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Berlin Brandenburg

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  • 1
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid, which has been shown to facilitate the transmission of HIV. H. ducreyi is likely exposed to multiple sources of antimicrobial peptides in vivo. APs are small, cationic molecules with both bactericidal and immunomodulatory functions. Because H. ducreyi is able to establish and maintain an infection in an environment rich with antimicrobial peptides, we hypothesized that the bacterium was resistant to the bactericidal effects of these peptides. Using a 96-well AP bactericidal assay, we examined H. ducreyi susceptibility to eight human APs likely to be encountered at the site of infection, including the α-defensins human neutrophil peptide-1, human neutrophil peptide-2, human neutrophil peptide-3, and human defensin 5, the β-defensins human β defensin-2, human beta defensin-3, and human beta defensin-4, and the human cathelicidin, LL-37. H. ducreyi survival was compared to the survival of Escherichia coli ML35, a strain known to be susceptible to several antimicrobial peptides. H. ducreyi was significantly more resistant than E. coli ML35 to the bactericidal effects of all peptides tested. Furthermore, we found that representative class I and class II strains of H. ducreyi were each resistant to APs of each functional category, indicating that resistance to antimicrobial peptides could represent a conserved method of pathogenesis for H. ducreyi as a species. The H. ducreyi genome contains a homolog for the Sap influx transporter. To study the role of the H. ducreyi Sap transporter in AP resistance, we generated an isogenic sapA mutant and used the 96-well AP bactericidal assay to compare the AP susceptibility profiles of wild-type H. ducreyi, the sapA mutant and the sapA trans-complement to α-defensins, β-defensins, and LL-37. We observed a 25% decrease in the survival of the sapA mutant when it was exposed to LL-37. These findings suggest that the H. ducreyi Sap transporter plays a role in H. ducreyi resistance to LL-37, but it is likely that other AP resistance mechanisms co-exist within the bacterium.
    Keywords: Antimicrobial Peptides ; Chancroid ; Hnp-2 ; Ll-37 ; Haemophilus Ducreyi ; Hnp-2 ; Hnp-3 ; Hd-5 ; Hbd-2 ; Hbd-3 ; Hbd-4 ; Haemophilus Ducreyi ; Chancroid ; Sexually Transmitted Diseases ; Peptide Antibiotics
    Source: Networked Digital Library of Theses and Dissertations
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  • 2
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.
    Keywords: Haemophilus Ducreyi ; Phosphoethanolamine Transferase ; Antimicrobial Peptide Resistance
    Source: Networked Digital Library of Theses and Dissertations
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  • 3
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Localization of flavocytochrome b to the phagosome is essential for microbial killing, yet the subcellular distribution of flavocytochrome b in macrophages and how it is incorporated into macrophage phagosomes is not well characterized. In neutrophils, flavocytochrome b localizes primarily to specific granules that are rapidly mobilized to the phagosome upon stimulation. In contrast to neutrophils, macrophages do not contain specific granules, and trafficking of membrane proteins to the phagosome is more dynamic, involving fission and fusion events with endosomal compartments. We hypothesized that in macrophages, flavocytochrome b localizes to both plasma membrane and endosomal compartments that deliver flavocytochrome b to the phagosome. We generated fluorescently tagged versions of both p22phox and gp91phox, and rigorously verified their functionality in Chinese Hamster Ovary cells. Localization of flavocytochrome b was then examined in both RAW 264.7 murine macrophages and primary murine bone marrow derived macrophages (BMDM) in the presence and absence of interferon gamma (IFNg). We found that in “resting” macrophages, flavocytochrome b localizes primarily to the Rab11-positive endosome recycling compartment that recycles to the plasma membrane. In addition, phagocytosis assays showed flavocytochrome b is incorporated into the phagocytic cup and colocalized with Rab11 at the base of the cup, suggesting Rab11-positive endosomes may be involved in trafficking of flavocytochrome b between intracellular membranes and forming or nascent phagosomes. However, in IFNg activated macrophages, flavocytochrome b was localized predominantly in the plasma membrane, with little present in endosomal compartments. This shift in flavocytochrome b distribution occurred following sustained exposure to IFNg and correlated with increased flavocytochrome b protein expression and increased extracellular production of superoxide. Taken together, our results suggest the IFNg-induced redistribution of flavocytochrome b may be important for enhancing the production of superoxide at the cell surface and may be a potential new mechanism by which IFNg enhances antimicrobial activity in macrophages.
    Keywords: Host Defense ; Superoxide ; Superoxide
    Source: Networked Digital Library of Theses and Dissertations
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  • 4
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Globally, protein malnutrition remains problematic, adversely affecting several systems including the immune system. Although poorly understood, protein restriction severely disrupts host immunity and responses to infection. Induction of high-affinity, long-lasting immunity depends upon interactions between B and T lymphocytes. B lymphocytes exploit several pathways including endocytosis, macroautophagy, and chaperone-mediated autophagy to capture and deliver antigens to the endosomal network. Within the endosomal network antigens are processed and loaded onto major histocompatibility complex (MHC) class II molecules for display and recognition by T lymphocytes. To examine the effect of macronutrient malnutrition on MHC class II antigen presentation, we grew B lymphocytes in media containing amino acids, sugars and vitamins but lacking serum, which contains several types of macronutrients. Our studies show macronutrient stress amplified macroautophagy, favoring MHC class II presentation of cytoplasmic antigens targeted to autophagosomes. By contrast, macronutrient stress diminished MHC class II presentation of membrane antigens including the B cell receptor (BCR) and cytoplasmic proteins that utilize the chaperone-mediated autophagy pathway. The BCR plays a critical role in MHC class II antigen presentation, as it captures exogenous antigens leading to internalization and degradation within the endosomal network. While intracellular protease activity increased with macronutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of high molecular mass macronutrients restored endocytosis and antigen presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Cytosolic chaperone HSC70 has been shown to play a role in endocytosis, macroautophagy, chaperone-mediated autophagy and proteolysis by the proteasome, potentially connecting distinct routes of antigen presentation. Here, altering the abundance of HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and antigen presentation suggesting macronutrient deprivation alters the availability of HSC70. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating distinct membrane trafficking pathways during cellular stress. These results offer a new explanation for impaired immune responses in protein malnourished individuals.
    Keywords: Mhc Class Ii ; Hsc70 ; Antigen Presentation ; Nutrient Deprivation ; Malnutrition ; Protein Deficiency ; Immunity - Nutritional Aspects ; Nutrition Disorders ; Deficiency Diseases ; Lymphocytes ; Antigens ; Nutrient Interactions ; B Cells
    Source: Networked Digital Library of Theses and Dissertations
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  • 5
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease. Chlamydia spp. are all obligate intracellular organisms that undergo a biphasic developmental cycle within a vacuole termed the inclusion. Infectious, non metabolically active elementary bodies (EBs) are endocytosed and differentiate into non infectious, metabolically active reticulate bodies (RBs) before re-differentiating back into EBs. The chlamydial factors that mediate these differentiation events are mostly unknown. Comparative genomics revealed that Chlamydia spp. have small, highly conserved genomes, suggesting that many of their genes may be essential. Genetic manipulation strategies for Chlamydia spp. are in their infancy, and most of these cannot be used to inactivate essential genes. We generated a clonal ethyl methanesulfonate (EMS)-mutagenized C. trachomatis library and screened it for temperature sensitive (TS) mutants that produced fewer inclusions at either 32°C or 40°C compared to 37°C. Because EMS mutagenesis elicited multiple mutations in most of the library isolates, we also developed a novel lateral gene transfer strategy for mapping mutations linked to TS phenotypes. We identified TS alleles of genes that are essential in other bacteria and that are involved in diverse biological processes including DNA replication, protein synthesis, carbohydrate metabolism, fatty acid biosynthesis, and energy generation, as well as in highly conserved chlamydial hypothetical genes. TS DNA polymerase (dnaEts) and glutamyl-tRNA synthestase (gltXts) mutants were characterized further. Both the dnaEts and gltXts mutants failed to replicate their genomes at 40°C but exhibited unique signs of stress. Chlamydial DNA replication begins by 12 hpi and protein synthesis begins by 2 hpi. However, inclusion expansion and replication of both of the mutants could be rescued by shifting to them to 37°C prior to mid-late development. Since gltXts is likely unable to produce aminoacyl-tRNAs at 40°C, our observation suggests that de novo chlamydial translation uses a pre-existing pool of aminoacyl-tRNA in EBs. Genetic suppressor analysis indicated that the inability of the dnaEts mutant to replicate its genome at 40°C might be linked to an inability of mutant DnaE to bind the DNA template. The tools and mutants we have identified will be invaluable assets for investigating many essential aspects of chlamydial biology.
    Keywords: Chlamydia ; Essential Genes ; Genetics ; Mutagenesis
    Source: Networked Digital Library of Theses and Dissertations
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  • 6
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Borrelia burgdorferi, a bacterium which causes Lyme disease, is maintained in nature through a cycle involving two distinct hosts: a tick vector and a mammalian host. To adapt to these two diverse environments, B. burgdorferi undergoes dramatic alterations in its surface lipoprotein. Two essential lipoproteins, outer surface protein A (OspA) and outer surface protein C (OspC), are reciprocally regulated throughout the B. burgdorferi lifecycle. Very little is known about the regulation of OspA. These studies elucidate the regulatory mechanisms controlling the expression of OspA. Various truncations of the ospA promoter were created and then studied in our novel in vitro model of ospA repression or grown within the host-adapted model. A T-Rich region of the ospA promoter was determined to be a cis-element essential for both the full expression and full repression of ospA.
    Keywords: Borrelia Burgdorferi ; Ospa ; Outer Surface Lipoprotein A ; Borrelia Burgdorferi -- Research ; Lyme Disease ; Lyme Disease -- Molecular Aspects ; Spirochetes -- Molecular Aspects ; Lipoprotein A ; Host-Bacteria Relationships ; Bacteria -- Physiology ; Bacterial Cell Walls ; Bacterial Cell Surfaces
    Source: Networked Digital Library of Theses and Dissertations
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  • 7
    Description: Indiana University-Purdue University Indianapolis (IUPUI) At the present time, gentamicin is used in the treatment of both Gram-negative and Gram-positive bacterial infections. However, the poorly understood side effect of nephrotoxicity is a serious problem and is one of the dose-limiting factors in the use of gentamicin. In our model system, Saccharomyces cerevisiae, which is relatively resistant to gentamicin, at least 20 genes are required for gentamicin resistance. Inspection of the physical and genetic interactions of the gentamicin sensitive mutants reveals a network centered on the ARF pathway which plays a key role in the regulation of retrograde trafficking. Our studies show that arf1ts arf1Δ arf2Δ cells, gea1ts gea1Δ gea2Δ cells, and gcs1ts gcs1Δ glo3Δ cells are all hypersensitive to gentamicin which indicates that impaired Arf1 function causes yeast cells to become hypersensitive to gentamicin. As evidence, cellular CPY trafficking and processing are blocked by the presence of gentamicin in some of these mutants. Interestingly, gentamicin can directly affect the level of the GTP-bound form of Arf1 in a cell growth phase-dependent manner; even though total Arf1 levels in S. cerevisiae are not affected. As predicted, we also find that gentamicin-bound resin can enrich both yeast Arf1-TAP protein and rat Arf1 protein in vitro. With the help of mass spectrometry, we also generated a gentamicin-binding protein list. Gentamicin hypersensitivity is also observed in S. cerevisiae double deletion strains that lack both ARF1 and ARF2 but are kept alive by the presence of hARF4 or bARF1. Increased -1 programmed ribosomal frameshifting efficiency is also observed in cells treated with gentamicin. Finally, a comparison of a gentamicin mixture and four of the gentamicin congeners reveals that gentamicin C1 is less toxic than other gentamicin congeners or the gentamicin total mixture.
    Keywords: Gentamicin ; Arf Protein Trafficking Pathway ; Gentamicin ; Bacterial Diseases -- Treatment ; Nephrotoxicology
    Source: Networked Digital Library of Theses and Dissertations
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  • 8
    Description: Indiana University-Purdue University Indianapolis (IUPUI) Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcerative disease that facilitates the transmission of HIV-1. H. ducreyi also causes non-sexually transmitted cutaneous ulcers in children in tropical regions. During human infection, H. ducreyi is subject to a variety of stresses. The stringent response is a bacterial stress response system induced by nutrient limiting conditions and mediated by guanosine tetra- and pentaphosphate [(p)ppGpp] and the transcriptional regulator DksA. (p)ppGpp and DksA jointly interact with RNA polymerase to regulate genes critical for bacterial survival. We hypothesized that the stringent response is required for H. ducreyi virulence in humans. A ΔrelAΔspoT mutant, which is unable to synthesize (p)ppGpp, was partially attenuated for abscess formation in human volunteers. Loss of (p)ppGpp increased bacterial resistance to phagocytosis and stationary phase survival; however, the mutant was more sensitive to oxidative stress. A ΔdksA mutant was also partially attenuated in humans. The ΔdksA mutant behaved like the (p)ppGpp mutant in stationary phase survival and sensitivity to oxidative stress, but exhibited decreased resistance to phagocytosis. Both mutants had decreased adherence to fibroblasts, but the mechanisms underlying the adherence defect were distinct. To better understand the roles of (p)ppGpp and DksA in regulating gene expression, we performed transcriptome analysis of the parent and mutant strains. (p)ppGpp and DksA deficiency resulted in dysregulation of multiple genes including several known virulence determinants. At stationary phase, (p)ppGpp and DksA targets were not identical but significantly overlapped; as the mutants were phenotypically distinct, this finding underscores both the unique and joint roles DksA and (p)ppGpp play in regulation of H. ducreyi virulence. We conclude that (p)ppGpp and DksA play significant roles in H. ducreyi pathogenesis. This is the first study to show that the stringent response has a direct role in the ability of a bacterial pathogen to cause disease in humans.
    Keywords: Haemophilus Ducreyi ; Humans ; Pathogenesis ; Stringent Response ; Virulence ; Haemophilus Ducreyi ; Chancroid -- Etiology ; Sexually Transmitted Diseases ; Haemophilus Infections ; Rna Polymerases ; Transcription ; Mutation -- Genetics
    Source: Networked Digital Library of Theses and Dissertations
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  • 9
    Description: Indiana University-Purdue University Indianapolis (IUPUI) The Lyme disease agent, Borrelia burgdorferi, has a complex system that allows it to thrive in the harsh and distinct environments of its tick vector and mammalian host. Although it has been known for some time that the Borrelia oxidative stress regulator protein (BosR) plays a necessary role in mammalian infectivity and functions as a transcriptional regulator of alternative sigma factor RpoS, very little is known about its mechanism of action, other than the suggestion that BosR activates rpoS transcription by binding to certain upstream regions of the gene. In our studies, we performed protein degradation assays and luciferase reporter assays for further understanding of BosR function. Our preliminary findings suggest that BosR is post-transcriptionally regulated by an unknown protease and may not need to bind to any rpoS upstream regions in order to activate transcription. We also describe the construction of luciferase reporter systems that will shed light on BosR’s mechanism of action. We postulate the provocative possibility that unlike its homologs Fur and PerR in other bacterial systems, BosR may not utilize a DNA-binding mechanism in order to fulfill its role as a transcriptional regulator to modulate virulence gene expression.
    Keywords: Lyme Disease ; Borrelia Burgdorferi ; Bosr ; Rpos ; Lyme Disease -- Research -- Analysis ; Borrelia Burgdorferi -- Research ; Genetic Regulation ; Gene Expression -- Technique ; Genetic Transcription -- Regulation ; Protease Inhibitors ; Virulence (Microbiology) ; Rna Polymerases ; Polymerase Chain Reaction ; Ticks As Carriers Of Disease ; Messenger Rna -- Research -- Analysis -- Evaluation ; Microbial Genetics -- Technique ; Polyacrylamide Gel Electrophoresis
    Source: Networked Digital Library of Theses and Dissertations
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  • 10
    Description: Indiana University-Purdue University Indianapolis (IUPUI) CD1d is a cell surface glycolipid that, like Major Histocompatibility Complex (MHC) class I and MHC class II molecules, presents antigen. However, instead of peptides, CD1d presents lipids to Natural Killer (NK) T cells, a subset of T cells that express both NK cell markers and the T cell receptor and produces both T helper (Th) 1 and Th2 cytokines. Our lab focuses on the regulation CD1d-mediated antigen presentation. TGF beta is a known regulator of the immune system, such as controlling MHC class II antigen presentation. Further, TGF beta can activate the mitogen activated protein kinase (MAPK) p38, a known negative regulator of CD1d-mediated antigen presentation. Therefore, we hypothesized that TGF beta would be a negative regulator of CD1d-mediated antigen presentation, and our results showed a decrease in antigen presentation by CD1d in response to TGF beta treatment. However, this inhibition was not through p38 activation, as indicated by the absence of a rescue of CD1d-mediated antigen presentation in, TGF beta-treated, p38 dominant negative-expressing cells. Alternatively, the Smad pathway, the canonical pathway activated by TGF beta, was investigated through a lentivirus shRNA-mediated knockdown of Smad2, Smad3 and Smad4 proteins. Smad2 shRNA-expressing cells showed in an increase in CD1d-mediated antigen presentation, suggesting an inhibitory role for Smad2. In contrast, Smad3 shRNA-expressing cells did not differ from control cells. However, as in the case of Smad2, CD1d+ cells in which Smad4 was knocked down, were substantially better at CD1d-mediated antigen presentation than control cells, suggesting that it also negatively regulates antigen presentation. Overall, these studies demonstrate that the canonical TGF beta/Smad pathway regulates an important part of the host’s innate immune response, vis-à-vis CD1d-mediated antigen presentation.
    Keywords: Antigen Presenting Cells -- Regulation ; Glycolipids -- Regulation ; Immune System -- Regulation ; Transforming Growth Factors-Beta ; T Cells
    Source: Networked Digital Library of Theses and Dissertations
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