Throughout nature cells use peptides as a source of nutrition. For microbes, an ability to utilize peptides is especially important in nitrogen-poor environments, as peptides can be catabolized for their use as a nitrogen source. The yeast Saccharomyces cerevisiae imports di/tripeptides from the environment using the peptide transporter Ptr2p. Cellular levels of Ptr2p are highest under poor-nitrogen conditions. Here we report that the addition of a rich nitrogen source to the growth medium results in a down-regulation of Ptr2p, wherein plasma membrane Ptr2p is ubiquitinated, endocytosed, and delivered to the vacuole for destruction. We report evidence that the N-terminal portion of Ptr2p that is cytoplasmic is involved in mediating this effect. Mutations to known phosphorylation sites (Y37, S39, and S45) and suspected ubiquitination sites (K27 and K34) on Ptr2p’s Nterminal region greatly impair both the normal turnover of Ptr2p and its down-regulation in response to a rich nitrogen source. The data presented support the notion that Ptr2p turnover from the cytoplasmic membrane requires phosphorylation and ubiquitination of a discrete N-terminal cytoplasmic domain.

Buruli ulcer is a necrotizing skin infection and is the third most important mycobacterial disease in immune competent individuals after tuberculosis and leprosy in humid tropical countries. The causative agent Mycobacterium ulcerans is unlike other mycobacterial pathogens in that it appears to maintain an extracellular location during infection. Another unusual feature of the bacterium is that it is the only mycobacterium known to produce a dermo-necrotic polyketide toxin called mycolactone. A single Buruli ulcer, which can cover 15% of a person's skin surface, contains huge numbers of extracellular bacteria. The infection is characterized by massive necrosis at the site of infection followed frequently by debilitating disfiguration. Despite their abundance and extensive tissue damage, there is a remarkable absence of acute inflammatory response to the bacteria, and lesions are often painless. Though there is extensive literature on interaction of other mycobacterial species with innate immune cells, information concerning interaction of M. ulcerans with macrophages and neutrophils is scarce and requires further investigation. Research in this dissertation was geared towards understanding the poor innate immune response generated following M. ulcerans infection. One hallmark of most diseases caused by mycobacteria including M. tuberculosis, M. bovis, M. leprae, M. marinum, M. haemophilum is the ability of the bacilli to grow within host cells and cause granulomas. In contrast, M. ulcerans primarily forms extracellular microcolonies within necrotic tissues and is rarely found within host cells. The role of mycolactone in the extracellular location of the bacteria was investigated using a macrophage infection model. Experiments using a panel of mycolactone negative (myc-) and wild type (WT) M. ulcerans strains showed that presence or absence of mycolactone determines whether the bacteria are extracellular or intracellular. Exposure of macrophages to high concentrations of mycolactone interfered with their phagocytic ability. These observations that a mycolactone mutant is better phagocytized than the wild type strain is consistent with the presence of almost exclusively extracellular bacteria in Buruli ulcer patients. Experiments studying the effect of mycolactone concentrations on fibroblast cell lines showed that mycolactone-mediated apoptosis and necrosis was concentration dependent. Mycolactone caused necrosis at high concentrations and apoptosis at low concentrations. Chemotaxis assays using human neutrophils showed that neutrophils do not respond to M. ulcerans (WT or myc-) or mycolactone. Mycolactone treatment resulted in rapid necrosis of human neutrophils in a dose dependent manner in vitro. These data could be relevant in vivo in human infections where toxin gradients produced by a pool of extracellular M ulcerans may cause apoptosis or necrosis of inflammatory cells trying to move into the focus of infection and clear the bacteria. Lack of an inflammatory reaction during the necrotizing stage of Buruli ulcer could be due to abrogation of production of inflammatory cytokines (e.g. TNF-a, lL-l, lFN-y), chemokines (IL-8) and lowered expression of cell-adhesion molecules (e.g. lCAM-l, selectins, VCAM) which help inflammatory cells reach the site of infection. TNF-a and lL-8 are key players in immuno-inflammatory responses. Studies regarding TNF-a response to bacterial infection and mycolactone treatment in vitro showed that WT M. ulcerans and mycolactone did not induce TNF-a production while myc- M. ulcerans did. Interestingly, LPS mediated TNF-u production was inhibited by WT M. ulcerans and mycolactone. Both WT and myc- M. ulcerans as well as mycolactone did not induce IL-8 production. WT M. ulcerans and mycolactone induced expression of the cell adhesion molecule ICAM-I was less than that induced by myc- M. ulcerans or LPS. Microarray analysis of genes modulated by mycolactone yielded interesting information. Genes that were significantly upregulated by mycolactone included those related to transcriptional repressors, cytoskeletal rearrangement, cell cycle control/proliferation, apoptosis, G-protein receptors, tumor supression and immune response. Genes downregulated by mycolactone included those related to DNA repair, inactivation of complement and metalloproteinases, immune response, leukotrienes production, receptors for collagen and laminin. Data from the present study provide new insight into the effect of mycolactone on macrophages, fibroblasts, neutrophils and host gene-expression pathways induced or repressed by mycolactone. Knowledge obtained from the present study can be expected to contribute to a better understanding of the role of mycolactone in host-pathogen interactions as well as pathophysiology of the disease.

Coronaviruses are a family of positive-sense, single-stranded, 5’-capped and 3’- polyadenylated RNA viruses that replicate entirely in the cell cytoplasm. Replication of the viral genome requires translation to produce proteins used for RNA synthesis and virion assembly. The 5’- and 3’- untranslated regions of the coronavirus genome have been found to contain cis-acting elements that are required for replication of the genome and a defective interfering RNA. Presumably, both viral and cellular proteins interact with these elements and serve as trans-acting factors in genome translation and replication. Of interest is the functional significance of a 5’-proximal cis-acting 397-nucleotide region which includes the 210-nucleotide 5’ untranslated regions and the 5’ proximal 187 nucleotides of the coding region for the first nonstructural protein, p28. Here, research examining the structural requirements in this region and in the 3’ untranslated region for bovine coronavirus replication is presented. The following features were discovered: (i) A second higher order structure within the coding region of p28 is required for replication but not translation of the defective interfering RNA. Although no viral proteins were found to bind this region, two cellular proteins of ~60 and ~100kDa were found which might prove to be essential trans-acting factors. (ii) p28, the first synthesized protein in the genome is an RNA binding protein that interacts with cis-replication stem-loops in the 5’ and 3’ untranslated regions. p28 may therefore function through these interactions to regulate genome translation or replication. (iii) In constructs with a renilla luciferase reporter containing only the genomic 5’ and 3’ untranslated regions, in vitro translation in the absence of viral proteins revealed a 5’ and 3’ end interaction that mediates a repression of cap-dependent translation. Therefore, translation of the coronavirus genome may require the assistance of viral proteins.

Although conventional therapies for metastatic cancers have made significant progress in recent years, they are relatively nonspecific and have many deleterious side-effects. Recently, novel therapies, including adoptive cellular immune therapies have had sporadic, but spectacular success in cancers such as malignant melanoma and renal cell carcinoma: tumors in which an immune response has been demonstrated. However, other physiological mechanisms, such as blood coagulation inhibit the immune response against cancers. Our previous work has shown that one of these coagulation proteins, soluble fibrin (sFn), inhibits unstimulated and activated lymphocyte adherence to tumor cells by blocking leukocyte integrin (CD11a/CD18) binding to tumor cell CD54, suggesting that sFn is an immunosuppressive agent in cancer. Since these receptors are also involved in lymphocyte/endothelial cell adherence and diapedesis (a necessary step in the immune response to cancer), it was hypothesized that sFn inhibits these functions, and that blockade of this inhibition using specific peptides would restore these immune responses. Fluorescently labeled lymphocytes and Interleukin-2 activated lymphocytes (LAK cells) were incubated with sFn (or its components; fibrinogen, Gly-Pro-Arg-Pro, or thrombin) in the presence or absence of specific blocking peptides. Lymphocyte and LAK cell adherence to endothelial cell monolayers was measured by perfusion at physiological shear rates in a microscope-mounted closed perfusion chamber, followed by image analysis using Image Pro Plus software. Diapedesis was measured by detection of fluorescence in 24-well microplates following immune cell incubation (18 h) with endothelial cell monolayers grown in transwells. SFn inhibited lymphocyte (54.1 + 11.3 %) and LAK cell (43.9 + 4.4 %) adherence to sFn pretreated endothelial cells, and intermediate values were obtained from sFn pre-treatment of only one cell type. Adherence was restored by peptide mediated blockade of sFn/CD54 binding, but not by CD11b blocking peptides. Diapedesis was also inhibited by sFn (lymphocyte 29.6 + 7.7 %; LAK 12.2 + 4.9 %) and restoration was observed using blocking peptides. These results confirm the stated hypotheses, and if physiologically relevant, suggest that sFn is an etiological agent in tumor growth and metastasis, and that blockade using fibrin specific peptides may enhance the effectiveness of adoptive immunotherapies.

Detection of pathogenic bacteria in the environment and food products is of increasing importance especially in light of recent outbreaks of Escherichia coli O157:H7. I describe here a bacteriophage based bioluminescent bioreporter method for E. coli O157:H7 detection that combines the specificity bacteriophage have for their host, quorum sensing, and lux based bioluminescence from Vibrio fischeri. This new method for detection of E. coli utilizes the luxI/luxR quorum sensing present in V. fischeri which uses N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) as an autoinducer (Miller & Bassler 2001). Once the concentration of OHHL is high enough it binds the LuxR protein initiating transcription of luxCDABE and additional luxI, leading to the production of light. This bioluminescent bacteriophage bioreporter method uses 2 components, first, the bioreporter cell called E. coli OHHLux which carries the complete lux cassette (luxCDABE) along with the transcriptional regulator luxR (Ripp et al. 2006). The OHHLux (lambda phage resistant) detects the diffusible OHHL produced by the second component, the luxI engineered phage which when infecting target E. coli cells produces autoinducer. The initial feasibility of the phage based reporter assay was tested using the well characterized lambda phage and E. coli K12 model (Ripp et al. 2006). Once the initial feasibility of this bacteriophage based bioluminescent bioreporter system was confirmed, an E. coli O157:H7 specific phage, PP01, was constructed with a luxI insert. This PP01- luxI phage was used in conjunction with E. coli OHHLux to test for E. coli O157:H7 in pure culture, apple juice, ground beef, tap water, and spinach rinsates. This method has the potential to be a sensitive, rapid, and non-invasive method of screening for E. coli O157:H7 contamination. The system provided rapid and sensitive detection with results in well under 24 h even at E. coli O157:H7 concentrations as low as 1 CFU/ml (Brigati et al. 2007, Ripp Submitted 2007). Our system is self-sufficient and could be adapted to be fully-automated and capable of high throughput screening in a production line setting.

The Roseobacter clade is an abundant and biogeochemically relevant group of marine bacteria. Physiological and ecological traits identified in specific representatives of the clade are often universally attributed to all Roseobacter group members, however, culture-dependent studies utilizing phylogenetically distinct members are rare. Other attributes often associated with this clade include motility, biofilm formation and surface attachment, chemotaxis and quorum sensing. This study compared a collection of 13 diverse Roseobacter strains both pheno- and genotypically on the basis of these traits. Motility was determined for seven previously uncharacterized strains, with five of the strains demonstrating motility. Microscopic analysis using both phase contrast and transmission electron microscopy supported this finding. A crystal violet assay was used to assess biofilm formation on plastic and glass surfaces with a range of surface properties and yielded a wide array of phenotypic responses. Taking into account the variety of surface types and media types tested approximately half (54%) of the strains showed pronounced biofilm formation and all motile strains were capable of forming biofilms. Degenerate primer sets were designed to probe strains for which no genome sequence is currently available for genes involved in flagellar synthesis and chemotaxis. Two strains that demonstrated no signs of motility in the laboratory were found to possess a necessary gene for flagellar formation and a flagellar-associated chemotaxis gene. Genome analysis including other sequenced Roseobacter strains revealed that flagellar, chemotaxis and quorum sensing operons are abundant in members of this lineage, with 89% possessing flagellar and chemotaxis operons and 78% possessing genes believed to be involved in quorum sensing. This study underscores the diversity of this clade and emphasizes the difficulty of assigning phenotypic capabilities to all lineage members.

In traditional cuban medicine, pomegranate fruits have been used to treat acidosis, dysentery, microbial infections, diarrhoea, helminthiasis; haemorrhage and respiratory pathologies [Vuorela et al., 2003; Roig, 1974; Jimenez et al., 1979; Seoane, 1984].Pomegranates contain high levels of Polyphenolic compounds, which are largely responsible for the fruit’s antioxidant properties. A number of studies have demonstrated that polyphenolic complexes derived from other plants have antiviral effects, suggesting that antiviral activity may also reside in the polyphenol (PP) fraction of pomegranates. The decay of organic matter generates an extremely heterogeneous mixture of organic molecules referred to as humic substances. They are sub-classified on the basis of solubility characteristics. The Fulvic Acid (FA) fraction of humic substances includes a variety of low molecular acidic molecules that are soluble in water under all pH conditions. Antiviral activity of fulvic acid containing Secomet V against poxviruses and SARS has been demonstrated [Kotwal et al., 2006]. The current study was undertaken to evaluate the direct anti-influenza virus activity of pomegranate constituents present in Pomegranate Extracts: Pomegranate Juice (PJ) Pomegranate Liquid Extract (POMxl), Pomegranate Polyphenol enriched Powder Extract (POMxp) and Fulvic Acid (FA). Both Pomegranate Extracts and Fulvic Acid had anti-influenza activity. With regard to Pomegranate Extracts, all of the extracts had rapid antiviral activity when combined with influenza virus. The acidity of PJ and POMxl solutions contributed to anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that brief treatment at room temperature with > 200 ìg/ml PPs substantially reduced the infectivity of H1N1, H3N2, and H5N1 influenza viruses. Generally, the loss of infectivity was accompanied by loss of hemagglutinating activity. Electron microscopic examination of influenza particles neutralized by PP treatment identified a coating of amorphous material and some damage to virion integrity. Reassortant H5N1 viruses derived from avian isolates were less affected by PP treatment, indicating that PP susceptibility is modulated by small changes in surface glycoproteins. Our analysis supports the development of pomegranate-derived PPs as natural, rapidly active, broad-spectrum anti-influenza agents. Our finding demonstrates rapid anti-influenza virus activity in Pomegranate PPs and the Fulvic Acid.

Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 134-150). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 143-172). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Antimicrobial activity of some plant extracts