Thesis (PH.D.) -- Syracuse University, 2006 / "Publication number AAT 3251783."

abstract: Coccidioidomycosis (valley fever) is caused by inhalation of arthrospores from soil-dwelling fungi, Coccidioides immitis and C. posadasii. This dimorphic fungus and disease are endemic to the southwestern United States, central valley in California and Mexico. The Genome of Coccidioidies has been sequenced but proteomic studies are absent. To address this gap in knowledge, we generated proteome of Spherulin (lysate of Spherule phase) using LC-MS/MS and identified over 1300 proteins. We also investigated lectin reactivity to spherules in human lung tissue based on the hypothesis that coccidioidal glycosylation is different from mammalian glycosylation, and therefore certain lectins would have differential binding properties to fungal glycoproteins. Lectin-based immunohistochemistry using formalin-fixed paraffin-embedded human lung tissue from human coccidioidomycosis patients demonstrated that Griffonia simplificonia lectin II (GSL II) and succinylated wheat germ agglutinin (sWGA) bound specifically to endospores and spherules in infected lungs, but not to adjacent human tissue. GSL II and sWGA-lectin affinity chromatography using Spherulin, followed by LC-MS/MS was used to isolate and identify 195 proteins that bind to GSL-II lectin and 224 proteins that bind to sWGA lectin. This is the first report that GSL II and sWGA lectins bind specifically to Coccidioides endospores and spherules in infected human tissues. Our list of proteins from spherulin (whole and GSL-II and sWGA binding fraction) may also serve as a Coccidioidal Rosetta-Stone generated from mass spectra to identify proteins from 3 different databases: The Broad Institutes Coccidioides Genomes project, RefSeq and SwissProt. This also serves as a viable avenue for proteomics based diagnostic test development for valley fever. Using lectin chromatography and LC MS/MS, we identified over 100 proteins in plasma of two patients and six proteins in urine of one patient. We also identified over eighty fungal proteins isolated from spherules from biopsied infected lung tissue. This, to the best of our knowledge, is the first such example of detecting coccidioidal proteins in patient blood and urine and provides a foundation for development of a proteomics based diagnostic test as opposed to presently available but unreliable serologic diagnostic tests reliant on an antibody response in the host. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2015

<p>Sulfur-cycling microbial mats found around near-shore hydrothermal vents at White-Point, Palos Verdes are highly diverse communities that show variation in community structure over small spatial scales. This study investigated the within-site variability of the mats by comparing community structure, using 16S rRNA gene sequences, between mat samples collected in the intertidal and subtidal habitats at two different time points; March and June of 2015. The mats were dominated by two sulfur-oxidizers Sulfurovum and Arcobacter. Analysis showed that many of the same microbes were found across sample types; however, mat community structure was significantly different between the intertidal and subtidal habitats and between the two intertidal time points. Changes in community structure correlated to different environmental conditions across the four sample types, suggesting that community structure is driven by environmental selection over dispersal capabilities within this vent site.

A Chinese hamster ovary (CHO) fibroblast, transfected with murine major histocompatibility complex (MHC) class II genes, inefficiently simulated CD4+ Th cells specific for ovalbumin (OVA), hen egg lysozyme (HEL), and pork insulin which contains disulfide bonds. However, the fibroblasts elicited a T cell response to λ-repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented, suggesting that the processing defect is a recessive trait. Three distinct processing phenotypes were observed among twenty-eight hybrid clones analyzed for their ability to process a suboptimal concentration of OVA suggesting that a limited number of genes mediates the defect of WAB4 cells. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL either as effectively or more efficiently than a B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed conjugate consisting of 125I-tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage for the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. The WAB4 cells had normal intracellular levels of cysteine, however they possessed diminished levels of intracellular glutathione (GSH). Buthionine sulfoximine (BSO) - mediated reduction of intracellular levels of GSH decreased the ability of the hybrid line WALC to process HEL. Conversely, treatment of WAB4 cells with N-acetyl cysteine increased their efficiency in the processing of HEl. These findings indicate that the intracellular level of GSH influences the capacity of cells to process antigens with the disulfide bonds. Thus, the antigen processing defect exhibited by transfected CHO cells is probably caused by their impaired ability to reduce disulfide bonds which may be related to the diminished intracellular GSH level.

Sublines were derived from the U937 human monocytic cell line by limiting dilution techniques. Several of the sublines derived in this manner were found to constituitively express la, unlike most U937 cell lines previously examined. Thus the la+ and la- sublines were examined and characterized phenotypically and functionally. Morphologically the sublines, both la+ and la-, were found to be similar to the originally described U937 parent cell line. They were positive for production of non- specific esterase and expressed the HLA phenotype A(3,X), B(51,18), DR(2,X). The sublines were examined by immunofluorescence techniques with a large number of antibodies specific for cell surface structures and found to express MHC class I, MHC class II the major type being HLA-DR and perhaps HLA-DO in small amounts, Fc receptor, TA-1 structure typical of monocytes and T cells, endothelial antigens and CD4. The sublines and parent U937 cell line were found to express few cell surface antigens typical of mature monocytes/macrophages; however, they expressed myeloid antigens typical of early monocytic or promonocytic lineage. Expression of MHC class II by the sublines was confirmed by immunoprecipitation with monoclonal antibody to a framework determinant of human la and SDS-PAGE autoradiography which gave bands at p29:34 for Ia+ sublines but not for Ia- parent U937 cell lines. Cell surface expression of la was increased to a significant degree by treatment with gamma interferon which peaked at 24-48 hours of treatment. Functionally the parent U937 cell line and several Ia+ sublines were examined in several assays. The la- and Ia+ U937 cells were found to stimulate the generation of specific CTLs in CML assays to approximately the same degree. The Ia+ sublines were found to stimulate in MLR assay, although variable results were obtained and indicated that the U937 parent and subline cells produced factors which were found to be inherently immunosuppressive. The sublines were able to substitute for monocytes by reconstituting the C03 mediated T cell mitogenic response. The Ia+ sublines and the parent U937 cell line (also weakly Ia+) were found to present tetanus toxoid antigen to nylon-wool purified T cells following an overnight pulse with antigen, and this response was found to be significantly abrogated by addition of antibody specific for CD4 and MHC class II, but not MHC class I. Preliminary characterization of the immunosuppressive factor produced by the U937 cell line and the sublines revealed that it was strongly antiproliferative and affected lymphoid cells somewhat more than non-lymphoid cells; that its probable molecular weight was approximately 90,000, it was not inactivated by treatment with trypsin or chymotrypsin, was not immediately inactivated by freezing although dialysis and long term storage diminished its activity, and was partially inactivated by heat treatment at both 56°C and 80°C. Clear indication of soluble lL-1 production by the U937 parent cell line and the sublines was problematic due to the strong inhibition of proliferative assays by supernatants; however, partial inactivation of inhibitory activity by heat treatment as well as partial removal of the inhibitor fraction by gel filtration indicated that lL-1 or a cytokine with lL-1 activity was constituitively produced by the cells. Membrane lL-1 was detected in very low amounts in unstimulated cells and was significantly increased by treatment with phorbol esters but not other immunomodulators. Preliminary examination of Northern blots of HLA-DR alpha and HLA-DQ alpha mRNA production by the parent U937 cells and the sublines revealed that both HLA-DR alpha and HLA-DQ alpha mRNA were detectable for |a+ sublines E11, G4 and G11, that only trace amounts were detectable for relatively Ia- subline E9 and that surprisingly, HLA-DQ alpha was detectable for the 2-1 parent cell line but no HLA-DR alpha mFiNA was detectable. The results of mRNA analysis following gamma interferon treatment, as well as treatment with LPS and phorbol esters, were variable for the different sublines and indicate that in some sublines gamma interferon treatment appears to augment la specific mRNA and in others the levels are decreased indicating that the expression of MHC class II specific mRNA may be differentially regulated in the different sublines.

<p> <i>Chromobacterium violaceum</i> is a Gram-negative bacillus that is a facultative anaerobic, oxidase positive, glucose fermenting, and non-lactose fermenting. <i>C. violaceum</i> is free living which can be found in the soil and water. <i>C. violaceum</i> produces a possible antioxidant called violacein a purple pigment that is considered to be one of the significant characteristics of <i>C.violaceum. Chromobacterium violaceum</i> one of the Gram negative bacteria which use Quorum Sensing (QS) occurs within a single bacterial species, and regulates a lot of different processes ,essentially serving as a simple communication network so that QS is the accumulation of signaling molecule that enable a single cell to sense the communication from other cells .The density and bioluminescence through quorum sensing is manipulated via the signaling molecule N-acyl-homoserine (AHLs) that is produced by <i>C. violaceum.</i> The major objective in this work is to determine the reason why 14N1 cannot produce the pigment on LB media in cultures using heavy cell inoculum and high concentration of AHL on all the <i>C. violaceum</i> strains. <i>C. violaceum </i> 14N23 and 14N1 were isolated from copper basin, Tennessee and (ATCC) 12472 and (ATCC) 31532 obtained from American Type Culture Collection (ATCC), and CV026. 14N1 produces a pigment after 24 hours by using high inoculation 20% which is 10 ml overnight culture of 14N1 to 40 ml LB broth media with different shaker's speed started from (in static culture , 150,175,225). On the AHLs 14N1 did not produce a pigment and that the same with 14N23. However, 12472 did produce a pigment after 24 hours, and the positive control (ATCC) 31532 produced and the negative control CV026 did not.</p>

Clostridium scindens is an obligate anaerobe isolated from the normal intestinal flora of man. It is the only bacterium known at present to synthesize both steroid-17,20-desmolase and 20α-hydroxysteroid dehydrogenase (ZOα-HSDH). This study was undertaken to characterize these neutral steroid transforming reactions in extracts of C. scindens; to purify and characterize the enzymes responsible for side-chain cleavage and 20α- hydroxysteroid oxidoreduction and to clone the genes encoding these enzymes with a view towards studying the mechanism of induction by steroids in a prokaryotic system. Both enzymes were found to be co-inducible in cells cultured in the presence of 17α-hydroxysteroids. In cell extracts, only 17α,21- dihydroxysteroids served as substrates for both activities. Desmolase has been partially purified by conventional DEAE- cellulose chromatography. Although the desmolase reaction was stimulated by NAD+ and bivalent metal cations in cell extracts, the partially purified enzyme was only reactive with coenzyme B12. The observed difference in cofactor requirements in crude cell extracts and partially purified preparations is not understood at present. 20α-HSDH has been purified 252-fold to apparent homogeneity by a method involving ammonium sulfate fractionation, conventional DEAE-cellulose chromatography, elution from Cibacron blue agarose, gel filtration HPLC and DEAE HPLC. The pH optimum for reduction of the 20-ketone of cortisol with NADH is between pH 6-6.5. The pH optimum for oxidation of the 20α-hydroxyl of 20α-dihydrocortisol with NAD+ is approx. pH 8. Michaelis constants were determined: Km for NADH (8 μM); cortisol (32 μM); cortisone (22μM); NAD+ (526μM): 20α-dihydrocortisol (41 μM). The native enzyme is apparently a tetramer comprised of subunits with a Mr 40,000. Although two bands with a slightly different charge (pI approx. 6.1) were observed on two-dimensional PAGE, a single N-terminus was obtained by gas phase sequencing. A computer aided search (FASP) showed that the partial amino acid sequence (1-11) was highly homologous with glyceraldehyde-3-phosphate dehydrogenases (GAPDH) isolated from a variety of sources. Residues 4-8 of GAPDH are involved in binding of the pyridine nucleotide cofactor. On Western blots of cortisol induced and uninduced cell extracts, two bands differing in size by approximately 2000 daltons were observed. The presence of immunoreactive proteins with the same molecular weight as the purified 20α-HSDH in uninduced cell extracts may be due to cross-reactivity with constitutive GAPDH (Mr, 39,000). Using a 32P-labelled oligonucleotide probe corresponding to amino acids 3-8, two discrete bands were seen on Southern blots of EcoR1 digests of C. scindens genomic DNA. Efforts to clone these fragments in lambda gt11 were unsuccessful.

<p> The microbiota is an important contributor to host health and fitness, impacting all aspects of life, from development and metabolism to immunity and behavior. Substantial work has been done to characterize the factors that shape the microbiota, and have demonstrated the importance of both environmental and host factors. However, much of the multifaceted relationship between the environment, the host, and the microbiota remains to be elucidated. My dissertation attempts to demonstrate the ways in which hosts can shape their microbiota through a taxonomic and functional evaluation of microbiotas within two experimental systems. </p><p> In chapter 1, I examine the relative contributions of the host and the environment to microbiota composition. To characterize the gut microbiota of C. elegans, I used 16S rDNA-targeted sequencing to measure the gut bacteria of worms grown in natural-like environments. By taking advantage of the availability of genetically-homogenous worm populations to reduce noise and average out inter-individual variation, and thus better discern shared features of the C. elegans microbiota, I demonstrate that the worm gut microbiota assembly is a deterministic process. My results suggest a dominant contribution of the host to microbiota composition, and further suggest a role for negative interactions between microbiota members. </p><p> In chapter 2, I further examined the contribution of host genetics to microbiota composition by identifying differences in microbiotas assembled in worms of different genotypes spanning 200-300 million years of nematode evolution. Using 16S rDNA- targeted sequencing, I demonstrate a significant contribution of host genetics to microbiota composition. However, experimental variables affected worm microbiota composition more than the worm genotype, and hindered my identification of host-specific taxa. In an attempt to overcome this, I isolated members of the Enterobacteriaceae core microbiota family from C. elegans and C. briggsae. This, however, did not identify phylogenetic distinctions between commensals of the two species.</p><p> In chapter 3, I surveyed the roles of specific host factors in shaping the C. elegans gut microbiota by examining the gut microbiota of C. elegans mutants deficient in feeding and immune function. When selecting mutants, I considered selection during feeding and selection within the intestine as two general ways a host could shape its microbiota. Altered feeding could affect colonization by changes in food choice, food uptake, and food grinding. Immune responses can significantly affect the colonization of pathogens, and it is likely that these immunity and stress response pathways could affect the colonization of non-pathogenic bacteria. </p><p> In chapter 4, I utilized the tomato plant and P. syringae system to examine the ability of the phyllosphere microbiome to confer resistance to P. syringae infection of tomato leaves. In plants, the phyllosphere (above ground) microbiome is likely to be important for resistance to pathogens that infect the aerial portions of plants; however, relative to the below-ground microbiome, these communities are understudied and we do not yet know how the protective effects of this microbiome might vary as a function of bacterial density or resource availability. My results suggest that the presence of a phyllosphere microbiome does decrease pathogen colonization success, as plants sprayed with a microbiome had significantly less pathogen growth than plants sprayed with sterile buffer. I additionally show that the dose of microbiome spray has an effect on protection, and that this effect is dependent on the composition of the microbial community, but not microbial diversity. Finally, my results suggest that microbe-microbe competition (resource community dynamics) may play an important role in protection, as the addition of fertilizer abolishes the observed microbiome-mediated protection. Together, my results may alter our understanding of microbiome-mediated protection within agricultural settings and the use of plant probiotics. (Abstract shortened by ProQuest.)</p><p>

<p> Epstein-Barr Virus (EBV) is a herpesvirus responsible for approximately 1% of cancers worldwide, including African Burkitt lymphoma, Hodgkin lymphoma, lymphomas in immunosuppressed patients, and nasopharyngeal and gastric carcinoma. Interestingly, the development of different cancers is mainly due to expression of the latent EBV proteins, although expression of lytic genes has recently been shown to play a role. Many of the molecular mechanisms behind EBV reactivation have been revealed, however, the contribution of chromatin dynamics to EBV reactivation is not well understood. A better understanding of how the switch between the latent and lytic cycle is regulated could have implications in treating disease. Here we investigate the contribution of chromatin dynamics to EBV reactivation in the context of i) nuclear localization and contacts with the human genome, and ii) the chromatin-reading protein BRD4. We used in situ Hi-C to show that the Epstein-Barr virus associates with repressive compartments of the nucleus during latency, and non-repressive compartments of the nucleus during reactivation. This adds 3D re-localization as a novel component to the molecular events that occur during EBV reactivation. Furthermore, we show that the protein BRD4 plays an important role in EBV lytic reactivation. BRD4 binds to the lytic origins of replication and inhibition of BRD4 by JQ1 inhibits the lytic cycle at two different steps. In summary, this work has led to a better understanding of how the latent-lytic switch of EBV is regulated.</p><p>

<p> Microbial life represents the majority of the diversity of life on planet earth. Microbes are found in all ecosystems. The microbial community of an ecosystem can be an indicator of its health and the foundation of the ecosystem function. Thus, an understanding of the microbial community of an ecosystem is vital to understanding the ecosystem itself. To fully grasp the microbial community structure, it is essential to understand the factors that shape the community composition and diversity of the ecosystem. This works focusses on the primary drivers of microbial composition in three ecosystems: an estuary, the gut of <i>Drosophila melanogaster,</i> and walnut grove soil. </p><p> In the benthic estuarine environment of Trunk River, physical perturbations in the water column above decaying seagrass altered the composition of the microbial community causing a visible microbial bloom. To understand the microbial community progression in the bloom, we simulated perturbed sites in the river and studied four different depths in the water column for two weeks. We found the bloom was largely made up of <i>Prosthecochloris vibrioformis,</i> a phototrophic sulfur oxidizer. The bloom appears to be driven by pH, salinity, and sulfide gradients, forming at a depth of &ap; 25cm beneath the surface of the water. </p><p> For the third chapter, we explored the effect of host diet on its gut microbial community. We created a controlled experiment in <i>Drosophila melanogaster,</i> a model organism. A population of <i>D. melanogaster </i> preconditioned on a balanced lab diet was split into two treatment diets, a high-sugar diet and a high-yeast diet. The microbial communities in the fecal matter of the flies were sampled for 4 days to understand their compositional changes. We found that a shift in the diet of <i>D. melanogaster </i> changed the phylogenetic, taxonomic, and functional compositions of the microbial communities. Each dietary change led to a distinct taxonomic, phylogenetic, and functional composition by the end of the experiment. The functional diversity of both treatment groups decreased, indicating a shift away from a diverse set of metabolic capabilities when subjected to a more comprehensive nutrition to a more specific set of metabolic capabilities adapted for the main nutrient, either sucrose or yeast extract.</p><p> In the fourth chapter, Pesticide-treated orchard soils were used to understand the effects of deliberate intervention (external factors) on microbial ecosystems. We also studied the potential of pathogens to colonize soil that had been exposed to such external factors. In a controlled experiment, soil was subjected to different fumigation treatments with and without subsequent amendment. It was then inoculated and incubated with <i>Agrobacterium tumefaciens. </i> The results indicate a fumigant-specific shift in the phylogenetic, taxonomic, genic, and functional composition of the soil. The low diversity fumigated soil was also better colonized by the <i>A. tumefaciens.</i> However, post-fumigation amendment with vermicompost increased the diversity, shifting the compositions towards non-fumigated vermicompost and suppressing <i> A. tumefaciens</i> colonization.</p><p> With this work, we have been able to implicate some of the important factors at play in the determination of microbial composition in various ecosystems. Additionally, we showed that the influence of these factors on microbial community is measurable on different metrics of composition.</p><p>