Matching Items (41)
Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
Description
Immunosignature is a technology that retrieves information from the immune system. The technology is based on microarrays with peptides chosen from random sequence space. My thesis focuses on improving the Immunosignature platform and using Immunosignatures to improve diagnosis for diseases. I first contributed to the optimization of the immunosignature platform by introducing scoring metrics to select optimal parameters, considering performance as well as practicality. Next, I primarily worked on identifying a signature shared across various pathogens that can distinguish them from the healthy population. I further retrieved consensus epitopes from the disease common signature and proposed that most pathogens could share the signature by studying the enrichment of the common signature in the pathogen proteomes. Following this, I worked on studying cancer samples from different stages and correlated the immune response with whether the epitope presented by tumor is similar to the pathogen proteome. An effective immune response is defined as an antibody titer increasing followed by decrease, suggesting elimination of the epitope. I found that an effective immune response usually correlates with epitopes that are more similar to pathogens. This suggests that the immune system might occupy a limited space and can be effective against only certain epitopes that have similarity with pathogens. I then participated in the attempt to solve the antibiotic resistance problem by developing a classification algorithm that can distinguish bacterial versus viral infection. This algorithm outperforms other currently available classification methods. Finally, I worked on the concept of deriving a single number to represent all the data on the immunosignature platform. This is in resemblance to the concept of temperature, which is an approximate measurement of whether an individual is healthy. The measure of Immune Entropy was found to work best as a single measurement to describe the immune system information derived from the immunosignature. Entropy is relatively invariant in healthy population, but shows significant differences when comparing healthy donors with patients either infected with a pathogen or have cancer.
ContributorsWang, Lu (Author) / Johnston, Stephen (Thesis advisor) / Stafford, Phillip (Committee member) / Buetow, Kenneth (Committee member) / McFadden, Grant (Committee member) / Arizona State University (Publisher)
Created2018
Description
Clean water for drinking, food preparation, and bathing is essential for astronaut health and safety during long duration habitation of the International Space Station (ISS), including future missions to Mars. Despite stringent water treatment and recycling efforts on the ISS, it is impossible to completely prevent microbial contamination of onboard water supplies. In this work, we used a spaceflight analogue culture system to better understand how the microgravity environment can influence the pathogenesis-related characteristics of Burkholderia cepacia complex (Bcc), an opportunistic pathogen previously recovered from the ISS water system. The results of the present study suggest that there may be important differences in how this pathogen can respond and adapt to spaceflight and other low fluid shear environments encountered during their natural life cycles. Future studies are aimed at understanding the underlying mechanisms responsible for these phenotypes.
ContributorsKang, Bianca Younseon (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Hepatitis C virus (HCV) is a globally prevalent infection which is a main contributor to the global burden of liver disease. Due to its ability to establish a chronic infection, and the lack of usefulness of traditional neutralizing antibody vaccine design in producing a protective immune response, a preventative vaccine has been notoriously difficult to produce. To overcome this, a vaccine using non-structural protein 3 (NS3) as a target to elicit a T cell specific immune response is thought to be a possible strategy for eliciting a protective immune response against hepatitis C infection. In this paper, a recombinant strain of measles virus (MV) that expresses HCV NS3 protein was analyzed. The replication fitness of this recombinant virus also indicates that this construct replicates at a higher rate than parental measles strain. It is also demonstrated through western blot analysis of protein expression and immunofluorescence that this recombinant virus expresses both the inserted HCV NS3 protein, as well as native measles proteins.
ContributorsWoell, Dana Marie (Author) / Reyes del Valle, Jorge (Thesis director) / Nickerson, Cheryl (Committee member) / Julik, Emily (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
Description
Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can bind double-stranded RNA and phosphorylate eukaryotic translation initiation factor, eIF2α, shutting down protein synthesis and halting the viral life cycle. To combat host defenses, vaccinia virus encodes E3, a potent inhibitor of the cellular anti-viral eIF2α kinase, PKR. The E3 protein contains a C-terminal dsRNA-binding motif that sequesters dsRNA and inhibits PKR activation. We demonstrate that E3 also interacts with PKR by co-immunoprecipitation. This interaction is independent of the presence of dsRNA and dsRNA-binding by E3, indicating that the interaction is not due to dsRNA-bridging.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
ContributorsFoster, Clayton (Co-author) / Alattar, Hamed (Co-author) / Jacobs, Bertram (Thesis director) / Blattman, Joseph (Committee member) / McFadden, Grant (Committee member) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The International Space Station (ISS) utilizes recycled water for consumption, cleaning and air humidity control. The Environmental Control and Life Support Systems (ECLSS) have been rigorously tested at the NASA Johnson Space Center. Despite the advanced engineering of the water recovery system, bacterial biofilms have been recovered from this potable water source. Microbial contamination of potable water poses a potential threat to crew members onboard the ISS. Because astronauts have been found to have compromised immune systems, bacterial strains that would not typically be considered a danger must be carefully studied to better understand the mechanisms enabling their survival, including polymicrobial interactions. The need for a more thorough understanding of the effect of spaceflight environment on polymicrobial interactions and potential impact on crew health and vehicle integrity is heightened since 1) several potential pathogens have been isolated from the ISS potable water system, 2) spaceflight has been shown to induce unexpected alterations in microbial responses, and 3) emergent phenotypes are often observed when multiple bacterial species are co- cultured together, as compared to pure cultures of single species. In order to address these concerns, suitable growth media are required that will not only support the isolation of these microbes but also the ability to distinguish between them when grown as mixed cultures. In this study, selective and/or differential media were developed for bacterial isolates collected from the ISS potable water supply. In addition to facilitating discrimination between bacteria, the ideal media for each strain was intended to have a 100% recovery rate compared to traditional R2A media. Antibiotic and reagent susceptibility and resistance tests were conducted for the purpose of developing each individual medium. To study a wide range of targets, 12 antibiotics were selected from seven major classes, including penicillin, cephalosporins, fluoroquinolones, aminoglycosides, glycopeptides/lipoglycopeptides, macrolides/lincosamides/streptogramins, tetracyclines, in addition to seven unclassified antibiotics and three reagents. Once developed, medium efficacy was determined by means of growth curve experiments. The development of these media is a critical step for further research into the mechanisms utilized by these strains to survive the harsh conditions of the ISS water system. Furthermore, with an understanding of the complex nature of these polymicrobial communities, specific contamination targeting and control can be conducted to reduce the risk to crew members. Understanding these microbial species and their susceptibilities has potential application for future NASA human explorations, including those to Mars.
ContributorsKing, Olivia Grace (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Sustainability (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
Blood donations today undergo extensive screening for transfusion transmitted infections (TTI) since the discovery of the first infectious agent in the early 1900s. Nucleic Acid Testing (NAT) is a serological test used widely in disease detection. NAT is known to rapidly and effectively detect pathogenic genomic material in blood by reducing the "window period" of infection. However, NAT produces false negative results for disease positive samples posing a risk of disease transmission. Therefore, NAT is used in conjunction with the Enzyme-Linked Immunosorbent Assay (ELISA) to mitigate these risks. However, the ELISA assay also poses the same risk as NAT. This study proposes immunosignaturing as an alternative serological test that may combat this risk and investigates whether it would be more effective than other standardized serological tests in disease detection. Immunosignaturing detects antibodies by utilizing a microarray of randomized peptide sequences. Immunosignaturing provides information about an individual's immune health from the pattern of reactivity of antibody-peptide binding. Unlike ELISA and NAT, immunosignaturing can be programmed to detect any disease and detect multiple diseases simultaneously. Using ELISA, NAT, and immunosignaturing, immune profiles of asymptomatic patients were constructed for 10 different classes of blood borne diseases. A pattern of infection was identified for each disease and the sensitivity and specificity of these assays were assessed relative to each other. Results indicate that immunosignaturing can be a viable diagnostic tool in blood testing. Immunosignatures demonstrated increased sensitivity and specificity compared to ELISA and NAT in discerning disease positive and negative samples within and across different classes of disease.
ContributorsSharma, Megumi (Author) / McFadden, Grant (Thesis director) / Nickerson, Cheryl (Committee member) / Green, Alex (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Despite the safe and effective use of attenuated vaccines for over fifty years, measles virus (MV) remains an insidious threat to global health. Problematically, infants less than one year of age, who are the most prone to severe infection and death by measles, cannot be immunized using current MV vaccines. For this dissertation, I generated and performed preclinical evaluation of two novel MV vaccine candidates. Based on data from clinical trials that showed increasing the dosage of current MV vaccines improved antibody responses in six-month-old recipients, I hypothesized that increasing the relevant antigenic stimulus of a standard titer dose would allow safe and effective immunization at a younger age. I generated two modified MVs with increased expression of the hemagglutinin (H) protein, the most important viral antigen for inducing protective neutralizing immunity, in the background of a current vaccine-equivalent. One virus, MVvac2-H2, expressed higher levels of full-length H, resulting in a three-fold increase in H incorporation into virions, while the second, MVvac2-Hsol, expressed and secreted truncated, soluble H protein to its extracellular environment. The alteration to the virion envelope of MVvac2-H2 conferred upon that virus a measurable resistance to in vitro neutralization. In initial screening in adult mouse models of vaccination, both modified MVs proved more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic in the gold standard MV-susceptible mouse model. Remarkably, MVvac2-H2 better induced protective immunity in the presence of low levels of artificially introduced passive immunity that mimic the passive maternal immunity that currently limits vaccination of young infants, and that strongly inhibited responses to the current vaccine-equivalent. Finally, I developed a more physiological infant-like mouse model for MV vaccine testing, in which MV-susceptible dams vaccinated with the current vaccine-equivalent transfer passive immunity to their pups. This model will allow additional preclinical evaluation of the performance of MVvac2-H2 in pups of immune dams. Altogether, in this dissertation I identify a promising candidate, MVvac2-H2, for a next generation measles vaccine.
ContributorsJulik, Emily (Author) / Reyes del Valle, Jorge (Thesis advisor) / Chang, Yung (Committee member) / Blattman, Joseph (Committee member) / Hogue, Brenda (Committee member) / Nickerson, Cheryl (Committee member) / Arizona State University (Publisher)
Created2016
Description
Many Fic domain proteins, through catalyzing post translational modifications (PTM) of protein substrates, functionally contribute to bacterial pathogenesis and the regulation of bacterial growth. Furthermore, one form of Fic-mediated regulation is the Fic toxin-antitoxin system, whereby an antitoxin interacts with and inhibits the Fic toxin. This study sought to determine the functional importance of Mycobacterium tuberculosis Fic and its putative antitoxin protein, Rv3642c. Using M. tuberculosis H37Rv genetic deletion mutants, fic and Rv3642c were demonstrated to promote intracellular survival in human THP-1 macrophage-like cells. Unlike other Fic toxins, of Fic toxin-antitoxin systems, Fic did not inhibit bacterial growth in vitro in the absence of Rv3642c. Notably, Fic demonstrated in vitro AMPylation of a THP-1 cell extract protein as shown by immunodetection. Fic also exhibited auto-AMPylation activity. Interestingly, a mutation of the conserved histidine in the Fic domain motif, a residue previously shown to be critical for AMPylation, had no effect on Fic-mediated ATP hydrolysis or AMPylation activity. Rv3642c was demonstrated to form a complex with Fic when co-expressed in Escherichia coli, indicating a toxin-antitoxin interaction. Screening M. tuberculosis protein fractions and culture filtrate with α-Fic and α-Rv3642c rabbit antisera did not detect monomers of Fic or Rv3642c, thus the cellular localization of Fic and the Rv3642c-Fic complex remains unclear. The results of this study provide insight into the function of M. tuberculosis Fic, and suggest that Fic and Rv3642c are important for M. tuberculosis survival in the intracellular macrophage environment. Furthermore, these findings challenge the current dogma that Fic domain catalysis is dependent on the conserved histidine of the Fic motif.
ContributorsLaMarca, Ryan (Author) / Haydel, Shelley (Thesis advisor) / Lake, Douglas (Committee member) / Nickerson, Cheryl (Committee member) / Arizona State University (Publisher)
Created2017
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013