Department of Medicine Theses and Dissertations
http://hdl.handle.net/10675.2/321355
2024-03-06T12:23:10Z96 plays a role in the virulence of C. jejuni
http://hdl.handle.net/10675.2/560614
96 plays a role in the virulence of C. jejuni
Rathbun, Kimberly M
Campylobacter jejuni is a gastrointestinal pathogen of humans but part of the normal flora of poultry. C. jejuni therefore grows well at both 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 37°C of CJ0596, a predicted periplasmic chaperone that is similar to proteins found to be involved in outer membrane protein (OMP) folding and virulence in other bacteria. The cj0596 gene was highly conserved in multiple strains and species of Campylobacter (24 in total), implying the importance of this gene. To study the role CJ0596 plays in Campylobacter pathogenesis, a mutant derivative of strain 81-176 was constructed in which the cj0596 gene was precisely deleted. This mutant was complemented by restoring the gene to its original chromosomal location. The mutant strain demonstrated a decreased growth rate and lower final growth yield, yet was more motile than wild-type. The cj0596 mutant also showed altered levels of several outer membrane proteins (OMPs), and changes in membrane-associated characteristics (antimicrobial sensitivity, autoagglutination, and biofilm formation). In either single or mixed infections, the mutant was less able to colonize mice than wild-type. Purified, recombinant CJ0596 had peptidyl-prolyl cistrans isomerase (PPIase) activitty, but did not functionally complement an E. coli surA mutant. These results suggest that C. jejuni CJ0596 is a PPIase and loss of CJ0596 alters phenotypes that have been shown to be related to the pathogenesis of the bacterium.
2009-05-01T00:00:00ZMechanisms of ET-1-mediated 02~ production in the rat aorta
http://hdl.handle.net/10675.2/367842
Mechanisms of ET-1-mediated 02~ production in the rat aorta
Loomis, E. D.
The objectives of this project were to test the hj^othesis that in the rat aorta endothelin-I (ET-I) binds to the ETa receptor stimulating superoxide (O2’ ) production. Furthermore, we wanted to identify the mechanism through which ET-1-mediates O2' production. Chemiluminescent detection of O2” production using probes such as lucigenin has been widely used with enzyme systems, leukocytes, and vascular tissues. Our first goal was to develop a microplate high-throughput protocol for lucigeninamplified chemiluminesence detection of 0 2 'L We have developed a novel adaptation to lucigenin-based assays that allows up to 36 samples to be counted at virtually the same time. Recent studies have shown that NOS 3 can become uncoupled and produce O2* when deprived of its cofactor BH4 . In addition several authors have shown that ONOOoxidizes BH4 in vitro. Using the high-throughput lucigenin assay and dihydroethidine (DHE) staining we have shown that (1) ET-1 is able to stimulate 0 2 "^ production in both endothelium-intact and -denuded vessels through the ETA-receptor, (2) ET-1 stimulates O2’ production through both NAD(P)H oxidase and an endothelial source of NOS, and (3) addition of exogenous tetrahydrobiopterin (BH4 ) and inhibition of peroxynitrite (ONOO-) inhibit ET-I-mediated 0 2 *^ production. Therefore our data have led us to hypothesize that ET-I stimulates O2* production by activating NAD(P)H oxidase through the ETa receptor. O2' production by NAD(P)H oxidase leads to the formation of ONOO- and the degradation of BH4. The loss of BH4 leads to uncoupled NOS which then contributes to ET-1-mediated production. In addition, we have found that (1) ET-1 increases the production of interleukin- 6 (IL-6 ) and (2) ET-1-mediated O2' production adversely affects vascular contractility. Although these consequences do not appear to be due to NOS uncoupling, they help support the role of ET-1 in vascular dysfunction.
2004-06-01T00:00:00ZRole of GCN2-dependent metabolic stress in regulating myeloid cell activation and differentiation.
http://hdl.handle.net/10675.2/346287
Role of GCN2-dependent metabolic stress in regulating myeloid cell activation and differentiation.
Liu, Haiyun
Amino acid metabolism is a pivotal regulator of innate and adaptive immunity. During inflammation, myeloid cells expressing enzymes such as indoleamine 2, 3-dioxygenase (IDO) and arginase 1 (ARG1) that degrade the amino acids L-tryptophan (L-Trp) and L-arginine (L-Arg), respectively. This serves a critical role in controlling cellular survival, development, and function. Therefore, it is important to understand the metabolic stress sensing pathways that regulate immune cell behavior, which further control the overall inflammatory environment. General Control Non-depressible 2 (GCN2) is an integrated stress response (ISR) kinase activated by intracellular amino acid limitation. Activated GCN2 phosphorylates eukaryotic initiation factor 2 α (eIF2α), which leads to global translation repression while up-regulating various stress-associated transcription factors. We found that in a murine LPS-induced endotoxemia model, IDO expression in macrophages depleted L-Trp that activated the GCN2. GCN2 signaling promoted macrophage cytokine production (IL-6, IL-12), increased CHOP expression and NF-κB activation. GCN2 knockout (GCN2KO) mice showed significantly lower serum and splenic cytokine levels compared to wild-type (WT) mice, and were protected from septicemia induced mortality. In the murine EG7 tumor model, GCN2 signaling was also activated in myeloid-derived suppressor cells (MDSCs) mediated by ARG1 depletion of L-Arg. We found that GCN2 was required for transcription factor C/EBPβ induction and monocytic bone marrow MDSC (BM-MDSC) development. GCN2KO BM-MDSCs showed significantly reduced ARG1 activity and failed to suppress antigen-specific cytotoxic T lymphocytes (CTLs) in vitro and in vivo. GCN2KO mice also exhibited increased efficacy in eliminating tumor cells after adoptive CTL transfer therapy. These data suggest that myeloid cells actively deplete intracellular amino acids to regulate their own cellular behavior. Different amino acid metabolic stress signals converge on the GCN2 pathway which serves as a secondary messenger to modulate downstream transcription factors. Depending on the type of inflammation, GCN2 can either promote pro-inflammatory responses or the immunosuppressive function of myeloid cells. Thus, targeting the GCN2 pathway in myeloid cells may have great potential in clinical therapy.
2014-12-01T00:00:00ZRegulation of Virulence in the Human Pathogen Campylobacter jejuni by the RNA Binding Protein CsrA
http://hdl.handle.net/10675.2/345280
Regulation of Virulence in the Human Pathogen Campylobacter jejuni by the RNA Binding Protein CsrA
Fields, Joshua A
Campylobacter jejuni is a leading bacterial cause of gastroenteritis in both the industrialized and developing world and has been associated with the onset of long term, debilitating sequelae such as Guillain-Barre Syndrome and reactive arthritis. The RNA binding protein CsrA (carbon storage regulator A), one of the relatively few regulatory elements in the C. jejuni genome, has been shown to regulate a number of processes in several other bacterial species including metabolism and virulence characteristics. We proposed the hypothesis that CsrA globally regulates C. jejuni pathogenesis via post-transcriptional repression or activation of virulence associated proteins. We created a csrA mutant in the C. jejuni strain 81-176 to investigate the role of CsrA in the virulence and physiology of the organism. In the absence of CsrA, we found that C. jejuni was no longer able to resist oxidative stresses, form biofilms, or adhere to intestinal epithelial cells in vitro in comparison to the wild type. We also found that C. jejuni was less motile than its parent strain and was defective in autoagglutination and fibronectin binding in vitro and mouse colonization in vivo. When we compared the proteome of the mutant strain to that of the wild type, we found that CsrA acted mostly upon the expression of proteins in stationary phase. In the absence of CsrA proteins responsible for various steps in C. jejuni metabolism, motility, oxidative stress responses, and epithelial cell adherence were differentially expressed. Finally, to further understand the molecular mechanisms of C. jejuni CsrA, we expressed it in a csrA mutant strain of E. coli. By heterologously expressing the C. jejuni protein in strain in which CsrA had been thoroughly characterized, we were able to show by complementation that C. jejuni CsrA was capable of both activating and repressing known targets of E. coli CsrA indicating that the molecular mechanisms of the two proteins are inherently the same.
2011-10-01T00:00:00Z