, few bona fide factors have been reported in the literature.  This is primarily due to two reasons.  In most cases, the genes responsible for production of the espoused virulence factors (e.g. gliotoxin, fumagillin, phthoic acid) have not been found and, therefore, mutants are not available for examination.  In a few cases, disruption of a gene encoding a postulated virulence factor has yielded a fully virulent pathogen. Additionally, it is likely that many of the factors which render A. fumigatus a devastating pathogen are unknown.  Here we present how insight into processes required for toxin formation and sporulation in the genetic model A. nidulans can be utilized in identifying potential A. fumigatus virulence factors.  We will focus on the identification of A. fumigatus secondary metabolite mutants and their phenotype and virulence attributes.

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When: Monday, March 22, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Lindsey Moser, MMI (Schultz-Cherry lab)

Title: Cellular Biology of Astrovirus-Induced Diarrhea.

Abstract: While astroviruses have been shown to be a major cause of childhood diarrhea, little research has been conducted on the pathogenesis of infection. We are currently investigating the effect of infection on intestinal barrier function. Preliminary data by our lab indicates that astrovirus induces a decrease in intestinal integrity as well as a disruption of tight junctions. These findings are important in elucidation of the pathogenesis of astrovirus infection, but also have implications in human diseases which result from the disruption of cellular contacts.

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When: Monday, February 23, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Carolyn Lipke, Bacteriology (Goodrich-Blair lab)

Title: Xenorhabdus nematophila secreted proteases and their role in virulence in insects.

Abstract: Xenorhabdus nematophila (X.n.) is a gram-negative bacterium capable of causing virulence in insects. Study of the function and regulation of X.n. virulence factors may contribute to methods for insect biological control, as well as help to elucidate the insect innate immune response, which closely parallels that of mammals. Secreted proteases have been implicated as virulence factors in other pathogens, but their role in X.n. virulence has not been previously investigated. My talk will sumarize our work to date on determining what genes encode the two known X.n. secreted proteases, how they are regulated, and what role they play in virulence in their insect hosts.

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When: Monday, October 27, 2003, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Holly Hamilton, Medical Microbiology & Immunology (Dillard lab)

Title: Type IV Secretion & Host Cell Interactions of Neisseria gonorrhoeae.

Abstract: The gonococcal genetic island (GGI) of Neisseria gonorrhoeae encodes a type IV secretion system that secretes DNA and is involved in host cell interactions. Mutants of type IV secretion system genes are deficient in DNA secretion and display a delay in adherence and aggregation phenotype during infection of primary human cervical cells.

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When: Monday, January 26, 2004, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Rich Proctor, Medical Microbiology & Immunology and Medicine

Title: Why Staphylococcus aureus has suddenly become so aggressive.

Abstract: Some of the new information about why S. aureus has suddenly become so aggressive and is killing healthy young people. This would be more clinical and epidemiological, but perhaps also of great interest to the group. The average age is 14 yo and the mortality rate is 70%!

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When: Monday, December 1, 2003, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Richard Stein, Dept. of Biochemistry & Molecular Genetics, University of Alabama at Birmingham (Klein postdoctoral candidate)

Title: Using Site-Specific Recombination to Examine Bacterial Chromosome Structure and Dynamics.

Abstract: Chromosomal DNA is negatively supercoiled and organized into independent supercoiled loops in vivo. Our lab uses site-specific recombination as an assay to characterize supercoil diffusion in vivo and to examine the dynamics of the bacterial chromosome. In this assay, two res sites, originating from the __ transposon, inserted into the bacterial chromosome, delete the interval between them. This recombination has three requirements: 1) the presence of the two res sites in direct repeat; 2) expression of the gd resolvase protein encoded by the tnpR gene of the __ transposon, and 3) negative supercoiling of the bacterial chromosome. As the wild-type __ resolvase is long-lived (over 40 minutes in log-phase cells), we generated an ssrA-tagged resolvase (tnpR-ssrA) that has a much shorter half-life (about 5 minutes). Point mutations were made in the ssrA tag to generate two additional resolvases with intermediate half-lives: tnpR-ssrA L9D and tnpR-ssrA A8D (half-lives of about 30 minutes and 15 minutes, respectively). The four resolvases were used to examine supercoil diffusion in the 43-45 min region of the Salmonella typhimurium chromosome and to characterize the half-life of the stochastic barriers to supercoil diffusion within this chromosomal region. Our results show that a first-order decay of resolution efficiency with distance persists, even when the resolution time is reduced. The short half-life proteins allow the evaluation of short-lived domain boundaries.

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When: Monday, November 24, 2003, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Adel Talaat, Animal Health & Biomedical Sciences

Title: Imaging the transcriptional profile of tuberculosis and paratuberculosis during infection.

Abstract: Between infection and the appearance of the first symptoms of the disease, bacteria interact with different microenvironments within the host. The outcomes of such host-pathogen interactions are in large part due to selective gene expression at different phases of the infection. Therefore, understanding bacterial gene expression in vivo is central to our understanding of how bacteria colonize, invade and disrupt the normal host cell functions and eventually produce disease. In this regard, Mycobacterium tuberculosis and paratuberculosis have a proven record for adaptation to different host microenvironments. No available vaccine or drug can reverse this trend at this time. Toward this end, we adapted the microarrays technology to analyze the gene expression profile of M. tuberculosis or M. paratuberculosis during infection on a genome-wide level. The microarray-based analyses revealed unique expression profiles of Mycobacteria once inside the host and identified clusters of genes that were exclusively expressed in vivo depending on the immune status of the host. In this seminar, we will discuss the impact of our findings on the molecular pathogenesis of tuberculosis and paratuberculosis.

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When: Monday, April 28, 2003, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Donna Paulnock, Medical Microbiology & Immunology

Title: Regulation of innate immune responses during African trypanosomiasis.

Abstract: The work in our laboratory is focused on understanding the role of the innate immune response in resistance to infection with the protozoan parasite Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness. Macrophage activation is one of the hallmarks of infection with the African trypanosomes. Activating factors delivered to macrophages during trypanosome infection include both interferon-gamma(IFN-g), produced by host Th1 cells and linked to host resistance, and substituents of the glycosylphosphatidylinositol anchor of the trypanosome variant surface glycoprotein (GIP-sVSG), a parasite coat component that is enzymatically released in large amounts during periods of parasite growth. We have proposed that the timing and magnitude of VSG and IFN-g exposure, and the subsequent signaling pathways generated by such exposure, determine the ultimate activation phenotype of the macrophage and thus the resistance status of the host. To test this hypothesis, we have embarked on a series of studies designed to characterize GIP-sVSG- dependent and IFN-g-dependent macrophage activation events and to determine the nature of the interaction between GIP-sVSG and the macrophage membrane. Our results to date demonstrate that GIP-sVSG-mediated activation does not occur by the same pattern of intracellular signal pathway activation as that associated with microbial compounds such as bacterial lipopolysaccharide (LPS). In addition, we have demonstrated that GIP-sVSG stimulation is associated with reduction in the level of STAT1 phosphorylation, an event required b yIFN-g-induced macrophage activation. Thus, GIP-sVSG appears able to modulate the IFN-g-mediated host response through regulation of intracellular phosphorylation events. These results will help to elucidate how aspects of macrophage activation determine host resistance to trypanosome infection.

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When: Monday, February 24, 2003, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Lyric Bartholomay, Christensen lab, Dept. of Animal Health and Biomedical Sciences

Title: Innate immune responses of the mosquito, Aedes aegypti.

Abstract: Mosquitoes employ both cellular and humoral innate immune responses to resist foreign invaders. The existing dogma is that antimicrobial peptides represent the first line of defense against infection in mosquitoes largely because these polypeptides exhibit bactericidal and/or fungicidal activities in vitro. Our studies of the immune peptide defensin in the mosquito, Aedes aegypti, challenge this assertion. Furthermore, ongoing ultrastructural and molecular characterization of circulating blood cells or hemocytes demonstrates their capacity to very rapidly and effectively manage bacterial infections.

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When: Monday, January 27, 2003, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Rob Striker, Depts. of Medicine and MMI

Title: Hepatitis C Viral Polymerase: Implications for Drug Design and Pathogenesis.

Abstract: RNA viruses exist as "quasispecies clouds" which has implications for pathogenesis, and treatment. The size and heterogeneity of these clouds are controlled in part by the viral RNA dependent RNA polymerase. We are investigating correlations between enzymatic properties....activity, drug susceptibility, fidelity, processivity with pathogenic/clinical outcomes... replication, selection of mutants, immune evasion, and recombination.

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When: Monday, November 25, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Katy Forest, Dept. of Bacteriology

Title: The Structural Basis of Bacterial Type IV Pilus Function.

Abstract: Hairy filaments decorate the surface of many Gram negative bacteria. These Type IV pili are used by pathogens and non-pathogens alike to attach to hosts, mediate natural transformation, slither along surfaces, and facilitate biofilm formation. Several Type IV pilin subunit structures have now been solved. These reveal a shared overall secondary structure arrangement and 3-D scaffold with important species-specific differences. With some understanding of the pilin subunit structures, and how these predict the assembled structure of the filament, our current efforts are on the mechanism of pilus-based surface motility. We have purified and characterized the presumed motor, PilT, showing that it is an oligomeric ATPase in vitro. We have crystallized PilT, and plan to solve its structure.

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When: Monday, October 28, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Karl W. Boehme; Department of Oncology

Title: Toll-like Receptor-Mediated Innate Immune Responses to Human Cytomegalovirus.

Abstract: Cytomegalovirus (CMV) infection results in the initiation of a number of cellular signal transduction pathways that alter the transcriptional program of the cell in a profound way. Among the genes most strongly induced by CMV are many indicators of innate immunity, including inflammatory cytokines and interferon-stimulated genes (ISGs). Furthermore, similar responses are invoked by a soluble form of the major viral envelope protein, glycoprotein B (gB), suggesting that mere cell contact by CMV particles is sufficient to trigger these events. Research in our laboratory has focused on identifying the cell surface component(s) that are responsible for the innate "sensing" of CMV. The Toll-like receptors (TLRs) are cell surface molecules that function to detect microbial pathogens and induce secretion of inflammatory cytokines and interferons. We have found that the pattern recognition receptors CD14 and TLR 2 recognize CMV virions and trigger inflammatory cytokine production. These findings will contribute significantly to our understanding of CMV pathogenesis, as well as the complex relationship between CMV and the host immune system.

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When: Monday, September 23, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Darby Brown, Allen lab, Dept. of Plant Pathology

Title: Using Tryptophan Auxotrophy to Identify Host-Induced Genes in Ralstonia solanacearum.

Abstract: Ralstonia solanacearum is a Gram negative soil inhabitant, and the cause of bacterial wilt, one of the most devastating crop diseases world-wide. This bacterium has an extremely wide host range and has the capability to overwinter in weedy species as well as persist in the soil for many years, making eradication efforts difficult.

The goal of our work is to identify bacterial genes that are specifically expressed in the host plant environment. To achieve this goal, we are using in vivo expression technology, or IVET. The requirements for a successful IVET screen include: (1) a deletion mutant in a gene that is absolutely necessary for bacterial growth in planta, (2) a library containing putative promoter fragments cloned upstream a promoterless copy of the deleted gene and (3) a method to screen against constitutive genes.

We have shown that trpEG, which codes for the enzyme anthranilate synthase, is absolutely necessary for bacterial growth and disease in planta, furthermore, we have shown that a heterologous promoter (PpilA) can drive the expression of a promoterless copy of trpEG resulting in restoration of virulence in the deletion mutant. Preliminary use of our IVET screen has identified 28 putative plant-induced genes.

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When: Monday, June 10, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Marcel Wüthrich, Klein Lab, Department of Pediatrics.

Title: Absolute and dispensible requirements for vaccine immunity against pulmonary blastomycosis.

Fungal infections have emerged as a significant medical problem, but there are no vaccines available against fungal pathogens. Understanding fundamental mechanisms of immunity against fungi will be needed for proper use and optimization of vaccines. Recent work in our lab has led to development of a fungal vaccine. Vaccination with a genetically engineered, live, attenuated strain of Blastomyces dermatitidis carrying a targeted deletion at the BAD1 locus confers sterilizing immunity against experimental lethal pulmonary infection. In studying vaccine mechanisms, we found that ab T-cells are requisite for durable vaccine immunity, whereas other T-cells and B-cells are dispensable. In immune-competent animals, CD4+ T-cell derived cytokines TNF-a and IFN-g mediate vaccine immunity. Surprisingly, however, these factors are dispensable in immune-deficient animals, which rely on alternate mechanisms for robust vaccine immunity, yet still require O2- production rather than generation of NO. Our recent findings have clarified the cellular and molecular bases behind a novel genetically engineered fungal vaccine. They also illustrate a sharp difference in vaccine mechanisms between immune-competent and immune-deficient hosts, which underscores the plasticity of residual immune elements in compromised hosts, and points to the feasibility of developing vaccines against invasive fungal infection in this fast growing patient population. 

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When: Monday, April 22, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Paula Roesch, Postdoctoral Trainee, Welch Lab, Dept. of Medical Microbiology & Immunology

Title: Escherichia coli Uropathogenesis: A Case of Needing the Right Serine?

E. coli is a diverse bacterium with clonal types capable of a commensal intestinal lifestyle while other strains cause human diseases such as diarrhea, dysentery, meningitis and urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) present a complex ecological case among the pathogenic E. coli. UPEC can colonize the human intestinal tract without causing disease, yet they can sequentially colonize the perineum, urethra, bladder and kidney. The goal of our research is to identify genes specific to UPEC that permit replication, survival and disease in the urinary tract. Comparison of the E. coli K-12 (MG1655), enterohemorrhagic E. coli (EHEC) EDL933 and uropathogenic E. coli (CFT073) genome sequences permitted identification of genes unique to UPEC. The differences among the strains at the MG1655 53 minute region led to an investigation of the dsdCXA locus. In MG1655 and CFT073 the locus is complete, but in EDL933 a large portion has been replaced with genes for sucrose utilization. The dsd locus is responsible for the detoxification of D-serine. D-serine is inhibitory for growth of E. coli in glucose or glycerol minimal medium at concentrations > 50 mg/ml because it blocks L-serine and pantothenate biosynthesis. In our studies, we address the question of why would some strains of E. coli need a tightly regulated system to catabolize a single, presumably rare D-amino acid. Furthermore, a new model is proposed where UPEC use D-serine as an environmental signal to modulate colonization of the urinary tract.

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When: Monday, March 18, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Bill Weidanz, Dept. of Medical Microbiology & Immunology

Title: Genetic disection of the immune response to Plasmodium chabaudi blood-stage infection.

Abstract: Malaria, causing substantial morbidity and mortality, remains a major health problem for nearly half of the earth's population. How the host defends itself against the parasite and the cells and molecules involved in mediating protection have been the focus of our research for many years. During the early 1990s, KO mice became available to the research community. These novel animals with consistent and well-characterized deficiencies have provided unique opportunities to study host immune responses to a great variety of infectious agents. I will summarize how we have utilized KO mice to examine the immune elimination of blood-stage malaria parasites. This will include data indicating i) that experimental infections caused by Plasmodium species are suppressed by cell- and antibody-mediated mechanisms of immunity, ii) that type 1 cytokines but not type 2 cytokines play a key role in suppressing parasitemia, iii) that CD28 co-stimulation is crucial for the expression of CMI against the parasite, and iv) that neither ROI or RNI are crucial for the destruction of blood-stage parasites.

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When: Monday, February 25, 2002, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Joe Dillard, Dept. of Medical Microbiology & Immunology

Title: Mutations in the gonococcal genetic island affecting transformation and interaction with host cells.

Abstract: Neisseria gonorrhoeae is a gram-negative bacterium and the causative agent of the sexually-transmitted infection gonorrhea. Gonococcal infections can also cause pelvic inflammatory disease, disseminated gonococcal infection, and neonatal blindness. Horizontal gene transfer occurs frequently in the gonococcal population, by the process of natural transformation. This transformation results in the antigenic variation of surface molecules as well as rapid spread of antibiotic resistance. My laboratory is focussing on the role of genes in the gonococcal genetic island (GGI) in pathogenesis and genetic transformation. The GGI is a 57 kb region of the chromosome that is found in 80% of N. gonorrhoeae strains. The GGI appears to have been horizontally acquired; it has a lower G+C content than the rest of the chromosome and contains very few copies of a gonococcal repeat sequence. The island encodes 60 genes, 22 of which constitute a type IV secretion system. In other bacteria, type IV secretion systems transport substrates directly from the bacterial cytoplasm to the cytoplasm of the host cell during infection, or transport DNA between bacteria during conjugation. We have made multiple mutations in the type IV secretion system genes. The mutants are defective in the ability to donate DNA for natural transformation. The mutants also exhibit a delay in attachment to primary cervical cells, suggesting the type IV secretion system is involved in interaction with host cells.

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When: Monday, November 26, 2001, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Jay Bangs, Dept. of Medical Microbiology & Immunology

Title: Down to the crossroads: intersection of secretion and endocytosis in African trypanosomes.

Abstract: African trypanosomes are the causative agent of Sleeping Sickness in humans, which is a re-emerging disease throughout sub-Saharan Africa. My laboratory focuses on the basic cell biology of these protozoa; and specifically on intracellular trafficking of lysosomal and cell surface proteins as key aspects of the host:parasite relationship. The trypanosome lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector; each stage has a unique protein surface coat that forms the first line of contact with the host. These coat proteins are anchored in membranes by glycosylphosphatidylinositol (GPI) anchors and are essential for survival in each stage. Consequently, correct protein targeting to the cell surface is critical to the success of the parasite. Also, endocytic and lysosomal functions are greatly up-regulated in the pathogenic bloodstream stage for both nutritional and defensive purposes. Three distinct areas of research in my laboratory will be presented: 1) GPI-dependent targeting of surface coat proteins; 2) novel cell surface metalloproteases in surface coat exchange during life cycle differentiation, and as drug targets in the pathogenic bloodstream stage; and 3) stage-specific targeting of lysosomal membrane protein. Our ultimate goal is to define aspects of trypanosomal secretory processes that may provide novel avenues to chemotherapeutic intervention.

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When: Monday, September 24, 2001, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Paul Lambert, Dept. of Oncology

Title: P53-INDEPENDENT ACTIVITIES OF THE HUMAN PAPILLOMAVIRUS TYPE 16 E6 ONCOGENE IN CARCINOGENESIS: TRANSGENIC ANIMALS STUDIES.

Abstract: Anogenital papillomaviruses, particularly human papillomavirus type 16 (HPV-16), are associated with the majority of human cervical cancers. In these cancers, two papillomaviral proteins, E6 and E7, are commonly expressed. These viral proteins bind to and inactivate the tumor suppressors p53 and pRb, respectively. Transgenic mice expressing HPV-16 E6 and E7 in the skin display epithelial hyperplasia and, late in adult life, spontaneously develop tumors. These observations demonstrate the carcinogenic potential of E6 and E7 and implicate these HPV-16 proteins as causative agents in human cervical cancers. We have now utilized our HPV-16 transgenic mouse model to assess the mechanism of action of the E6 oncogene in cancer. The phenotypes of the K14E6 transgenic mice can not solely be accounted for by E6's capacity to inactivate p53. Using a mutant of E6, I128T, that is reduced in its capacity to bind the ubiquitin ligase, E6AP, and thereby reduced in its capacity to inactivate p53, we have evaluated the p53-independent effects of E6 in cancer. K14E6I128T mice display a subset of the phenotypes seen in K14E6 wild type mice including epithelial hyperplasia and synergy with chemical carcinogens in inducing frank cancer. These data support the hypothesis that p53-independent activities of E6 contribute to its oncogenic activities in vivo. Studies now in progress are attempting to ascertain the role of additional E6 interacting cellular factors in mediating E6's p53-independent properties.

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When: Monday, August 27, 2001, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Sally Leong, Dept. of Plant Pathology

Title: Comparative Genomics as a Tool to Understand the AVR1-CO39-Pi-CO39 (t) Interaction.

Abstract: The genomes of members of the Graminae family, which includes the many hosts of Magnaporthe grisea (Pyricularia grisea), are highly syntenic and can be considered as essentially one genome. Disease resistance gene homologs have been mapped to syntenic locations across grass genomes. It will be interesting to learn whether homologs of disease resistance genes to blast are found in the same genomic location of other monocot hosts of M. grisea and how these genes functionally relate. This information is also essential to the design of resistance to rice blast disease that is based on expression of AVR genes in host plants and may lead to the identification of new disease resistance loci in these allied monocot hosts. As noted above, homologs of AVR1-CO39, AVR2-YAMO and PWL2 are broadly distributed in subspecific groups of M. grisea. In some cases these have been shown to be functional and to exhibit the same host or cultivar specificity as AVR2-YAMO or PWL2. Whether other alleles confer new host/cultivar specificities will be important to learn. By analogy, homologs of the corresponding disease resistance genes may specify novel resistances that are recognized by these cultivar and host specificity gene alleles. The availability of cloned cultivar and host specificity genes from M. grisea and the corresponding disease resistance genes provides an experimental avenue to test this hypothesis. Work from my laboratory along with collaborators at the University of Wisconsin, the University of Kentucky, Kobe University, the John Innes Centre, and the University at Bangalore explore this relationship in the AVR1-CO39-PiCO39 (t) interaction across grass hosts and M. grisea pathotypes.

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When: Monday, July 23, 2001, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Gerry Byrne, Dept. of Medical Microbiology & Immunology

Title: Pathogenesis of Chlamydial Infections: Evolving Concepts of Chronic Diseases.

Abstract: Chlamydia trachomatis is well recognized as a causative agent in several important chronic human infectious diseases and C. pneumoniae has recently gained prominence due to its association with atherosclerosis and cardiovascular disease. Numerous fundamental questions about chlamydiae and the diseases they cause have not been adequately answered. These include such basic concepts as (a) what constitutes a chlamydial strain, (b) what is the nature of chlamydial virulence factors and how do they contribute to pathogenesis of chlamydial diseases, and (c) what sort of growth options are available to chlamydiae within the infected host cell. We have been examining how unique chlamydial growth patterns may play a role in the transition from acute to chronic disease states and have focused on the expression of stress response proteins as chlamydial virulence factors. Results will be presented that relate the struggle for intracellular chlamydial survival with host cell changes that are consistent with the involvement of C. pneumoniae in atherosclerotic lesion progression and destabilization. New research directions will be discussed with an emphasis on using genomic information to study chlamydial gene expression patterns in various cell culture models and in vivo.

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When: Monday, April 23, 2001, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Gary Splitter, Dept. of Animal Health and Biomedical Sciences

Title: Facultative intracellular bacterium: Brucella abortus intracellular adaptation and host response.

Abstract: Brucella spp. is a Gram negative facultative intracellular bacterium that induces chronic infectious disease by direct contact or by consumption of animal products. Brucella is considered a potential pathogen for bioterrorism. Our long-term goal is to develop a Brucella vaccine. Little is known regarding Brucella genes encoding proteins that contribute to intracellular survival and virulence. Recently, a Brucella promoter trap system has been engineered using the promoterless green fluorescence protein (GFP) gene to identify Brucella promoters and associated genes that are activated following intracellular infection. A library of Brucella genes activated within 4 hours of macrophage infection have been identified and additional genes are being determined. Also, our previous evidence indicates that CD4+, CD8+ T cells and IFN-gamma are prominent during clearance of acute infection; but, little data exist to indicate the immunologic features critical to disease resolution. However, a Brucella memory response is a likely foundation for successful vaccination, and immunologic memory is hypothesized to play a key role in protection. Now, strategies will be created to protect interferon regulatory factor-1 gene knockout (IRF-1-/-) mice that die within 7- 10 days from virulent Brucella using attenuated Brucella mutants as vaccine candidates, and evaluate the cytokines and cell phenotypes of immune cells to understand the mechanism of protection. In addition, IRF-1-/- mice can mount a protective immune response if vaccinated with certain attenuated mutants.

Our findings are anticipated to help define host-pathogen interactions and provide greater understanding of how Brucella produces disease.

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When: Monday, March 26, 2001, 4:00-5:00 PM

Where: 408 SMI (MMI Conference Room)

Speaker: Peter McNamara, Dept. of Medical Microbiology & Immunology

Title: Staphylococcus aureus Virulence Factor Regulation.

Abstract: Staphylococcus aureus can cause a range of infections, syndromes, and toxicosis that vary in their severity, duration, and pathogenic mechanism. One common factor affecting the establishment and progression of staphylococcal disease is the production of the requisite cell-surface and extracellular proteins.  S. aureus can produce upward of 40 molecules that augment virulence. These factors are regulated in a specific manner. The bacteria use signal transduction systems to sense the environmental conditions they encounter during the course of the pathogenic process and a regulatory RNA along with various transcriptional activators and repressors to optimize the expression of many genes to enhance survival new milieus. I will provide an overview of the staphylococcal regulatory loci, discuss our work on the identification and characterization of new transcriptional regulators, and within the limits of what is known, provide a model for the regulatory circuit that governs the production of virulence factors in S. aureus.

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Date: January 22, 2001

Speaker: Matyas Sandor

Title: Granuloma - a localized battlefront between the host and infectious agents.

Abstract: Granulomas are local inflammatory lesions that can control infectious or innflammatory agents and and insulate the healthy tissue from the degrading effects of inflammation. CD4⁺ T cells play a crucial role in the initiation and regulation of granulomatous reactions. Using liver granulomas induced by infecting mice with Schistosoma mansoni, Leishmania chagasi, or Mycobacterium bovis, we explore how T cells induce granulomas. The requirements of cytokines, costimulatory molecules for granuloma formation, the role of T cell specificity in the T cell homing into granulomas, the extent of intragranuloma T cell repertoire, the role of specific and non-specific T cells in granuloma formation will be discussed.

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Date: November 27, 2000

Speaker: Heidi Goodrich-Blair

Title: Xenorhabdus nematophilus as a model for host-bacterium interactions.

Xenorhabdus nematophilus is a Gram-negative bacterium that is a mutualist of Steinernema carpocapsae nematodes and a pathogen of larval-stage insects. Our lab employs this organism as a model of host-microbe interactions to ask which functions bacteria require for mutualism, pathogenesis, or both. X. nematophilus is particularly well suited for experimental studies of host-microbe interactions because: a) it is genetically and technically tractable; b) it can be cultivated under laboratory conditions in the absence of its eukaryotic hosts; and, c) its mutualistic and pathogenic interactions with its hosts can be separately and easily assayed. Thus, each aspect of the host-microbe interaction can be dissected and analyzed individually. Furthermore, the hosts of X. nematophilus provide several advantages over other host-microbe systems. Insect hosts such as Galleria mellonella (wax moth) and Manduca sexta (tobacco hornworm) are inexpensively and rapidly propagated. Nematodes are also easily reproduced; millions of nematodes can be generated from the infection of a single insect host, and bacteria-free nematode eggs can be isolated and grown on selected lawns of bacteria in the absence of an insect host. Current research in the lab will be discussed, including characterization of X. nematophilus mutants that are defective either in nematode colonization or insect pathogenesis.

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Date: October 23, 2000

Speaker: Curtis Brandt

Title: A new way of looking at viral virulence: a system to study the contributions of multiple genes.

Abstract: Studies of how viral genes affect virulence have remained true to the reductionist philosophy in that one focuses on a single gene. With Herpes simplex virus (HSV), early studies relied on isolating mutant viruses with large changes in virulence (eg. LD₅₀ changes from 10 pfu to 10⁷ pfu) followed by the usual strategy of mapping and sequencing the mutation. More recently, one can choose an orf, engineer a mutation and then determine the effect. Many labs including ours have used this approach to identify virulence determinants but again, large effects are sought. If a mutation does not dramatically affect disease, it is not a serious contender. Gene chips are now being used to look at multiple changes in expression during infection but viruses are fundamentally different from other pathogens in that they carry few if any "extra" genes. For lytic viruses, the entire program of gene expression is used so differential gene expression is unlikely to be very informative. For chronic or latent viruses, differential gene expression occurs but relatively few genes are involved. Identification of such genes however still does not inform us as to function and effect on virulence. Realistically, it is the "constellation" of genes in a given virus that determine the virulence phenotype, with multiple genes ultimately contributing. Each contribution could be small yet critical. We have devised a system that allows us to identify and study the contributions of multiple genes. This system will be described along with current results from work in the lab.

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Date: September 25, 2000

Speaker: Jon Woods

Title: Histoplasma capsulatum pathogenesis.

Abstract: Our laboratory studies pathogenic mechanisms of the dimorphic fungus Histoplasma capsulatum using a variety of molecular approaches.  H. capsulatum is a major cause of respiratory and systemic mycosis in the United States and worldwide, particularly in immunocompromised people such as those with AIDS. It is a facultative intracellular pathogen that survives and replicates in the harsh environment of the host macrophage phagolysosome to cause disease. We focus on environmental gene regulation, virulence determinants, and interactions with the mammalian host. We have used various techniques (IVET, differential display, cDNA RDA) to identify fungal genes upregulated during infection of host cells or animals; we are interested in their regulatory mechanisms and their functions and effects. One of these genes appears to be an antisense downregulator of a protein kinase gene. Another gene encodes a cell wall and secreted protein that may be an EGF homolog involved in attachment or signaling. Other genes include homologs of a peptide transporter and iron acquisition genes. In separate but related work, we identified iron-regulated secreted ferric reductase activity in this organism, that may provide an iron acquisition mechanism that is additional, alternate, or interactively complementary to siderophore production. We are also examining a secreted catalase that is a major immunogen in the mammalian host and may serve as a defense against host oxidative responses.