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Coursey, TamiStudying Host Mechanisms for Suppressing Geminivirus Infection through Chromatin Regulation
Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology
Geminiviruses are viral DNA plant pathogens responsible for crop loss around the world. Within the host these small single-stranded DNA (ssDNA) genomes form double-stranded DNA intermediates and are template for extra-chromosomal viral minichromosomes. Nucleosomes on viral minichromosomes contain post-translational modifications (PTMs) that demarcate open, actively transcribed chromatin from compact, transcriptionally repressed chromatin. These histone PTMs are also often accompanied by correlative changes in repressive methylation on viral DNA. As geminiviruses do not integrate into the host and are transmitted as a ssDNA, these viruses present a truly de novo chromatin model. Using this model previous work has determined some of the key host pathways and players responsible for repressing geminivirus virulence and has identified functions for previously uncharacterized proteins. The use of the geminivirus de novo model is particularly relevant for testing pathways and identifying proteins involved in chromatin regulation. In this dissertation repressive chromatin formation present on the viral genome has been linked to viral chromatin compaction and decreased transcriptional output. Host recovery from geminivirus infection induced a target specific “hypersuppression” of host transposable elements (TEs). Changes in both viral chromatin and host TE expression was dependent on host DNA dependent RNA Polymerase V (Pol V), a Pol II relative. Pol V is a key component of the RNA-directed DNA Methylation (RdDM) pathway responsible for repressing TE and viral gene expression through DNA and histone methylation. Pol V-dependence for hypersuppression was not correlated with classical RdDM outputs (e.g. changes in histone and DNA methylation) at hypersuppressed loci. These results suggest Pol V-mediated suppression is associated with addition functional outputs outside of classic RdDM. In the second part of this dissertation, histone reader proteins have been identified as critical viral chromatin regulators. Histone readers recognize and bind histone PTMs. Their association with a PTM induces changes in the chromatin landscape that can further alter DNA access to transcriptional machinery. In plants the number of identified readers is limited. In chapter 3 of this dissertation plant Emsy-Like (EML) 1 and 3 were tested for histone reader activity and effects on geminivirus virulence. Both EML proteins associate with histones and specifically recognize the histone 3 lysine 36 (H3K36) residue. Trimethylated H3K36, one of the histone PTMs bound by EML1 and 3, is present on viral chromatin. Additionally both EML1 and 3 bind viral chromatin. EML1 activity was further linked to viral transcriptional repression and decreased Pol II accessibility on viral chromatin. This work has thus identified two new plant histone readers, their targeted histone PTM, and has identified an EML1-mediated method of viral suppression.

Committee:

David Bisaro (Advisor); Richard Slotkin (Committee Member); Deborah Parris (Committee Member); Mark Parthun (Committee Member)

Subjects:

Biology; Microbiology; Molecular Biology; Virology

Keywords:

Geminiviruses; chromatin; RdDM

Cook, Gregory PANTARCTIC CHLAMYDOMONAS STRAINS C. SP. UWO241 AND ICE-MDV EXHIBIT DIFFERENTIAL RESTRUCTURING OF THE PHOTOSYNTHETIC APPARATUS IN RESPONSE TO IRON
Master of Science, Miami University, 2018, Microbiology
As an integral cofactor for many redox-associated processes, iron (Fe) homeostasis is crucial in order to produce sufficient energy for the organism. Fe limitation, or excess, can cause major alterations in the function and structure of the photosynthetic apparatus. Photosynthetic psychrophiles grown under permanent low temperatures exhibit novel adaptations in their photosynthetic apparatus to deal with this permanent stress. The ice- covered lakes of the McMurdo Dry Valleys harbor many species of cold-adapted algae, including Chlamydomonas sp. UWO241 (UWO241). As a consequence of adaptation to multiple permanent extreme conditions, UWO241 exhibits a remodeled photosynthetic apparatus for maintaining redox poise. One unusual characteristic of UWO241 is the absence of a PSI-associated 77K fluorescence emission under a wide range of growth conditions. This phenotype resembles Fe deficiency in other model organisms such as C. reinhardtii. We hypothesized that adaptation to permanent iron deficiency in its native environment may contribute to this unusual phenotype. We compared the effect of Fe availability on the physiology and photobiology of UWO241 with the model C. reinhardtii as well as a second psychrophilic alga, Chlamydomonas sp. ICE-MDV (ICE). The impacts of a restructured photosynthetic apparatus on the unique Fe-associated phenotype in UWO241 will be discussed.

Committee:

Rachael Morgan-Kiss, Dr. (Advisor)

Subjects:

Microbiology

Neudeck, Michelle JoanTolerance of Planktothrix agardhii to nitrogen depletion
Master of Science (MS), Bowling Green State University, 2018, Biological Sciences
Sandusky Bay is plagued by recurrent cyanobacterial harmful algal blooms (cHABs). A persistent bloom occurred in 2015 from June into October even though the levels of nitrogen dropped to near undetectable levels due to high rates of both assimilatory and dissimilatory nitrate reduction. Planktothrix agardhii is the main organism of the cHABs despite being non-diazotrophic. This cyanobacterium produces microcystin, a hepatotoxin. The Sandusky Bay waters were sampled every two weeks. RNA was extracted from the samples and sequenced. The metatranscriptomes were analyzed for genes pertaining to nitrogen storage and retrieval. P. agardhii stores N as cyanophycin, a polymer of arginine and aspartic acid. This is produced by cyanophycin synthetase which is encoded by cphA. P. agardhii has two paralogs of this gene. cphA is associated with cphB, the gene that encodes cyanophycinase that hydrolyzes cyanophycin into arginine-aspartic acid dimers. cphA' is monocistronic. N was replete from June until mid-July, while the discharge of the Sandusky River was high, cphA' was transcribed at this time. When the N levels dropped to 0, cphB and cphA were transcribed. Degradation of the phycobilisome is another source of N that can be scavenged by P. agardhii. A small protein that triggers the degradation of the phycobilisome is encoded by nblA. The transcripts from P. agardhii demonstrated that nblA was not transcribed until after cphB was transcribed. This indicated that P. agardhii utilized the N stores of cyanophycin before utilizing the N sequestered in the phycobilisome. The Sandusky Bay was turbid throughout the sampling season which is ideal for low light tolerant Planktothrix. The transcripts of hliA, which encodes for a protein that protects the photosynthetic apparatus from oxidative damage, was almost continuously present in the samples.

Committee:

George Bullerjahn, Ph.D (Advisor); R. Michael McKay, Ph.D (Committee Member); Paul Morris, Ph.D (Committee Member)

Subjects:

Biology; Microbiology

Keywords:

cyanophycin; phycobilisome; Planktothrix agardhii; nitrogen stress; highlight stress

Heisler, David BruceRole of Actin and Actin-binding Proteins in the Pathogenesis of Actin-targeting Bacterial Toxins
Doctor of Philosophy, The Ohio State University, 2017, Biochemistry Program, Ohio State
Proteinaceous bacterial toxins are among the deadliest compounds on earth; as little as a single copy of a toxin can be sufficient to compromise a host cell. Most toxins amplify their toxicity by targeting low abundant, essential host proteins or by altering signaling cascades. However, toxins that directly target actin, one of the most abundant proteins in the cell, seem doomed to inefficacy. Upon delivery to the cytoplasm of host cells, the actin cross-linking domain, from pathogenic strains of Vibrio and Aeromonas spp., catalyzes the covalent cross-linking of actin monomers into non-functional, non-polymerizable actin oligomers. The original hypothesis of ACD pathogenicity required the passive cross-linking of a bulk population of the actin cytoskeleton and to cause a subsequent morphological change in cell shape. In contrast to this hypothesis, we found that low doses of the actin oligomers bind to monomeric actin binding domains to potently inhibit actin regulatory proteins. Through a multivalent interaction with the formin homology 1 domain’s polyproline rich stretches and the formin homology 2 domains, the actin oligomers bind with low nanomolar affinity, preventing the nucleation and elongation of actin by formins. This novel “gain-of-function” mechanism of toxicity exerted by ACD converts actin monomers into potent secondary toxins to disrupt the actin homeostasis of a host cell. In addition to formins, we predicted the existence of other targets of the ACD-produced oligomers due to the organization of many actin binding proteins, which consist of several closely positioned G-actin-binding domains. We identified a diverse pool of actin-binding proteins that contain a short, G-actin-binding Wiskott-Aldrich syndrome homology motifs 2 (WH2) as additional targets of the oligomers: Ena/VASP, Spire, and activator of the Arp2/3 complex N-WASP. We predict that other nucleation promoting factors the Arp2/3 complex will also serve as targets. Herein, we identify the oligomers as universal inhibitors against WH2 domain-containing proteins. Next, we identified a bacterial effector protein acting on the host cytoplasm that share properties with eukaryotic proteins and is potently inhibited by the oligomers. The Vibrio effector VopF/L is a dimeric protein that brings six WH2 domains in close proximity to nucleate filaments in vitro. We have tested and confirmed the inhibition of VopF/L in vitro and used single-molecule speckle (SiMS) microscopy to demonstrate a potential mechanism of effector domain regulation utilized by bacteria during the infection of host cells. Additionally, we provide the first evidence of the dynamic behavior of VopF/L in living cells, raising questions concerning the reorganizing functional activities of VopF/L. Finally, similar to ACD, actin-targeting ADP-ribosylating toxins (ART) would have to overcome problems associated with targeting a highly abundant substrate. Despite low sequence homology, all but one actin-specific ARTs covalently ADP-ribosylate arginine-177 on monomeric actin, illustrating its strategic, but likely underappreciated role in toxicity. We found that profilin, a monomeric actin binding protein, enhances the modification rate of some but not all ARTs via a profilin binding sequence in the ART. Altogether, these results demonstrate a previously unappreciated connection between the pathogenic mechanisms of bacteria and the proteins that regulate the actin cytoskeleton.

Committee:

Dmitri Kudryashov (Advisor); Comert Kural (Committee Member); Jeffrey Parvin (Committee Member); Jian-Qiu Wu (Committee Member)

Subjects:

Biochemistry; Biology; Microbiology

Keywords:

bacterial toxins; ACD; actin;

Agu, Chidozie VictorUse of process design and metabolic engineering to enhance bioconversion of lignocellulosic biomass and glycerol to biofuels
Doctor of Philosophy, The Ohio State University, 2016, Animal Sciences
Recent efforts to reduce dependency on food-based substrates for industrial applications aim towards the use of inexpensive and readily available non-food based substrates such as lignocellulosic biomass (LB) and biodiesel-derived glycerol. Interestingly, the utilization of lignocellulosic sugars for biofuel production is contingent on the disruption of recalcitrant LB cell wall structure prior to enzyme hydrolysis. Disruption and hydrolysis processes generate lignocellulose-derived microbial inhibitory compounds (LDMIC) including acids, aldehydes and phenolics. Additionally, fermentation of glycerol to butanol, a next-generation biofuel, is hampered by the inability of Clostridium beijerinckii NCIMB 8052, a butanol fermentation workhorse, to efficiently metabolize glycerol. Therefore, this study investigated novel strategies for enhancing butanol and ethanol production through process design and metabolic engineering. Towards process design, the bacterium Cupriavidus basilensis ATCC®BAA-699 was used to detoxify 98% of the LDMIC present in acid-pretreated Miscanthus giganteus (MG) lignocellulosic biomass hydrolysates. Fermentation of the detoxified MG hydrolysates by C. beijerinckii resulted in 70%, 50%, and 73% improvement in acetone-butanol-ethanol (ABE) concentration, yield and productivity, respectively, when compared to the fermentation of undetoxified MG hydrolysates. The second objective was to explore metabolic engineering strategies to enhance glycerol utilization by C. beijerinckii and improve butanol production in the presence of LDMIC. To realize this objective, genes that encode glycerol dehydrogenases (Gldh) and dihydroxyacetone kinase (Dhak) in a hyper-glycerol utilizing bacterium (Clostridium pasteurianum ATCC 6013) were systematically cloned into C. beijerinckii. By over-expressing two C. pasteurianum Gldh genes (dhaD1+gldA1) as a fusion protein in C. beijerinckii, we achieved 50% increase in cell growth, ABE production (up to 40%), and enhanced rate of furfural detoxification (up to 68%) during the fermentation of furfural-challenged (4 to 6 g/L) glucose+glycerol medium. Further, co-expression of dhaD1+gldA1 resulted in significant payoff in cell growth (57%), glycerol consumption (14%), and ABE productivity (27.3%) compared to over-expression of a single Gldh. In parallel, while co-expression of dhak and gldA1 in C. beijerinckii improved glycerol consumption by 37% relative to the plasmid control, over-expression of all three genes (dhaD1+gldA1+dhak) improved butanol production by >50% in the presence of 5 and 6 g/L furfural relative to the plasmid control. Objective 3 aimed to develop a high-throughput alcohol dehydrogenase (ADH)-dependent assay for screening hyper- or hypo- butanol producing C. beijerinckii mutant libraries. Screening of the activities of ADHs from different microorganisms showed that Thermotoga hypogea derived ADH has ~7-fold activity towards butanol than ethanol. It was rationalized that T. hypogea ADH can be used to selectively quantify butanol in the presence ofethanol (e.g., in ABE broth). Objective 4 aimed to use allopurinol to inhibit xanthine dehydrogenase/oxidase and improve ethanol fermentation of LB hydrolysates by Saccharomyces cerevisiae. Allopurinol increased S. cerevisiae growth (19%), ethanol titer (21%), ethanol productivity (20%), ethanol yield (24%), and the chronological lifespan of S. cerevisiae (>16 h) during the fermentation of 100% corn stover hydrolysate. Taken together, this study encompasses novel strategies to enhance LB and glycerol utilization and potentially improve the economics of biobutanol and bioethanol production.

Committee:

Thaddeus Ezeji (Advisor); Jeff Firkins (Committee Member); Zhongtang Yu (Committee Member); Macdonald Wick (Committee Member); Gireesh Rajashekara (Committee Member)

Subjects:

Agriculture; Animal Sciences; Biochemistry; Biology; Microbiology; Molecular Biology

Devine, Racheal ARegulatory Features of the 5' Untranslated Leader Region of aroL in Escherichia coli K12 and the sRNA, ryhB, in Shewanella oneidensis MR-1
Doctor of Philosophy, Miami University, 2018, Microbiology
RNA is an important regulator of gene expression within bacterial, eukaryotic, and archaeal cells. This work focuses on two aspects of RNA regulation: the first half investigates the role of regulatory features within the 5’ untranslated leader region (UTR) of the E. coli aroL mRNA and the second half focuses on an sRNA in S. oneidensis MR-1. The 5’UTR of mRNAs contain information necessary for ribosome recognition and subsequent translation initiation. Translation initiation is a prominent part of gene expression, as it is the rate-limiting step of translation. The 70S ternary initiation complex contains initiator tRNA and the mRNA’s start codon positioned in the P-site of the 70S ribosome. The Shine-Dalgarno (SD) sequence within the 5’UTR of the mRNA is an important feature that helps facilitate the initial interaction between the mRNA and the 30S subunit. Translation of mRNAs lacking an SD has been reported and suggests that alternative mechanisms of mRNA-30S interactions exist. The aroL mRNA contains a short open reading frame within its 5’UTR. Ribosome binding and expression assays showed that this open reading frame gets translated, and its translation affects aroL translation downstream. The upstream open reading frame binds 30S subunits in the absence of a canonical SD sequence. In this work, we have shown that multiple signals in the mRNA (upstream and downstream of the AUG) contribute to 30S binding to and translation from the AUG start codon. In this work we have also characterized an sRNA, a ryhB homologue, in S. oneidensis MR-1. sRNAs contain regulatory features in their sequence and structure that help regulate translation of bacterial mRNAs in response to environmental cues. Similar to what has been reported in E. coli, the sRNA is regulated in response to iron limitation in S. oneidensis MR-1 and may have a regulatory role in iron metabolism.

Committee:

Mitch Balish (Advisor)

Subjects:

Microbiology; Molecular Biology

Keywords:

translation initiation; non-canonical translation initiation; Shine-Dalgarno; non-Shine-Dalgarno led mRNA;

Mojica, Maria FernandaACTIVE SITE LOOPS AND SUBSTRATE PROFILE IN CLASS B METALLO-ß-LACTAMASES: FROM MECHANISTIC STUDIES TO THERAPY DEVELOPMENT
Doctor of Philosophy, Case Western Reserve University, 2017, Biochemistry
ß-lactams are the most important class of antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria. ß-lactams are characterized by their four membered cyclic amide ring, and act by inhibiting transpeptidases involved in cell wall synthesis. In particular, carbapenems, the “last resort” ß-lactam antibiotics, are used to treat the most resistant Gram-negative pathogens. Unsurprisingly, bacteria developed several mechanisms to resist the action of ß-lactams, including efflux, reduced permeability, altered transpeptidases, and most importantly, inactivation by ß-lactamases, enzymes that selectively hydrolyse the ß-lactam ring, rendering the antibiotics ineffective against their natural targets. Metallo-ß-Lactamases (MßLs) are metal-dependent enzymes of particular interest and concern given i) their ability to hydrolyse and provide resistance to virtually all ß-lactams including carbapenems; and ii) unavailability of clinically useful MßL inhibitors. Structurally, these enzymes feature a aß/ßa fold, in which the Zn-containing active site is positioned within a shallow groove formed by the interface of the a and ß domains, and is surrounded by active site loops (ASL). Herein, we study the effects that amino acid substitutions in the ASLs of two clinically relevant MßLs, VIM-2 and NDM-1, have on substrate specificity, enzyme catalysis and stability. We show that the substitution Arg228Leu at the ASL-10 of VIM-like enzymes expands the substrate spectrum to include 3rd and 4th cephalosporins, while changes at the ASL-3 in NDM-1 not only affect substrate catalysis but also alter the metal coordination center. We also introduce a novel class of mechanism-based MßL inhibitors, the bisthiazolidines, and demonstrate their in vitro effectiveness at restoring the susceptibility toward carbapenems in MßL-producing gram negative bacteria. We then investigate the sequence diversity of the L1 MßL of Stenotrophomonas maltophilia, an emerging multi-drug resistant bacterium for which antibiotic treatment options are limited. Based upon our knowledge of the mechanism and biochemistry of MßL and other ß-lactamases, we show that a novel antibiotic combination, ceftazidime-avibactam and aztreonam, is effective against S. maltophilia and other MßL-producers. Lessons learned during the development of this thesis provide valuable information concerning the catalytic mechanism of these enzymes, towards the development of effective MßL inhibitors and to the testing of novel combination therapies.

Committee:

BONOMO ROBERT (Advisor); MERRICK WILLIAM (Committee Chair); van den AKKER FOCCO (Committee Member); CAREY PAUL (Committee Member); VILA ALEJANDRO (Committee Member)

Subjects:

Biochemistry; Microbiology

Guzzo, Marissa BlaireMolecular Mechanisms Underlying the Intrinsic Sulfonamide Resistance in Bacteria
Doctor of Philosophy, Case Western Reserve University, Molecular Biology and Microbiology
The increasing frequency of MDR- and XDR-bacterial infections continues to pose a serious threat to global public health. This concern has not only intensified with the reduced efficacy of currently available treatments but with the significant decline in drug development and discovery. Through knowledge and understanding of bacterial intrinsic molecular resistance mechanisms, an emerging concept of “targeting resistance," or potentiation, has gained much interest as a means of re(sensitizing) pathogenic bacteria to available antibiotics. With a renewed interest in reviving the use of antifolates, such as sulfonamides, in the treatment of Mycobacterium tuberculosis (Mtb) infections, our lab developed a chemo-genomic screening approach for determining antifolate resistance determinants in mycobacterial species. We found that disruption of 5,10-methenyltetrahydrofolate (5,10-CH+-H4PteGlun) synthetase (MTHFS), an enzyme responsible for the conversion of 5-formyltetrahydrofolate (5-CHO-H4PteGlun, folinic acid) to 5,10-CH+-H4PteGlun in the folate-dependent one-carbon (1C) metabolic network led to severe defects in cellular folate homeostasis thus weakening the intrinsic antifolate resistance in both Mycobacterium smegmatis and Escherichia coli resulting in enhanced sensitivity to sulfonamides. Further investigation into folate mediated 1C metabolism led to the discovery of a novel mechanism of intrinsic sulfonamide resistance referred to as the methylfolate (MF) trap. Until now, this phenomenon has only been characterized in mammals as a means of explaining the relationship of Vitamin B12 (VB12), folate, and homocysteine (Hcy) homeostasis in many medical conditions such as anemia, neural tube defects, and Alzheimer’s dementia. This trap occurs as a result of defects in the multi-cycling reaction catalyzed by the B12-dependent methionine synthase (MetH). We demonstrate that in the presence of sulfonamide, MF trap induction, either through VB12 deficiency and/or mutations affecting MetH enzymatic activity, causes impaired homeostasis of folate and related metabolites as well as the cytotoxic accumulation of Hcy-thiolactone. This metabolic blockage ultimately leads to thymineless death rendering pathogenic bacteria, such as M. tuberculosis, E. coli, Pseudomonas aeruginosa, and Salmonella typhimurium, more susceptible to existing sulfonamides. We also provide proof-of-concept demonstrating that chemical induction of the MF trap by inhibiting VB12 uptake represents a novel method of potentiation by enhancing the antimicrobial activity of available, clinically approved sulfonamides.

Committee:

Arne Rietsch (Committee Chair); Liem Nguyen (Advisor); W. Henry Boom (Committee Member); Robert Bonomo (Committee Member)

Subjects:

Microbiology

Fyffe, Deanna LynneMethods to Monitor Lake Erie's Harmful Algal Blooms: A Fellowship with the Cooperative Institute for Great Lakes Research
Master of Environmental Science, Miami University, 2017, Environmental Sciences
To fulfill the professional experience requirement for a Master of Environmental Science degree at Miami University, I completed a fellowship with the Cooperative Institute for Great Lakes Research (CIGLR). My work involved investigating data trends of recent Lake Erie harmful algal blooms and potential monitoring methods. Data trends revealed monitoring stations closest to the mouth of the Maumee River had the highest average cyanobacteria concentrations. Bloom biomass distribution tended to favor the surface of the water column but was likely influenced by wind speed in 2016 and 2017. I also compared chlorophyll-a data from a CIGLR-owned bbe FluoroProbe to laboratory extraction data. The bbe FluoroProbe provided consistent results when used in the field and in the laboratory, but generally identified less chlorophyll-a than pigment extraction methods. Additionally, I performed field, laboratory, and analytical work to evaluate commercial in situ fluorometers. Due to proprietary reasons, the individual fluorometer data could not be presented in this report. I provided an example field deployment evaluation for the YSI EXO2 Multiparameter Sonde, a CIGLR-owned fluorometer that was used during the field tests for ancillary data. The EXO2 ultimately had low accuracy when compared to traditional laboratory methods, but both methods produced similar data trends.

Committee:

Suzanne Zazycki, JD (Advisor); Bartosz Grudzinski, PhD (Committee Member); Vanni Michael, PhD (Committee Member)

Subjects:

Aquatic Sciences; Environmental Health; Environmental Science; Environmental Studies; Freshwater Ecology; Microbiology; Water Resource Management

Keywords:

Lake Erie, Great Lakes, harmful algal bloom, HAB, water quality, in situ, fluorometer, cyanobacteria, biomass, microcystis, chlorophyll, phycocyanin, pigment, research, laboratory, EXO2, FluoroProbe, NOAA, GLERL, CIGLR, monitoring

Roberts, ErinCytokine expression, cytoskeleton organization, and viability of SIM-A9 microglia exposed to Staphylococcus aureus-derived lipoteichoic acid and peptidoglycan
Master of Science (MS), Wright State University, 2017, Microbiology and Immunology
In these experiments, SIM-A9 microglia were exposed to Staphylococcus aureus cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) at a concentration of 5 ug/mL for six, twelve, eighteen, and twenty-four hours and the cytokine expression, cytoskeleton organization, and cell viability of the cells were observed. Following LTA and PGN exposure, TNF-a; secretion increased at each time interval and was highest observed at 24 hours. No significant IL-10 secretion was detected. Over the 24 hour period, cell viability and cytotoxicity of LTA and PGN treatment groups were not significantly different from the control, indicating the observed inflammatory cytokine response was not due to cell death. These data suggest that LTA and PGN play a predominantly inflammatory role in the first twenty-four hours of S. aureus CNS infection.

Committee:

Nancy Bigley, Ph.D. (Advisor); Barbara Hull, Ph.D. (Committee Member); Dawn Wooley, Ph.D. (Committee Member)

Subjects:

Immunology; Microbiology

Keywords:

Staphylococcus aureus; lipoteichoic acid; LTA; peptidoglycan; PGN; microglia; CNS; TNFa; IL-10; tubulin; actin; viability; cytotoxicity;

LATTIN, ESTHER EPROFILE AND NITROGEN DYNAMICS OF MICROBIAL COMMUNITIES IN AGRICULTURAL INTERCROPPING SHRUB-CROP SYSTEMS OF SENEGAL
Master of Science, The Ohio State University, 2017, Environmental Science
In the face of widespread food insecurity and extreme population growth in sub-Saharan Africa, sustainable, resilient systems are needed to increase agricultural yields that can remediate degraded soils. Agroforestry is one of the suggested strategies and shows some promise. Piliostigma reticulatum and Guiera senegalensis are two indigenous woody species that currently grow interspersed in farmers’ fields in Senegal and throughout the Sahel. Shrub management consists of coppicing and burning the above ground biomass before planting. Previous research has shown that decomposition of above ground biomass contributes to soil organic matter and carbon, nitrogen content and phosphorus availability in soils. Hydraulic lift from the long tap roots redistributes moisture to upper soil horizon. In addition, root exudates and root turnover also create ideal habitats for soil microbes. The collective benefits of shrubs on soils results in `resource islands’ across the Sahalien landscape. Optimized intercropping with Piliostigma reticulatum and Guiera senegalensis have been shown to increase yields in groundnut (Arachis hypogaea) and pearl millet (Pennisetum glaucum) (Dossa et al., 2012, 20132; Bright et al., 2017). Research has shown that shrubs can promote microbial diversity and activity beneath the canopy of these shrubs over soil outside the influence of shrubs (Deidhou et al., 2009). However, little is known on whether this shift also occurs in the rooting zone of crops growing adjacent to shrubs. Therefore, the first objective of this research was to characterize the effect G. senegalensis and P. reticulatum have on soil microbial communities in a shrub-millet intercropping systems in farmers’ fields spanning across a rainfall gradient. A fatty acid methyl ester (FAME) extraction and MIDI microbial biomarker library were used to create a community profile on samples from shrub root zones, millet root zones within shrub influence and millet root zones outside of shrub influence in 6 farmers’ fields across the rainfall gradient. Samples were collected in 2013 and 2014. The microbial biomass from the fatty acid analysis ranged from 15.67-23.75 nmol g-1 soil in 2013 and from 15.77-20.87 nmol g-1 soil in 2013. Neither year demonstrated significant influence on microbial biomass or on the distribution of the FAME profile by the shrubs. The regional treatments caused significant changes in the FAME profile in the millet rooting zone treatments. This suggests that while agricultural management interfered with the shrubs ability to create distinctly heterogeneous resource islands, they were still able to maintain a stable soil habitat to sustain the microbial community. The second objective was to determine the potential of diazotrophs to fix atmospheric N within the canopy soil of G. senegalensis and P. reticulatum along synthetic fertilizer rate and precipitation gradients. An incubation study using the stable isotope, 15N in the form of dinitrogen gas (15N2) was used to investigate the activity of nitrogen fixing bacteria in shrub rhizosphere soil, soil treated with shrub litter cover and soil untreated by shrub from two long term research sites in the central and southern regions of Senegal. Samples were collected from both fertilized and not fertilized plots in the rainy and dry season of 2014. The optimized incubations estimate that BNF is capable of providing from 0.25 with fertilizer to 61.6 kg N ha-1 without at Nioro and 0.03 without fertilizer to 1.1 kg N ha-1 with fertilizer at Keur Matar. The high levels of estimated BNF during the dry season, ~50-100 kg N ha-1 at both sites, may have been obscured by the artificial moisture content. These estimations suggest that BNF would contribute significantly to the 9 kg N ha-1 and 22 kg N ha-1 recommended N fertilization for groundnut and Millet. Keur Matar soil diazotrophs were less significantly affected by nitrogen fertilization while both showed significantly more BNF activity during in the incubation of dry season soils. A study using field conditions would provide a more accurate estimation.

Committee:

richard dick (Advisor); nick basta (Committee Member); olli tuovinen (Committee Member)

Subjects:

Agriculture; Microbiology; Soil Sciences

Keywords:

soil; nitrogen; microbial community; FAME; stable isotope; diazotroph

Wu, XiaojunIdentification of two novel in vivo-upregulated Francisella tularensis proteins involved in metal acquisition and virulence
Doctor of Philosophy (PhD), University of Toledo, 2016, Biomedical Sciences (Infection, Immunity, and Transplantation)
Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal binding and uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. We have identified two new proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of those iron acquisition genes and demonstrated that both are upregulated during mouse infections. Based on sequence homology and in vivo upregulation, we hypothesized that FmvA and FmvB are OMPs involved in metal acquisition and virulence. Despite sequence similarity to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither fmvA nor fmvB mutants exhibited growth defects in iron limitation. However, among other metals examined in this study, magnesium limitation significantly induced fmvB expression, fmvB mutant was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on fmvB mutant in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of fmvB mutant revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in fmvB mutant, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in magnesium uptake, we demonstrated that FmvB was outer membrane-localized. Finally, both fmvA and fmvB mutants were found to be significantly attenuated in mice and cytokine analyses revealed that fmvB mutant-infected mice produced lower levels of pro-inflammatory cytokines, including GM-CSF, IL-3, and IL-10, compared with mice infected with wild-type F. tularensis. Taken together, these studies have characterized two previously-unstudied F. tularensis proteins, have shown that both play roles in F. tularensis virulence, and provide new insights into the importance of magnesium for intracellular pathogens.

Committee:

Jason Huntley (Committee Chair); Robert Blumenthal (Committee Member); William Maltese (Committee Member); Kevin Pan (Committee Member); R.Mark Wooten (Committee Member)

Subjects:

Biology; Biomedical Research; Health Sciences; Immunology; Microbiology

Keywords:

Francisella tularensis; outer membrane proteins; virulence; metal acquisition; hypothetical proteins

McDonald, Kristina MarieMycoplasma bovigenitalium qPCR Detection and Multilocus Sequence Typing Strain Differentiation
Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine
Mycoplasma bovigenitalium (Mbvg) is a common inhabitant of the reproductive tract of diseased and apparently normal cattle. The bovine mycoplasma has been associated with multiple reproductive tract disorders in both male and female cattle. Additionally, Mbvg can colonize the prepuce and distal urethra of bulls, often without producing disease, resulting in Mbvg contaminated semen. The use of Mbvg-contaminated semen for AI has been reported as a potential source of infection for the bovine female genital tract. Although the pathogenic nature of Mbvg has been reproduced experimentally, it is not evident what triggers certain animals to exhibit disease while others remain asymptomatic carriers. Therefore, it is critical to increase the sensitivity of Mbvg detection as well as improve our knowledge regarding the pathogenicity and epidemiology of Mbvg. The first objective of this study was to develop and validate a TaqMan qPCR for the rapid detection of Mbvg in bovine semen. The assay exclusively amplified 81 field and laboratory strains of Mbvg when tested against a panel 15 bovine mycoplasma species. Fresh bovine semen samples (n=77) were assessed for Mbvg by qPCR and culture. M. bovigenitalium was recovered in 57 (74%) semen cultures and detected by qPCR in 62 (80.5%) samples. The rapid nature of the assay (results obtained in 5 h) may provide an alternative diagnostic and research tool for the detection and quantification of Mbvg in bovine semen samples as opposed to traditional culture, which requires up to 10 days. The second objective of this study was to develop a Multilocus Sequence Typing (MLST) assay for Mbvg strain differentiation. The sequences of three targeted loci (ppa, tpiA, rpoB) were analyzed from a collection of 86 Mbvg isolates from around the world. A high degree of nucleotide variability was observed and the isolates were organized into 61 strain types (ST). A phylogenetic tree constructed from the concatenated sequences displayed coherent relationships between branching and the geographic and species origin of the isolates. The third objective of the study was to assess Mbvg in bovine semen using MLST to identify potentially pathogenic strains. Semen samples from 41 bulls were cultured for mycoplasma. A total of 33 Mbvg isolates were recovered (80.5%) and subjected to MLST. Despite isolates originating from the same location, a high degree of nucleotide variability was present resulting in 24 STs. Phylogenetic analysis of the concatenated sequences revealed four distinct clusters (strain family A-D). Fertility potential (SCR) and six months (± 3 months from collection date) semen quantity/quality data for each bull were compared to strain family. A statistically significant association was observed between strain family and SCR (P = 0.002). Further analysis revealed that bulls carrying strain family C had significantly lower SCR than bulls negative for Mbvg (P = 0.006). These findings may allow the development and refinement of additional hypotheses for future research studies to identify additional carriers of Mbvg strain family C, assess the impact of Mbvg on bull fertility, and intervention strategies to control and prevent its propagation in cattle.

Committee:

Thomas Wittum (Advisor); Gustavo Schuenemann (Advisor); Gregory Habing (Committee Member); Christopher Premanandan (Committee Member); James M. DeJarnette (Committee Member)

Subjects:

Animal Sciences; Epidemiology; Microbiology; Molecular Biology

Keywords:

Mycoplasma bovigenitalium; qPCR; MLST; bovine semen; genotyping; fertility

Agans, Richard ThomasModeling Effects of Diet on Human Gut Microbiota
Doctor of Philosophy (PhD), Wright State University, 2016, Biomedical Sciences PhD
The human gut microbiota is integrally involved in the metabolism of nutrients contained within the human diet. Studies into human nutrition have primarily been carried out using human and animal models. These studies are extremely important in our understanding of human nutrition, however, suffer from inherent limitations including unique microbial compositions between individuals, compliance in human studies, inability to carry out mechanistic studies, and inability to interrogate proximal regions of the gut without applying invasive techniques. In vitro gut simulator systems circumvent many of these limitations in animal and human models by allowing control of gut environmental conditions, decreasing variability observed between subjects, and enabling mechanistic investigations and interrogations of inaccessible regions of the gut. In this work a custom biofermentation system, the human gut simulator, was designed, validated, utilizing previously reported gut conditions, capable of temperature, pH, and atmosphere regulation, nutrient transit, and it allows real-time sampling of vessel contents or addition of exogenous agents. The human gut simulator was further employed to the study of gut microbiota response to dietary long chain fatty acids as a sole nutrient source, following stabilization on a rich `western’ medium. Microbiota showed rapid responses to the transition from western to fat medium; where a lack of carbohydrates and proteins resulted in decreased community density. Specific members of the microbiota were capable of utilizing long chain fatty acids, including Bilophila, Alistipes, and Escherichia/Shigella. Interestingly, members of the microbiota incapable of metabolizing long chain fatty acids included beneficial microbes Roseburia, Bifidobacterium, and Akkermansia. Ordination and principal response curves analyses highlighted a significant effect of medium change on shifts in microbial composition over time. In conjunction with in vitro studies, human volunteers were enrolled to assess responses of microbiota to diets high in proteins, carbohydrates, or fats. Microarray analysis revealed specific individual host responses to test diets with smaller community wide effects. Increasing the amount of protein in the diet had a positive impact on relative abundance of Akkermansia, Alistipes, Enterococcus, and Lactococcus, while higher carbohydrates and fats resulted in higher abundances of Bifidobacterium, and Alistipes and Escherichia/Shigella, respectively. Together these results indicate that the Human Gut Simulator allows for robust studies of the human gut microbiota, and offers a foundation for conducting nutritional interventions in human subjects.

Committee:

Oleg Paliy, Ph.D. (Advisor); Volker Bahn, Ph.D. (Committee Member); Nancy Bigley, Ph.D. (Committee Member); Mill Miller, Ph.D. (Committee Member); Lawrence Prochaska, Ph.D. (Committee Member)

Subjects:

Biomedical Research; Ecology; Microbiology; Nutrition

Keywords:

Microbiota, in vitro gut simulator, bioreactor, nutrition, fatty acid

Gandhi, RomaThe Natural Acquisition of the Oral Microbiome in Childhood: A Cross-Sectional Analysis
Master of Science, The Ohio State University, 2016, Dentistry
This cross-sectional study explored the development of the oral microbiome throughout childhood. Our previous studies of infants up to 1 year of age have shown early presence of exogenous species not commonly found in the oral cavity followed by rapid replacement with a small, shared core set of oral bacterial species. Following this initial colonization, we hypothesize that the complexity of the microbial community will steadily increase with advancing age as the oral cavity develops more intricate environmental niches for bacterial growth, and as children are exposed to new strains of bacteria and novel foods. We sampled 116 children and adolescents ranging from age 1 to 14 years and collected salivary, supragingival and subgingival samples. Bacterial community composition was analyzed at the level of species using rRNA gene amplicon sequencing. This data allowed us to determine commonality among core species and the relationship of age to microbial complexity and community composition. Understanding when the establishment of bacterial communities will occur will help us determine if species are acquired in a specific order and will provide clues as to whether some species require the presence of others to colonize. Taken together, insight will be provided into the reconstruction of the natural acquisition of the human oral microbiome from birth through the establishment of the permanent dentition. Changes in species complexity and the establishment of shared order of the oral microbiota have been examined in relation to age and site-specific samples. More specifically, our analyses suggest that the overall oral microbiome remains fairly stable after the first year of life, with very little influence from age, particular oral niches, and caries status.

Committee:

Ann Griffen, DDS (Advisor)

Subjects:

Dentistry; Microbiology

Hariharan, JananiPredictive Functional Profiling of Soil Microbes under Different Tillages and Crop Rotations in Ohio
Master of Science, The Ohio State University, 2015, Environmental Science
Food production and security is dependent on maintaining soil health and quality. Thus, the emphasis on sustainable and healthy soil function is a top priority for scientists and land managers. One of the most important factors that influences soil function is the microbial community. Recent advances have allowed us to quantify more accurately the composition of such communities, but there is still a knowledge gap with regard to the contribution of microorganisms to various processes occurring in the soil. Understanding this will facilitate the development of healthier agroecosystems. In this thesis, a predictive functional approach is used to elucidate bacterial species–function relationships. Bacterial community profiles were compared across two tillage systems and two crop rotations in Northern Ohio (Wooster and Hoytville). 16S rRNA gene-targeted sequencing was performed and the raw data obtained were filtered, denoised and processed using QIIME. Open-reference OTU picking and taxonomic assignment was performed using the Greengenes database. I then used a computational approach called PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to predict metagenomes and the most likely functions performed by individual species of bacteria. Sequence analysis reveals a large number of unidentified OTUs, which is consistent with our expectations of the soil ecosystem. Comparison of sequencing data from different platforms indicates that the dataset generated using Illumina sequencing provided better hits with the reference database than pyrosequencing, and was associated with a greater number of putative soil bacterial functions. PICRUSt allows an estimation of the level of involvement each OTU has with a specific gene function, which enables comparisons to be made across bacterial species and treatment conditions. Predicted functions of the bacterial community revealed a large number of proteins connected with metabolism and maintenance of natural organic molecules in soil as well as enzymes related to degradation of xenobiotics. Using this approach, I was also able to map specific OTUs to their functional potential. Bacterial enzymes implicated in the cycling of nitrogen, sulfur, carbon and methane through the soil were examined, as were enzymes that catalyzed the oxidative degradation of hydrocarbon compounds that are considered soil pollutants. Specialized groups of bacteria were linked to functions like nitrogen fixation and degradation of compounds like atrazine and chlorohydrocarbons. A broader range of OTUs was found to contain genes for carbon utilization and sulfur metabolism. These predictions are supported by previous ecological studies. There were other OTU-function relationships predicted in these studies that are novel and could be valuable in identifying commercially important microorganisms. These leads will require experimental validation. A clear difference was seen between the no-till and plow-till treatments, with no-till being functionally enriched for most major nutrient cycles. No such differences were observed between the different crop rotations. Proteobacteria, Actinobacteria and Acidobacteria were some of the most abundant phyla found in these soil samples, along with Nitrospirae, and Bacteroidetes. I concluded that long-term and continuous application of different tillage systems, and to a lesser extent crop rotation, result in unique bacterial communities that affect the overall functioning of the soil.

Committee:

Warren Dick (Advisor); Parwinder Grewal (Advisor); Margaret Staton (Committee Member)

Subjects:

Agriculture; Biogeochemistry; Bioinformatics; Ecology; Environmental Science; Microbiology; Soil Sciences

Keywords:

PICRUSt; soil metagenomics; soil bacteria; soil function; nutrient cycling

Ellis, David HaroldSilver nanoparticles: the immediate benefits of low bacterial resistance and the long-term risk of persistent stress in mammalian cells
Doctor of Philosophy (PhD), Wright State University, 2015, Biomedical Sciences PhD
Silver nanoparticles (AgNPs) are the fastest growing sector of nanotechnology, due mostly to their antibacterial properties. The antibacterial effectiveness of AgNPs is well known and derives from the shedding of silver ions which have multiple antibacterial targets in the bacterial cell. Due to their continuous release of ions and demonstrated antibacterial potency, some predict that AgNPs have a low potential for resistance development, which would make them a valuable asset in wound management. The ability for AgNPs to cause oxidative imbalance in mammalian cells is also well known, but the potential long-term impact of such a stress has not been studied despite its implication for negative outcomes in wound management. In this thesis, I demonstrate by using a stepwise increasing exposure protocol that Pseudomonas aeruginosa, but not Acinetobacter baumannii or Staphylococcus aureus could develop resistance to 10 nm, citrate-coated AgNPs. The potential for resistance development was lower than the antibacterial drug, ciprofloxacin, but not as low as silver nitrate to which none of the bacteria developed resistance. The resistance mechanism is not yet clear but appeared to involve the phenazine pigments produced by P. aeruginosa which can bind and reduce silver ions. In mammalian cells, I demonstrated the persistence and time-dependent oxidative stress of AgNPs in the A549, epithelial cell model, by using specialized imaging techniques and a common probe for oxidative stress. In addition, I showed that AgNPs can induce a senescent-like phenotype in A549 cells after an exposure that appears non-toxic in the typical viability assays used for assessing cytotoxicity. I confirmed that senescence was induced by showing an increase in senescence-associated, beta-galactosidase activity and the hypertrophic morphology of exposed cells, as well as a decrease in proliferation. The implication of this research for wound management is that AgNPs can be properly applied to wounds in order to inhibit bacterial colonization with little potential for resistant strains to emerge; however, the nanoparticles may persist in wound-associated, mammalian cells. There, the AgNPs will cause persistent oxidative stress with the potential to induce cellular senescence and reduce the long-term health and function of the surrounding tissue.

Committee:

Courtney Sulentic, PhD (Advisor); Saber Hussain, PhD (Advisor); Mill Miller, PhD (Committee Member); David Cool, PhD (Committee Member); Dan Halm, PhD (Committee Member)

Subjects:

Biology; Biomedical Research; Microbiology; Toxicology

Keywords:

silver nanoparticle; nanotechnology; toxicology; nanotoxicology; senescence; antibacterial; antibiotic resistance

Belyaevskaya, Anna VCharacterization of T box riboswitch gene regulation in the phylum Actinobacteria
Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology
Riboswitches are cis-acting RNA regulatory elements located in the 5' untranslated region of a gene. These elements modulate gene expression by structural rearrangements in response to an array of physiological signals. T box riboswitches regulate expression of amino acid-related genes by responding to the aminoacylation status of a specific tRNA that matches the amino acid identity of the regulated gene. Most T box RNAs function at the level of transcription attenuation. A terminator that prevents transcription of the downstream gene forms when aminoacylation of the cognate tRNA is high, whereas uncharged tRNA promotes stabilization of an antiterminator that prevents termination and therefore increases gene expression. T box riboswitches are typically composed of three conserved helical domains, designated Stem I, II, III, the Stem IIA/B pseudoknot, and the competing terminator and antiterminator elements; these domains include conserved primary sequence and several secondary structure elements. However, the predicted structure of many T box riboswitches from the phylum Actinobacteria differ from those found in other phyla. A major goal of this research was to characterize the unusual T box RNAs found in Actinobacteria. These riboswitches are present in ileS genes, and were divided into three groups based on the arrangement of the Stem I domain: canonical Stem I; Ultrashort Stem I (US); and Unusually Structured Stem I Region (USSR). The US and USSR domains lack conserved elements in the canonical Stem I that were previously thought to be essential for T box riboswitch regulation. In addition, most of T box riboswitches in Actinobacteria are predicted to regulate gene expression at the level of translation initiation instead of transcription attenuation. In the current study, we demonstrated that several T box RNAs from Actinobacteria are functional in vitro and undergo structural rearrangements and changes in ribosomal binding in response to uncharged tRNAIle that are consistent with regulation at the level of translation initiation. Results from fluorescence assays demonstrated that the US domain binds the cognate tRNAIle in the absence of other riboswitch sequences, and nuclear magnetic resonance spectroscopy revealed structural rearrangements in this unusual domain in response to interaction with the anticodon stem-loop of the cognate tRNAIle. The US riboswitch variant also was used to identify a novel tRNA interaction site between the conserved nts of the Loop E motif in the Stem II domain and the T arm of the tRNA. This work has investigated the molecular details of T box riboswitch gene regulation, explored the codon-anticodon interaction in the context of unusual RNA structures, and identified tRNA elements important for tRNA noncanonical role in gene regulation. Overall, the obtained information increased our understanding of RNA as a regulatory element.

Committee:

Tina Henkin (Advisor); Irina Artsimovitch (Committee Member); Venkat Gopalan (Committee Member); Anita Hopper (Committee Member)

Subjects:

Microbiology

Keywords:

Riboswitch, Gene Regulation, Actinobacteria

Kulkarni, Aditya S.Metabolic Studies of Albomycin Biosynthesis
Doctor of Philosophy (PhD), Ohio University, 2015, Biological Sciences (Arts and Sciences)
Albomycin is a sulfur containing metabolite produced by Streptomcyes sp. ATCC 700974. It is a structurally unique molecule with potent antibiotic activity. Unfortunately, it is produced in low amounts and this is thought to be the stumbling block for its commercial development. Cytosine is thought to be a precursor for albomycin production and experiments were done to increase intracellular cytosine to possibly increase albomycin production. Isolation of a codA mutant was attempted. Feeding cytosine in the growth medium and overexpressing a blsM gene (which generates cytosine) were found to have no effect on albomycin production. Experiments were also done to gain insights into the sulfur source utilized for albomycin biosynthesis by Streptomyces sp. ATCC 700974. Addition of iron and propargylglycine altered intracellular homocysteine and cysteine levels and albomycin production suggesting homocysteine as a precursor. Based on these results, sulfur amino acid metabolism of the albomycin producing strain was manipulated by overexpressing select pathways to observe effects on albomycin production. Albomycin production increased with overexpression of pathways producing homocysteine, namely the transsulfuration and direct sulfhydrylation pathways. No effect was observed on expression of the reverse transsulfuration pathway. Overexpression of the genes in the active methyl cycle leading to homocysteine was also found to increase albomycin production. AbmD is proposed to be the first enzyme of the albomycin biosynthetic pathway in Streptomyces sp. ATCC 700974 and to utilize cysteine or homocysteine as one of its substrates. Enzyme assays using purified AbmD demonstrated that homocysteine was utilized along with a co-substrate. Five pathway genes directing homocysteine biosynthesis along with the abmD gene were integrated into the Streptomcyes sp. ATCC 700974 genome to produce the strain SAK9. Albomycin production by SAK9 was found to be four times the wild type strain in glycerol containing media. These results suggest homocysteine is the precursor for albomycin biosynthesis. 13C labeling experiments conducted by feeding labeled cysteine and methionine were inconclusive. The seleno-amino acids selenomethionine and selenocystine were not incorporated into albomycin. Future directions for albomycin biosynthesis research are discussed.

Committee:

Donald Holzschu, Ph.D (Advisor); Erin Murphy, Ph.D (Committee Member); Tomohiko Sugiyama, Ph.D (Committee Member); Martin Tuck, Ph.D (Committee Member); Mark McMills, Ph.D (Committee Member)

Subjects:

Biochemistry; Microbiology; Molecular Biology

Keywords:

Albomycin; sulfur metabolism; secondary metabolite biosynthesis; metabolic engineering; Streptomyces

Richardson, Edward ThompsonRegulation of Macrophages by Mycobacterium tuberculosis and the ERK MAP Kinase Signaling Pathway
Doctor of Philosophy, Case Western Reserve University, 2015, Pathology
Mycobacterium tuberculosis, the cause of tuberculosis, survives for long periods in a latent state in infected individuals, and the immune system is typically able to control but not eliminate the bacteria. Latency is a complex phenomenon but involves, in part, interactions of the bacteria and its unique lipoproteins and lipoglycans with macrophages, the main cells that become infected. The purpose of this dissertation was to expand understanding of how M. tuberculosis engages with macrophages. In the first part, we characterized the lipoglycan binding function of M. tuberculosis lipoprotein LprG. We determined the binding properties of these M. tuberculosis lipoglycans to LprG using surface plasmon resonance. We also verified the presence of a non-acyl chain dependent binding mode to LprG, and determined that LprG also binds mannan. Finally, we determined that one function of LprG is to facilitate exposure of LAM on the bacterial cell surface for interaction with macrophages. LprG-deficient M. tuberculosis had reduced surface-exposed lipoarabinomannan, and had reduced ability to block phagolysosome maturation, a known immune evasion mechanism that requires lipoarabinomannan. These studies contribute to understanding of LprG, and develop increased knowledge of how M. tuberculosis lipoarabinomannan is exposed to macrophages to block phagolysosome fusion, a process involved in bacterial persistence and intracellular survival. In the second part, we studied the TLR2 signaling response of macrophages to M. tuberculosis. We determined that TLR2 was required for M. tuberculosis to trigger NF-κB and ERK, and that TLR2 signaling results in balanced downstream effects. NF-κB is required for expression of pro-inflammatory IL-12, and M. tuberculosis-stimulated Tpl2-ERK signaling suppressed IL-12 while inducing anti-inflammatory IL-10. These effects reduced CD4+ T cell responses against M. tuberculosis. Tpl2-deficient macrophages expressed IL-12 in response to M. tuberculosis, and were more potent at stimulating antigen-specific T cells, upon initial stimulation and recall. These findings contribute to understanding of the signaling triggered by M. tuberculosis, and the role of the macrophage-intrinsic ERK cascade in inhibiting T cell-mediated host defense. Together, these studies expand understanding of the regulation of macrophages by M. tuberculosis in ways that promote long-term survival of the bacteria, and may potentiate latent infection.

Committee:

Clifford Harding (Advisor); W Henry Boom (Advisor); Alex Huang (Committee Chair); Clive Hamlin (Committee Member); Liem Nguyen (Committee Member); Roxana Rojas (Committee Member); Pamela Wearsch (Committee Member)

Subjects:

Immunology; Microbiology

Keywords:

Tuberculosis; Mycobacterium tuberculosis; lipoprotein; LprG; lipoarabinomannan; Tpl2; MAP3K8; ERK; macrophages

Ohtola, Jennifer APneumococcal Vaccination in Aging HIV-Infected Individuals
Doctor of Philosophy (PhD), University of Toledo, 2015, Biomedical Sciences (Infection, Immunity, and Transplantation)
Advanced age and human immunodeficiency virus (HIV) infection are both risk factors for Streptococcus pneumoniae infections due to immunological dysfunction. The aging HIV-infected (HIV+) population may be at higher risk for pneumococcal disease due to the combination of these factors on humoral immunity. Current recommendations for pneumococcal vaccination in HIV+ adults include a priming dose of the 13-valent pneumococcal conjugate vaccine followed by one dose of the 23-valent pneumococcal polysaccharide vaccine 8 weeks later (PCV/PPV). We compared quantitative and qualitative antibody responses to PCV/PPV versus a single dose of PPV in HIV+ adults aged 50-65 years with CD4+ T cells/µl (CD4) >200 on antiretroviral therapy ≥1 year. We found that PCV/PPV did not demonstrate a clear immunological advantage to PPV alone, as serotype-specific IgG levels and functional titers postvaccination were similar between groups. In addition, these antibody responses were significantly reduced in HIV+ subjects vaccinated with PCV/PPV compared to age-matched, uninfected (HIV–) controls who received PCV/PPV. We also characterized the phenotype and surface expression of several receptors on serotype-specific B cells that may influence vaccine responses. HIV+ subjects vaccinated with PCV/PPV generated significantly reduced frequencies of circulating serotype-specific B cells postvaccination compared to those who received PPV only. However, phenotypic distributions of serotype-specific memory B cell subsets were similar between groups. Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)+ serotype-specific B cell percentages were significantly decreased in HIV+ PCV/PPV compared to PPV groups, indicating that prior PCV altered TACI expression. It remains unclear if this impact provides any benefit to vaccine responses. CD21+ serotype-specific B cells were also significantly reduced in HIV+ compared to HIV– PCV/PPV groups which may contribute to diminished antibody responses. Collectively, our findings suggest that continued efforts aimed at developing more effective vaccination strategies in susceptible adult populations are warranted, and further investigation into the immunological mechanisms that increase the risk of pneumococcal disease and induce potent vaccine responses are necessary.

Committee:

M. A. Julie Westerink, MD (Advisor); Deepak Malhotra, MD, PhD (Committee Member); Z. Kevin Pan, MD, PhD (Committee Member); Stanislaw Stepkowski, DVM, PhD, DSc (Committee Member); R. Mark Wooten, PhD (Committee Member)

Subjects:

Immunology; Microbiology

Keywords:

Streptococcus pneumoniae; pneumococcal conjugate vaccine; pneumococcal polysaccharide vaccine; HIV infection; aging; B cells; antibody

Njus, Kelsey AnneMolecular Techniques for the Identification of Commensal Fungal Populations on Cave Roosting Bats
Master of Science, University of Akron, 2014, Biology
Since the emergence of White-nose Syndrome (WNS) in 2006, North American bat populations have experienced devastating population declines. WNS is caused by the psychrophilic fungus Pseudogymnoascus destructans (Pd), which invades the wing tissues of bats during torpor, resulting in up to a 97% mortality rate. Yet some species, including the Indiana bat (Myotis sodalis) and the Virginia big-eared bat (Corynorhinus townsendii virginianus), appear to have reduced mortality from WNS, despite close proximity to infected bats. In an attempt to determine whether bats possess commensal fungal populations that confer resistance to WNS, we analyzed the culturable and non-culturable fungal communities of bat fur. To generate a library of the non-culturable fungal species, DNA was isolated from bat fur clippings, amplified by PCR of the internal transcribed spacer (ITS) region, sequenced, and identified using BLAST. Culturable fungal populations were obtained by swabbing and isolation in pure culture, and also identified using the ITS sequence. By comparing the communities of bats belonging to five species for four locations, we were able to determine that the species of the bat is the most important contributor to the composition of a bat’s fungal community, and this was best analyzed using molecular methods. While the majority of shared fungal species consisted of saprotrophic fungi, a small portion of these shared species consisted of yeast, the most abundant of which was Debaryomyces udenii; Virginia big-eared bats, which have a 0% mortality rate, have a fungal community that consists of 55% D. udenii. This data supports the hypothesis that bats harbor commensal fungal communities, and that these communities may confer resistance to WNS. Identifying whether commensal populations provide a mechanism of WNS resistance in bats could help in our understanding of the emergence of this pathogen, the future of the disease, and potential biological controls.

Committee:

Hazel Barton, Dr. (Advisor); James Holda, Dr. (Committee Member); Greg Smith, Dr. (Committee Member)

Subjects:

Biology; Ecology; Microbiology; Molecular Biology

Keywords:

White-nose Syndrome; commensal; fungi; bats; Pseudogymnoascus; PCR inhibition; Corynorhinus townsendii

Vadia, Stephen EListeriolysin O activates Listeria monocytogenes internalization into human hepatocytes through a novel pore-dependent mechanism
Doctor of Philosophy, The Ohio State University, 2014, Microbiology

The causative agent of listeriosis, Listeria monocytogenes, is a frequent contaminant of fruits, vegetables, cheeses, and processed foods. In certain high risk groups, L. monocytogenes can cause potentially fatal infections. The bacterium can traverse the intestinal epithelial barrier and spread throughout the body via the bloodstream and lymphatic system to infect the liver, cross the blood-brain barrier, and in pregnant women, the placental barrier. L. monocytogenes is able to cross these biological barriers due to its ability to proliferate within certain phagocytic and nonphagocytic cells. Bacterial invasins, most notably internalin (InlA) and InlB, bind to receptors on the surface of nonphagocytic cells to induce endocytosis of the bacterium through a zipper-like mechanism. L. monocytogenes can then escape from the endocytic vacuole and enter the cytosol through the activity of the pore-forming toxin listeriolysin O (LLO). In the cytosol, the bacterium can replicate and disseminate to adjacent cells by cell-to-cell spreading.

LLO belongs to the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming toxins. These toxins are secreted as monomers, oligomerize on cholesterol-rich membranes, and form large transmembrane pores. In addition to mediating vacuolar escape, LLO stimulates signaling pathways in host cells. We hypothesized that the signaling activity of extracellular LLO affects the intracellular lifecycle of L. monocytogenes. Using quantitative immunofluorescence microscopy to assess the importance of LLO for bacterial association to and internalization into epithelial cells, we found that LLO is required for efficient L. monocytogenes internalization into hepatocytes, and is sufficient to induce internalization of noninvasive bacteria and polystyrene beads. Using novel LLO variants that bind to host cells but are unable to form the pore complex, we demonstrated that pore formation was required for LLO-mediated internalization.

The CDC streptolysin O is known to stimulate a Ca2+-dependent membrane repair response that involves endocytosis of the toxin from the perforated plasma membrane. LLO stimulates a Ca2+-dependent membrane repair response, but this response is insufficient to account for L. monocytogenes internalization. LLO-mediated internalization and membrane repair both require the influx of extracellular Ca2+, but differ in their requirement for F-actin and K+ efflux. Surprisingly, using ionophores to stimulate Ca2+ and K+ fluxes, as happens when the plasma membrane is perforated by LLO, was sufficient to induce internalization of large particles. LLO mediates bacterial internalization in all human hepatocytes we tested and in additional epithelial cell lines. However, LLO does not induce bacterial internalization in all epithelial cells that it perforates.

In light of this data, LLO emerges as a significant invasion factor expressed by L. monocytogenes, which is coexpressed with InlA and InlB during infection. With the same approaches used to assess the role of LLO, we determined the contribution of InlA and InlB to bacterial association, internalization, and intracellular viability in nonphagocytic cells that express the InlA and InlB receptors. We found significant variability in the involvement of each of these invasins in bacterial association, internalization, and intracellular viability, demonstrating that the activity of invasins is not governed solely by the presence or absence of their receptors.

Committee:

Stephanie Seveau, PhD (Advisor)

Subjects:

Microbiology

Keywords:

Listeria monocytogenes; Listeriolysin O; Pore-forming toxin; Intracellular pathogen

Schlais, Michael J.Detection and Characterization of a Unique Ammonia Oxidizing Archaea; Cultured from Lake Superior
Doctor of Philosophy (Ph.D.), Bowling Green State University, 2014, Biological Sciences
In the past century Lake Superior has seen a 5-fold increase in nitrate levels. Previous research has shown this increase to be due to as of yet undescribed in-lake oxidation processes. It has recently been shown that ammonia oxidizing archaea (AOA) in marine and freshwater environments belonging to the phylum Thaumarchaeota have the ability to oxidize ammonium, and are specifically suited to the low NH4¿ /oligotrophic environment of Lake Superior. In this study isolation and enrichment of these unique freshwater ammonia oxidizing archaea from Lake Superior has enabled the direct measurement of NO2¯ and N2O production and NH4¿ metabolism. In the search for in-lake nitrifying organisms we have identified and described a novel group of freshwater ammonia oxidizing archaea using the ammonia monooxygenase alpha (amoA) subunit gene as a diagnostic tool for microbes capable of ammonia oxidation. Flow cytometry was used to determine cell abundances and ideal incubation times and temperatures for these AOA taken from these Lake Superior. These data along with nitrification measurements enabled us to determine per cell nitrification rates for our AOA enrichment cultures, which have shown that they do indeed account for a major component of in-lake nitrification processes. With the exception of the marine archaeon Nitrosopumilus maritimus, most research has been focused on culture-independent methods for the characterization of nitrifying microbes. This study is among the first of these culture dependent studies to describe methods for culturing a freshwater ammonia oxidizing archaea. iv

Committee:

George Bullerjahn, PhD (Advisor); Michael McKay, PhD (Committee Member); Scott Rogers, PhD (Committee Member); Paul Morris, PhD (Committee Member); John Snyder, PhD (Other)

Subjects:

Biology; Microbiology

Keywords:

ammonia oxidizing archaea; AOA; amoA; Nitrification

Pina-Mimbela, Ruby MelisaAssociation of Polyphosphate (poly P) Kinases with Campylobacter jejuni Invasion and Survival in Human Epithelial Cells
Master of Science, The Ohio State University, 2013, Veterinary Preventive Medicine
The foodborne and zoonotic bacterium Campylobacter jejuni, is one of the major causes of gastrointestinal disorders in humans worldwide. This human pathogen expresses a variety of carbohydrate, protein and lipid structures decorating its outer material (OM) which have been associated with virulence. On the other hand, inorganic polyphosphate (poly P), in many bacteria, is essential for stringent survival response and adaptation, and is considered as a virulence factor. Two kinases in C. jejuni have been associated with virulence: poly phosphate kinase 1 (PPK1) and poly phosphate kinase 2 (PPK2). PPK1 is responsible for poly P generation and PPK2 for GTP biosynthesis from poly P. GTP is a molecule involved in signaling processes as well as protein and capsule synthesis. In previous studies, we demonstrated that C. jejuni delta ppk1 shows reduced stringent survival response, adaptation, and in vivo colonization in chickens, whereas, delta ppk2, in addition to its stress tolerance defect, also displays reduced virulence properties. In the present study, the contribution of component(s) present in the outer material (OM) from the wild-type, delta ppk1 and delta ppk2 to C. jejuni invasion and intracellular survival in human epithelial cells in vitro was investigated. OM from C. jejuni wild type 81-176, delta ppk1 and delta ppk2 was extracted and fractionated into carbohydrates (lipoglycans, poly-oligosaccharides), lipids, and proteins. These fractions were tested for their contribution to C. jejuni invasion, intracellular survival and IL-8 production in INT-407 human embryonic intestinal cells. Different fractions from C. jejuni 81-176 wild type, delta ppk1 and delta ppk2 were pre-incubated with INT-407 human epithelial cells prior to infection with C. jejuni wild type. Results obtained imply that components present in the C. jejuni OM are associated to invasion and intracellular survival in INT-407 cells. Additionally, poly P kinases play a role in intracellular survival in epithelial cells. Alterations in OM from delta ppk1 and delta ppk2 are suggested to be important in C. jejuni intracellular survival in INT-407 cells. Our results suggested that OM proteins mediate C. jejuni invasion and intracellular survival to human epithelial cells whereas lipoglycans are associated with intracellular survival. In addition, alterations present in delta ppk2 lipids are associated with C. jejuni intracellular survival in INT-407 cells. On the other hand, poly- and oligo- saccharides fraction from delta ppk1 and delta ppk2 are likely to play a role in C. jejuni survival within epithelial cells. Further, we evaluated production of IL-8 in INT-407 cells by OM and its fractions from wild type, delta ppk1 and delta ppk2 strains. C. jejuni OM from wild type was not able to induce IL-8 in INT-407 cells, whereas IL-8 secretion was observed when these epithelial cells were exposed to OM from delta ppk1 and delta ppk2. Our results demonstrate that proteins, lipids, and lipoglycans present in C. jejuni OM are associated with its invasion and intracellular survival in human epithelial cells. Additionally, this study provides insights about the role of poly P in modulation of C. jejuni OM composition, thereby contributing to invasion and intracellular survival.

Committee:

Gireesh Rajashekara, Dr. (Advisor); Jordi B. Torrelles, Dr. (Committee Member); Chang-Won Lee, Dr. (Committee Member)

Subjects:

Biochemistry; Cellular Biology; Microbiology; Veterinary Services

Keywords:

Campylobacter jejuni; Poly P kinases; invasion; survival; epithelial cells; INT-407 cells

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