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Description
The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases.
ContributorsHolechek, Susan A (Author) / Jacobs, Bertram L (Thesis advisor) / Castillo-Chavez, Carlos (Committee member) / Frasch, Wayne (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers.
ContributorsSalamone, Damien Thomas (Author) / Jacobs, Bertram L (Thesis advisor) / Marsiglia, Flavio (Committee member) / Stout, Valerie (Committee member) / Johnson, Crista (Committee member) / Arizona State University (Publisher)
Created2011
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
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
Description
Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene.
ContributorsDelgado, Anca Georgiana (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Cadillo-Quiroz, Hinsby (Committee member) / Halden, Rolf U. (Committee member) / Rittmann, Bruce E. (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
Water contamination with nitrate (NO3−) (from fertilizers) and perchlorate (ClO4−) (from rocket fuel and explosives) is a widespread environmental problem. I employed the Membrane Biofilm Reactor (MBfR), a novel bioremediation technology, to treat NO3− and ClO4− in the presence of naturally occurring sulfate (SO42−). In the MBfR, bacteria reduce oxidized pollutants that act as electron acceptors, and they grow as a biofilm on the outer surface of gas-transfer membranes that deliver the electron donor (hydrogen gas, (H2). The overarching objective of my research was to achieve a comprehensive understanding of ecological interactions among key microbial members in the MBfR when treating polluted water with NO3− and ClO4− in the presence of SO42−. First, I characterized competition and co-existence between denitrifying bacteria (DB) and sulfate-reducing bacteria (SRB) when the loading of either the electron donor or electron acceptor was varied. Then, I assessed the microbial community structure of biofilms mostly populated by DB and SRB, linking structure with function based on the electron-donor bioavailability and electron-acceptor loading. Next, I introduced ClO4− as a second oxidized contaminant and discovered that SRB harm the performance of perchlorate-reducing bacteria (PRB) when the aim is complete ClO4− destruction from a highly contaminated groundwater. SRB competed too successfully for H2 and space in the biofilm, forcing the PRB to unfavorable zones in the biofilm. To better control SRB, I tested a two-stage MBfR for total ClO4− removal from a groundwater highly contaminated with ClO4−. I document successful remediation of ClO4− after controlling SO4 2− reduction by restricting electron-donor availability and increasing the acceptor loading to the second stage reactor. Finally, I evaluated the performance of a two-stage pilot MBfR treating water polluted with NO3− and ClO4−, and I provided a holistic understanding of the microbial community structure and diversity. In summary, the microbial community structure in the MBfR contributes to and can be used to explain/predict successful or failed water bioremediation. Based on this understanding, I developed means to manage the microbial community to achieve desired water-decontamination results. This research shows the benefits of looking "inside the box" for "improving the box".
ContributorsOntiveros-Valencia, Aura (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Torres, Cesar I. (Committee member) / Arizona State University (Publisher)
Created2014
Description
Microbial Electrochemical Cell (MXC) technology harnesses the power stored in wastewater by using anode respiring bacteria (ARB) as a biofilm catalyst to convert the energy stored in waste into hydrogen or electricity. ARB, or exoelectrogens, are able to convert the chemical energy stored in wastes into electrical energy by transporting electrons extracellularly and then transferring them to an electrode. If MXC technology is to be feasible for ‘real world’ applications, it is essential that diverse ARB are discovered and their unique physiologies elucidated- ones which are capable of consuming a broad spectrum of wastes from different contaminated water sources.
This dissertation examines the use of Gram-positive thermophilic (60 ◦C) ARB in MXCs since very little is known regarding the behavior of these microorganisms in this setting. Here, we begin with the draft sequence of the Thermincola ferriacetica genome and reveal the presence of 35 multiheme c-type cytochromes. In addition, we employ electrochemical techniques including cyclic voltammetry (CV) and chronoamperometry (CA) to gain insight into the presence of multiple pathways for extracellular electron transport (EET) and current production (j) limitations in T. ferriacetica biofilms.
Next, Thermoanaerobacter pseudethanolicus, a fermentative ARB, is investigated for its ability to ferment pentose and hexose sugars prior to using its fermentation products, including acetate and lactate, for current production in an MXC. Using CA, current production is tracked over time with the generation and consumption of fermentation products. Using CV, the midpoint potential (EKA) of the T. pseudethanolicus EET pathway is revealed.
Lastly, a cellulolytic microbial consortium was employed for the purpose ofassessing the feasibility of using thermophilic MXCs for the conversion of solid waste into current production. Here, a highly enriched consortium of bacteria, predominately from the Firmicutes phylum, is capable of generating current from solid cellulosic materials.
This dissertation examines the use of Gram-positive thermophilic (60 ◦C) ARB in MXCs since very little is known regarding the behavior of these microorganisms in this setting. Here, we begin with the draft sequence of the Thermincola ferriacetica genome and reveal the presence of 35 multiheme c-type cytochromes. In addition, we employ electrochemical techniques including cyclic voltammetry (CV) and chronoamperometry (CA) to gain insight into the presence of multiple pathways for extracellular electron transport (EET) and current production (j) limitations in T. ferriacetica biofilms.
Next, Thermoanaerobacter pseudethanolicus, a fermentative ARB, is investigated for its ability to ferment pentose and hexose sugars prior to using its fermentation products, including acetate and lactate, for current production in an MXC. Using CA, current production is tracked over time with the generation and consumption of fermentation products. Using CV, the midpoint potential (EKA) of the T. pseudethanolicus EET pathway is revealed.
Lastly, a cellulolytic microbial consortium was employed for the purpose ofassessing the feasibility of using thermophilic MXCs for the conversion of solid waste into current production. Here, a highly enriched consortium of bacteria, predominately from the Firmicutes phylum, is capable of generating current from solid cellulosic materials.
ContributorsLusk, Bradley (Author) / Torres, César I (Thesis advisor) / Rittmann, Bruce E. (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2015
Description
Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4 receptors in T cells and also in the budding of virions. But, there remain key structure/function questions regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis and thus, it makes for an attractive target to study the structural attributes of this protein by elucidating a structural model with X-ray crystallography. This study describes a multi-pronged approach of heterologous over-expression of Vpu. The strategies of purification and biophysical/ biochemical characterization of the different versions of the protein to evaluate their potential for crystallization are also detailed. Furthermore, various strategies employed for the crystallization of Vpu by both in surfo and in cubo techniques, and the challenges faced towards the structural studies of this membrane protein by characterization with solution Nuclear magnetic resonance (NMR) spectroscopy are also described.
ContributorsDeb, Arpan (Author) / Leket-Mor, Tsafrir S (Thesis advisor) / Fromme, Petra (Committee member) / Mason, Hugh (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016
Description
Creating sustainable alternatives to fossil fuel resources is one of the greatest
challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs.
challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs.
ContributorsZevin, Alexander Simon (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Vermaas, Willem Fj (Committee member) / Arizona State University (Publisher)
Created2015
Description
Invasive salmonellosis caused by Salmonella enterica serovar Typhimurium ST313 is a major health crisis in sub-Saharan Africa, with multidrug resistance and atypical clinical presentation challenging current treatment regimens and resulting in high mortality. Moreover, the increased risk of spreading ST313 pathovars worldwide is of major concern, given global public transportation networks and increased populations of immunocompromised individuals (as a result of HIV infection, drug use, cancer therapy, aging, etc). While it is unclear as to how Salmonella ST313 strains cause invasive disease in humans, it is intriguing that the genomic profile of some of these pathovars indicates key differences between classic Typhimurium (broad host range), but similarities to human-specific typhoidal Salmonella Typhi and Paratyphi. In an effort to advance fundamental understanding of the pathogenesis mechanisms of ST313 in humans, I report characterization of the molecular genetic, phenotypic and virulence profiles of D23580 (a representative ST313 strain). Preliminary studies to characterize D23580 virulence, baseline stress responses, and biochemical profiles, and in vitro infection profiles in human surrogate 3-D tissue culture models were done using conventional bacterial culture conditions; while subsequent studies integrated a range of incrementally increasing fluid shear levels relevant to those naturally encountered by D23580 in the infected host to understand the impact of biomechanical forces in altering these characteristics. In response to culture of D23580 under these conditions, distinct differences in transcriptional biosignatures, pathogenesis-related stress responses, in vitro infection profiles and in vivo virulence in mice were observed as compared to those of classic Salmonella pathovars tested.
Collectively, this work represents the first characterization of in vivo virulence and in vitro pathogenesis properties of D23580, the latter using advanced human surrogate models that mimic key aspects of the parental tissue. Results from these studies highlight the importance of studying infectious diseases using an integrated approach that combines actions of biological and physical networks that mimic the host-pathogen microenvironment and regulate pathogen responses.
Collectively, this work represents the first characterization of in vivo virulence and in vitro pathogenesis properties of D23580, the latter using advanced human surrogate models that mimic key aspects of the parental tissue. Results from these studies highlight the importance of studying infectious diseases using an integrated approach that combines actions of biological and physical networks that mimic the host-pathogen microenvironment and regulate pathogen responses.
ContributorsYang, Jiseon (Author) / Nickerson, Cheryl A. (Thesis advisor) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Ott, C Mark (Committee member) / Roland, Kenneth (Committee member) / Barrila, Jennifer (Committee member) / Arizona State University (Publisher)
Created2015