Doctor of Philosophy (PhD), Ohio University, 2014, Molecular and Cellular Biology (Arts and Sciences)

Ribo-regulators are defined as any RNA molecule that is capable of controlling the expression of a target gene(s) and/or the activity of a target protein. Ribo-regulators are found in every domain of life and, in bacteria, have been shown to have a role in many important processes including virulence, metabolism, iron-acquisition and stress responses. Ribo-regulators can be divided into two major groups, cis-encoded riboregulators and trans-encoded ribo-regulators. trans-encoded ribo-regulators are generally referred to as small RNAs (sRNAs) and consist of RNA molecules that bind target transcripts via complimentary base-pairing and those that sequester specific proteins. Shigella species are the etiological agents of shigellosis, severe bacillary dysentery that affects 165 million people worldwide each year and causes 1.1 million deaths. Infection by Shigella species requires the coordinated production of virulence factors, a process controlled largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional de-repressor of many Shigella virulence genes including those encoding the Type Three Secretion System. S. dysenteriae is known to produce several sRNAs that affect the expression of virulence genes including RyhB, a sRNA known to modulate the expression of virB. In Chapter 2 of this work the mechanism by which RyhB-dependent regulation of virB expression occurs is demonstrated to be independent of VirF activity. Additionally, it is shown that nucleic acid sequences within the virB open reading frame are necessary for RyhB-dependent regulation. In Chapter 3 of this work the first twin sRNAs unique to S. dysenteriae, RyfA1 and RyfA2, are characterized. RyfA1 and RyfA2 are nearly identical in sequence and are produced predominately during logarithmic growth. RyfA1 and RyfA2 are shown to have unique and reciprocal effects on the growth of S. dysenteriae. The twin sRNAs affect virulence in unique ways as meas (open full item for complete abstract)

Committee: Erin Murphy PhD (Advisor); Peter Coschigano PhD (Committee Member); Don Holzschu PhD (Committee Member); Sarah Wyatt PhD (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2024, Comparative Biomedical Sciences

Ehrlichia species are emerging obligate intracellular bacteria that are transmitted by ticks and can cause potentially fatal ehrlichiosis in humans. The biomolecular mechanisms of pathogenicity factors and tick transmission of Ehrlichia are inadequately understood. Ehrlichia japonica (Eja) infection in mice recapitulates the full spectrum of human ehrlichiosis and can be transmitted by Ixodes scapularis ticks, providing an excellent mouse model for fatal human ehrlichiosis and a transmission model for tick-borne diseases. To identify genes crucial for in vitro proliferation, in vivo survival, mouse virulence, and tick transmission of Ehrlichia, a library of insertional Eja mutants previously constructed was further expanded using Himar1 transposon-based random mutagenesis, resulting in a total of 407 insertional mutants isolated through antibiotic selection. 183 insertions were mapped in the coding regions of 129 distinctive genes. Three clonal intragenic mutants were studied in depth. The pathogenicity and host acquisition of wild-type (WT) Eja and an isogenic transposon mutant that lacks the tandem repeat protein 120 (∆TRP120) were compared. Both wild-type and ∆TRP120 Eja proliferated similarly in mammalian and tick cell cultures. Upon inoculation into mice, both wild-type and ΔTRP120 E. japonica multiplied to high levels in various tissues, with similar clinical chemistry and hematologic changes, proinflammatory cytokine induction, and caused fatal disease. However, the blood levels of ∆TRP120 Eja were almost undetectable within 24 h, whereas the levels of the wild-type increased exponentially. This led to much less ∆TRP120 Eja than wild-type Eja being acquired by larval ticks fed on the infected mice, and carried to nymphal stage, thus ∆TRP120 Eja failed to transmit from nymphs to naive mice. Mouse blood monocytes exposed to native TRP120 showed significantly reduced cell surface expression of the transmigration-related markers Ly6C and CD11b. Thus, TRP120 is re (open full item for complete abstract)

Committee: Yasuko Rikihisa (Advisor); Sanggu Kim (Committee Member); Amit Sharma (Committee Member); Estelle Cormet-Boyaka (Committee Member) Subjects: Biomedical Research; Cellular Biology; Microbiology; Molecular Biology

Bachelor of Science (BS), Ohio University, 2024, Biological Sciences

Staphylococcus aureus is an opportunistic bacterial pathogen that causes a wide variety of infections and disease in humans. The immense pathogenic ability of S. aureus is thanks to its wide arsenal of virulence factors. While there are many types of virulence factors, toxins are proteins with cytolytic properties toward human cells. This work focuses specifically on the α-type phenol-soluble modulins (αPSMs) which have been characterized to play a role in many different biological phenotypes. These protein toxins (PSMα1-4) play pivotal roles in lysis of human cells, formation of biofilms, and production of extracellular vesicles (EVs). Despite the extensive studies that have been on the αPSMs, significant gaps persist in the understanding of their individual roles. Traditional genetic approaches to studying the αPSMs, such as complete knockouts of the transcript and peptides or use of synthetic peptides, have limitations in interpreting specific mechanisms and roles of the individual αPSMs. There could be more complicated regulatory networks, or each peptide could act individually. In contrast, this thesis utilizes strains that produce the αPSM transcript, while selectively lacking individual peptides (or the group of four) through introduction of stop codons. Through biological assays, RNA-seq, and proteomic analysis, I worked to uncover the regulatory differences of the αPSM peptides versus the transcript that encodes them. Many unexpected results emerged through the analysis of Aim One, especially with showing increased hemolysis in a mutant that does not produce the αPSM peptides. There was also an increase in extracellular vesicle release and biofilm formation in individual mutants, challenging the known roles of these molecules in virulence. Aim Two focused on the regulatory aspects of the αPSM peptides and their transcript. I observed distinct transcriptomic and proteomic profiles in these comparisons, with alterations of many small regulatory (open full item for complete abstract)

Committee: Soichi Tanda PhD (Advisor); Ronan Carroll PhD (Advisor) Subjects: Biology

Master of Science, The Ohio State University, 2024, Animal Sciences

Listeria monocytogenes is an invasive, zoonotic, foodborne pathogen that causes human listeriosis. L. monocytogenes is a resilient pathogen capable of surviving and even proliferating under various environmental conditions. Its capacity to persist in soil, manure, water, and various farm environments necessitates an investigation to safeguard consumers and maintain the reputation of small specialty crop farms. Small specialty crop farms (SSCFs) are an important and growing sector for crop production in the USA. Currently, there is little information on the prevalence and genetic landscape of the L. monocytogenes from SSCFs. Bridging this knowledge gap would help understand potential public health risks associated with the L. monocytogenes coming from these farms. The goal of our study is to understand the prevalence and genomic diversity among the isolates in the SSCF niche by characterizing pangenome, serogroups, antimicrobial resistance genes, and virulence genes. We collected, tested, and processed dairy and poultry manure, soil, water, and produce samples (n = 1842) for L. monocytogenes from 15 SSCF between 2016 to 2020. Isolates were analyzed through Whole Genome Sequencing (WGS). The overall prevalence of L. monocytogenes on SSCF was 7.17% (132/1842 samples). Based on positive samples, the prevalence of L. monocytogenes was 23.2% (n=53/228) in dairy manure, 11.0% (n=14/127) in water, 6.2% (n=31/500) in produce, 3.6% (n=28/777) in soil, 2.9% (n=6/210) in poultry manure. From these 132 positive samples, 347 isolates were obtained and were subjected to whole genome sequencing (WGS). Pangenome analysis detected the presence of 2,036 core genes. In silico serotyping of L. monocytogenes detected the presence of four serotypes 1/2a, 1/2b, 1/2c, and 4b, implicated in human infections. Analysis of antimicrobial resistance (AMR) genes identified the presence of 10 genes: abc-f, fos(X), mecA, vanZ, mprF, mprB, vga(G), ampC, group_1242, and tetR. In addition, an arsenic- (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Joy Scaria (Committee Chair); Greg Habing (Committee Chair) Subjects: Animal Sciences; Microbiology; Public Health

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

Ehrlichiosis is a potentially life-threatening disease caused by infection with Ehrlichia sp., tick-borne bacterial pathogen. Infection of Ehrlichia japonica in the mouse provides a great disease model as it recapitulates full-spectrum, lethal human ehrlichiosis. We verified that our E. japonica transposon mutant ΔripE does not express resistance-inducing protein of Ehrlichia (RipE). While mutant ΔripE proliferates similarly to wild-type (WT) E. japonica in mammalian cell cultures in vitro, its virulence is attenuated in mouse infection. Although Ehrlichia spp. are obligate intracellular bacteria, they need to endure a brief extracellular stage before initiating a new cycle of infection. ripE mRNA was upregulated at the start of the exponential replication stage, and most RipE protein was found in smaller, CtrA-positive Ehrlichia that are primed to be liberated from host cells. Extracellular ΔripE contained significantly less ATP and lost infectivity more rapidly in culture compared with WT Ehrlichia. Complementation of ripE gene in ΔripE and overexpression of RipE in E. japonica significantly increased ATP reserves. More strikingly, RipE overexpressing E. japonica killed mice more rapidly than WT E. japonica. RipE is a bacterial surface-exposed protein and recombinant RipE-immunized mice produced Ehrlichia neutralizing antibodies which significantly prolonged the survival time following a lethal dose of Ehrlichia challenge. Our findings strongly suggest that RipE energizes Ehrlichia and confers resistance at the extracellular stage to promote Ehrlichia spreading and infection in demand in vivo, and hence, it stands out as a potential vaccine candidate and a therapeutic target against ehrlichiosis.

Committee: Yasuko Rikihisa (Advisor); Prosper Boyaka (Committee Member); Ian Davis (Committee Member); Stephanie Seveau (Committee Member) Subjects: Biomedical Research; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Pythium pathogens that can cause disease on leafy greens are well-adapted to hydroponic production systems due to their ability to produce swimming zoospores. These spores that rapidly disperse in the recirculating water. Current strategies to reduce disease losses primarily involve hygienic practices and UV sterilization of water when infections become problematic. Prior work had demonstrated that the Pseudomonas fluorescens isolate LE6_D7 was capable of contact-dependent killing of Pythium isolates from hydroponic greenhouse operations from California, Indiana, Ohio, and New Jersey. Whole genome sequencing revealed that this isolate possesses two complete Type Six Secretion Systems (T6SS). In this study, the focus was on proteins associated with T6SS, due to their role in delivering proteins into the oomycete cytoplasm, providing the simplest explanation of the contact-dependent phenotype. Bioinformatic analysis identified hcp_05421 as one of four genes in this strain that produced the nanotube of T6SSs. To determine the role of this gene in virulence against Pythium isolates, a DNA fragment of a truncated version of the target gene was synthesized through PCR, including upstream and downstream flanking regions. This DNA fragment was then ligated into the vector DONR1K18ms using a Gateway™ BP clonase reaction. The vector was introduced into LE6_D7 strain either by conjugation using the E. coli helper strain RHO5, or by electroporation in 10% glycerol. Among the ten vgrg1 type genes present in this P. fluorescens strain, the largest one (vgrg_00081) was selected for characterization using a similar approach. Insertional mutants resulting from a single crossover event after the integration of the plasmid into the genome were selected on antibiotic media. Insertional mutants of both Δhcp_05421 and Δvgrg_00081 genes resulted in the loss of contact-dependent killing in two tested strains of Pythium. Double crossover mutants of Δhcp_05421 and Δvgrg_00081 gene deletions (open full item for complete abstract)

Committee: Paul Morris Ph.D. (Committee Chair); Zhaohui Xu Ph.D. (Committee Member); Vipaporn Phuntumart Ph.D. (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2023, Molecular and Cellular Biology (Arts and Sciences)

Staphylococcus aureus is a Gram-positive opportunistic pathogen that chronically colonizes the anterior nares of ~30% of the global population. Colonization is often asymptomatic, however, it is associated with recurrent infections and increased risk of surgical site infections. These risks are attributed to the ability of the bacterium to transition from the nares to the deeper tissues of the body and blood stream. Transition of the bacterium can result in potentially fatal infections such as endocarditis, toxic-shock syndrome, and septicemia. The average temperature of the nares is roughly 34˚C while homeostatic body temperature is 37˚C. Given the opportunistic lifestyle and the temperature difference between the two niches we investigated the influence of temperature on S. aureus gene expression and pathogenesis. RNAseq and proteomic analyses were performed on S. aureus cultures grown at three different physiologically relevant temperatures; 34˚C, the temperature of the nares, 37˚C, body temperature, and 40˚C, representative of pyrexia. We find that small, but definite temperature differences are sufficient to induce large scale alterations in the transcriptome and proteome of S. aureus. Cross-reference analysis of the transcriptome and proteome provided evidence of temperature dependent post-transcriptional regulation, potentially mediated by sRNA regulatory elements. Through various phenotypic analyses, we also show that these changes manifest as biologically relevant phenotypes. In addition, this study is the first to demonstrate a decrease in host intracellular invasion by S. aureus at 34˚C compared to 37˚C. We also characterize the temperature dependent transcriptional regulation of the metalloprotease aureolysin. We first show that the observed temperature dependent expression of aur is a result of both differential aur transcript stability and aur synthesis. Additionally, we show that temperature dependent transcriptional regulation aur is mediated (open full item for complete abstract)

Committee: Ronan Carroll (Advisor); Jennifer Hines (Committee Member); Don Holzschu (Committee Member); Erin Murphy (Advisor) Subjects: Biology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2023, Biological Sciences (Arts and Sciences)

Staphylococcus aureus is a Gram-positive opportunistic pathogen capable of causing life threatening infections. A series of regulators controls expression of an array of virulence factors which allow for S. aureus to infect a host. During investigation into small protein encoding small RNAs, we identified a novel small protein regulator named ScrA. Overexpression of ScrA led to increased clumping, hemolysis, and biofilm formation, which was shown to be dependent upon the SaeRS two-component system, while mutation of scrA lead to alterations in host factor binding, increased membrane stability, and an overall decrease in virulence. Further investigation into the mechanism of ScrA activity revealed several amino acids essential to function residing within the transmembrane domain. Additionally, we identify the transmembrane domain as the key domain for ScrA function. Interaction partners were identified by both in vivo Co- Immunoprecipitations A pulldown of the ScrA promoter region identified the regulatory protein TcaR giving a potential link to the AlrR system. Taken together, these data reveal that ScrA acts as a novel regulator of virulence in S. aureus and may act as a niche specific link between the Arl and Sae systems.

Committee: Ronan Carroll (Advisor) Subjects: Biology; Microbiology

Doctor of Philosophy (PhD), University of Toledo, 2022, Biomedical Sciences (Medical Microbiology and Immunology)

Withheld at author's request

Committee: R. Mark Wooten Ph.D. (Advisor); Viviana Ferreira D.V.M., Ph.D. (Committee Member); Matam Vijay-Kumar Ph.D. (Committee Member); Randall Worth Ph.D. (Committee Member); Robert Blumenthal Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Experiments; Health Sciences; Immunology; Microbiology

Doctor of Philosophy, The Ohio State University, 2022, Food Science and Technology

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis), as the most relevant serovar associated to shell eggs, has caused numerous salmonellosis outbreaks. Salmonella Enteritidis exhibits great fitness in colonizing oviduct track of laying hen, leading to its predominance in shell eggs through vertical transmission route. Salmonella Enteritidis grown in egg yolk showed pronounced disease symptoms in a mouse model. However, the cause of the increasing virulence, attributed to the presence of the pathogen in egg environment, remains unrevealed. The high risk of S. enterica contamination prompted the food industry to develop egg pasteurization processes aiming to produce safer shell egg while maintaining the product's fresh qualities. Water-immersion heating is often used to pasteurize shell eggs. Recently, ozone in combination with moderate heat treatment was introduced as an alternative to the purely thermal pasteurization technology. Although such processing technologies are promising, it is not known how the physiology of Salmonella changes during the treatment or what safety risks these pasteurization technologies would cause. To full fill these research gaps, the goals of the current study are: (I) to understand the contribution of shell eggs, as a food vehicle, to the increased risk of salmonellosis; and (II) to reveal the role of egg pasteurization parameters in increasing or mitigating the salmonellosis risk. The hypotheses related to the first goal of this research are: (1) egg environment can serve as an evolutionary drive for Salmonella Enteritidis to acquire genetic advantages, contributing to the virulence of the pathogen; and (2) growth of Salmonella Enteritidis in egg yolk allows changes in the transcription of pathogen's virulence genes leading to increased virulence in a mouse model. To address these two hypotheses, the objectives under the first goal are: (1) using comparative genomic to explore whether egg environment provided Salmonella (open full item for complete abstract)

Committee: Ahmed Yousef (Advisor); Farnaz Maleky (Committee Member); Jiyoung Lee (Committee Member); V.M. Balasubramaniam (Committee Member) Subjects: Food Science; Microbiology; Molecular Biology

Master of Science (MS), Ohio University, 2022, Biological Sciences (Arts and Sciences)

Teg41 is a small RNA in Staphylococcus aureus that regulates the alpha phenol-soluble modulins, which are peptides known to contribute to virulence. Previous studies have suggested that Teg41 may have additional targets. In this study, various methods such as RNA-sequencing, β-galactosidase assays, and proteomics analyses were performed to identify and examine the regulatory targets of Teg41. Notably, there was a significant upregulation in the spa transcript as evident in both the RNA-seq and proteomics data in the Teg41 mutant strain. We hypothesize that the most likely target for Teg41 is another small RNA known as RNAIII due to its role in the regulation of spa and other virulence factors.

Committee: Ronan Carroll (Advisor); Donald Holzschu (Committee Member); Erin Murphy (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2021, Biological Sciences (Arts and Sciences)

Staphylococcus aureus is Gram-positive bacteria that can live as a harmless commensal or as a dangerous pathogen. Diseases caused by S. aureus range from mild, self-resolving skin infections to life-threatening endocarditis, osteomyelitis, and septicemia. S. aureus produces an arsenal of virulence factors that contribute to infection, and these virulence regulators are controlled by an intricate network of protein regulators and RNA-based regulators. In this work, we describe the function of a previously uncharacterized S. aureus sRNA, Teg41. Teg41 is a 205 nucleotide transcript directly adjacent to the αPSM transcript, which encodes four highly toxic peptides that cause host cell lysis, biofilm formation and spread, and proinflammatory cytokine production. In Chapter 2, we find that Teg41 regulates the αPSMs. We show that overexpression of Teg41 leads to increased hemolysis, which is dependent on the presence of the αPSMs. We also used in silico analysis to predict that the extreme 3′ end of Teg41 is necessary for interaction with the αPSM transcript. We created a strain with the 3′ end of Teg41 deleted (Teg41Δ3′) and found that this strain is attenuated in hemolysis, virulence, and PSM production. Finally, we show that the 3′ end of Teg41 is necessary for virulence and that a Teg41Δ3′ shows decreased Teg41 expression. In Chapter 3, we further characterize the interaction between Teg41 and the αPSMs by showing that Teg41-mediated hemolysis is dependent only on the αPSMs, and that a single chromosomally integrated copy of Teg41 restores virulence, hemolysis, extracellular vesicle production, and PSM production in a Teg41Δ3′ strain. We also show that substituting four nucleotides on the extreme 3′ end of Teg41 abolishes increased hemolysis in a Teg41 overexpression strain. Finally, we show Teg41 expression influences both the stability and abundance of the αPSM transcript, and, interestingly, that a Teg41Δ3′ strain also shows decreased stability in 50 transcri (open full item for complete abstract)

Committee: Ronan Carroll (Advisor); Nathan Weyand (Committee Member); Jennifer Hines (Committee Member); Erin Murphy (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and annotated its whole genome sequence. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated, 84 insertions were within the non-coding regions, and 74 insertions were in the coding regions of 55 distinct protein coding genes including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for intracellular multiplication in DH82 macrophages, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes conserved among Ehrlichia spp., but lacked homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IFN-γ, IL 1β, and IL-12 p40 in one clone. Thus, we identified two Ehrlichia genes responsible for non-macrophage infection-related virulence.

Committee: Yasuko Rikihisa PhD (Advisor); Amal Amer MD, PhD (Committee Member); Estelle Cormet-Boyaka PhD (Committee Member); Stephanie Seveau PhD (Committee Member) Subjects: Cellular Biology; Microbiology; Molecular Biology

Doctor of Philosophy (Ph.D.), University of Dayton, 2018, Biology

In order for enteric pathogens to be successful in causing their infection they must overcome various environmental factors including: low pH, decreased oxygen, high osmolarity, nutrient competition, and host immune system etc. One such pathogen, Listeria monocytogenes (L. monocytogenes), is well known for being extremely adaptable and avoids host immune defenses by causing its infection intracellularly. This unique infection route makes L. monocytogenes an attractive pathogen for gaining further understanding host immune responses to intracellular pathogens. However, the majority of studies involving L. monocytogenes take place in the presence of oxygen, which completely omits the anaerobic phase of gastrointestinal infection. The main goal of this study is to understand the effects of anaerobic growth on L. monocytogenes pathogenesis. Chapter I provides a brief introduction including background information on L. monocytogenes discovery, lifestyle, outbreaks, and pathogenesis. Following the introduction in Chapter II, I give a review on the role of oxygen in the pathogenesis of various other relevant enteric pathogens. Moving into Chapter III, I look at key morphological differences between aerobic and anaerobic growth conditions and explores how exogenous supplementation of key intermediates of the tricarboxylic acid cycle (TCA) affects subsequent pathogenesis. This transitions into Chapter IV, where I explore the role of respiratory activity in priming and sustaining intracellular pathogenesis. And finally, Chapter V evaluates the importance of menaquinone biosynthesis in L. monocytogenes growth and subsequent pathogenesis. Together these data support a regulatory role for metabolic activity in the success of L. monocytogenes infection.

Committee: Yvonne Sun Ph.D. (Advisor); Amit Singh Ph.D. (Committee Member); Jayne Robinson Ph.D. (Committee Member); Pothitos Pitychoutis Ph.D. (Committee Member); Donald Comfort Ph.D. (Committee Member) Subjects: Biology; Microbiology

Doctor of Philosophy, The Ohio State University, 2018, Plant Pathology

More than 700 million tons of rice is produced on an annual basis constituting roughly twenty-percent of all calories consumed by humans. This fact illustrates how the study of protecting and improving rice yield is acutely important to a growing world population. Outbreaks of rice blast disease, caused by the fungus Magnaporthe oryzae, have led to major losses with this disease being the main limiting factor in rice production in many regions. The devastation caused by this disease has inspired the intense study of the molecular basis of the disease, including avirulence and virulence factors. By using the gene-for-gene model, many resistance (R)-genes have been identified, allowing breeders to craft rice cultivars that are resistant to many strains of the fungus. However, much is still unknown concerning the cognate avirulence (Avr) and virulence genes. This disparity is due to the lack of recognized sequence patterns of these genes. One goal of this study is to identify fungal genes that are related to M. oryzae virulence in hopes of better understanding the relationship between this hemibiotrophic pathogen and its host. M. oryzae strains KJ201 and CHNOS60-2-3 were utilized after demonstrating a difference in pathotype on the rice cultivar C101A51 Previous studies in the Mitchell lab have identified five candidate genes coding for punitive virulence factors by performing random insertion Agrobacterium tumefaciens-mediated transformation (rATMT), infection assays, and TAIL-PCR. These genes were cloned into the strain CHNOS60-2-3, which is naturally compatible with the C101A51 rice. By infecting C101A51 plants with the CHNOS60-2-3 transformants, and assessing disease, we were able to determine that homoaconitase MOLYS4 is involved in virulence for M. oryzae. Learning assessments dictate the environment of our classrooms. With an ever-increasing pace of life, it is becoming more important that instructors find meaningful and efficient ways to use di (open full item for complete abstract)

Committee: Thomas Mitchell PhD (Advisor); Jason Slot PhD (Committee Member); Robert Tabita PhD (Committee Member); Guo-Liang Wang PhD (Committee Member) Subjects: Plant Pathology

Doctor of Philosophy (PhD), Ohio University, 2016, Biological Sciences (Arts and Sciences)

Shigella is a genus of Gram-negative pathogenic bacteria that causes shigellosis, a severe form of bacillary dysentery in human with an infectious dose of less than 100 cells. The global burden of shigellosis is estimated to be no less than 125 million infections, with the majority of both infection and resulting deaths occurring in children under the age of five. These facts, in combination with the lack of a vaccine or universally effective treatment makes understanding the molecular mechanisms underlying the pathophysiology of Shigella of utmost importance. To survive and successfully colonize in the host, bacterial pathogens regulate the expression of multiple virulence-associated factors in response to the changes of environmental cues. This study focused on two of the important virulence-associated processes in the infections of S. dysenteriae, the most virulent species in the genus of Shigella: 1) acquisition of essential nutritional iron via the Shigella heme uptake (Shu) system from the iron-limited environment within the human host, and 2) secretion of effector proteins required for the invasion processes through the type III secretion system (T3SS). Investigations presented here identify the host-associated environmental factors that regulate the expression of the specific factors required to complete the processes listed above, and characterize the molecular mechanisms underlying each regulation. Specifically, studies focused on the regulation of the Shu system demonstrate that its periplasmic binding component, ShuT, is subject to iron-dependent transcriptional regulation via the activity of the global transcriptional regulator Fur, and temperature-dependent post-transcriptionally mediated by an RNA thermometer located within the 5' untranslated region of the gene. Studies focused on the regulation of MxiG, a component of the T3SS, identify and characterize a functional RNA thermometer that mediates post-transcriptional temperature-dependent regulation. (open full item for complete abstract)

Committee: Erin Murphy (Advisor); Jennifer Hines (Committee Chair); Peter Coschigano (Committee Member); Tomohiko Sugiyama (Committee Member); Donald Holzschu (Committee Member) Subjects: Biology; Microbiology

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 (open full item for complete abstract)

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

Doctor of Philosophy, The Ohio State University, 2014, Comparative and Veterinary Medicine

Staphylococcus aureus (Staph.aureus) is the most common contagious pathogen causing intramammary infections (IMI) in cows worldwide. Although practices to control this organism have been advocated for decades, identification of risk factors is crucial in prevention and control of Staph.aureus. The objectives for Chapter 2 and Chapter 3 were to estimate prevalence of Staph.aureus in Ohio dairies and to determine the association of management practices with isolation of Staph.aureus in bulk tank milk (BTM). Questionnaire about herd characteristics, milking procedures, udder health/mastitis control, biosecurity and calf/heifer raising practices were mailed to 780 dairy producers, with a response rate of 49.2%. Staph.aureus prevalence was 48%, 64% and 69% when considering one, two or three BTM samples. Herds with somatic cell count lower than 150,000 cells/mL and milking routine practices such as pre-stripping, pre- and post-dipping and use of single towels per cow were associated with reduced detection of Staph.aureus. Dry-off practices such as abrupt cessation of milking, use of internal teat sealant or blanket dry treatment were associated with herd size. In addition to the presence of Staph.aureus in the infected udder and milk, the cow can also be colonized with the organism on body sites. The objective of the Chapter 4 was to assess the role of teat skin colonization by Staph. aureus in Staph.aureus IMI by evaluating genetic relatedness of Staph.aureus isolates from milk and teat skin of dairy cows using pulsed-field gel electrophoresis (PFGE) and characterizing the isolates based on the carriage of virulence genes. Cows in four known Staph.aureus positive herds were sampled and was found that quarters with teat skin colonized with Staph.aureus were almost five times more likely to be diagnosed with Staph.aureus IMI than quarters not colonized on teat skin. Three main clusters (A, B, C) were identified with PFGE using a cutoff at 80% similarity. All clusters conta (open full item for complete abstract)

Committee: Paivi Rajala-Schultz Dr. (Advisor) Subjects: Veterinary Services

PhD, University of Cincinnati, 2010, Medicine: Pathobiology and Molecular Medicine

Invasive infection by the fungus Aspergillus fumigatus has emerged as a leading cause of death among bone marrow and solid organ transplant recipients. The remarkable ability of A. fumigatus to thrive within the physically and nutritionally hostile conditions of its ecological niche, the compost, has likely translated to its capacity to grow rapidly within the mammalian lung. Accordingly, signal transduction pathways that relay environmental cues to the metabolic and stress responsive machinery are undoubtedly important virulence determinants in this organism, and may prove to be effective targets for novel antifungal therapies. Early studies by our laboratory, and others, have demonstrated the importance of the cAMP-dependent Protein Kinase A (PKA) pathway in A. fumigatus pathogenesis. This pathway becomes activated following an environmentally induced spike in intracellular cAMP, which causes the catalytic subunit (kinase) to be released from the inhibition of the regulatory subunit. The overarching goal of this dissertation work was to better understand the contributions of PKA to A. fumigatus growth and virulence. To do so, a combination of cellular and molecular analyses of mutants deficient in either the regulatory (pkaR) or catalytic (pkaC1) subunit genes was employed. Included in these studies is the first functional characterization of a second, divergent PKA catalytic isoform, PkaC2. The results of these studies have established a central role for PKA in nutrient signaling and utilization in A. fumigatus. For example, the deletion of either the PKA catalytic or regulatory subunit genes resulted in the loss of rapid spore germination in the presence of environmental nutrients. In addition, the PkaC1 and PkaC2 isoforms were shown to act in concert to regulate hyphal proliferation on carbohydrate substrates. More specifically, over-expression of pkaC2 enhanced growth of ΔpkaC1 when glucose or fructose served as the sole carbon source, whereas deletion of pka (open full item for complete abstract)

Committee: Judith Rhodes PhD (Committee Chair); David Askew PhD (Committee Member); Kathryn Wikenheiser-Brokamp MD, PhD (Committee Member); Alan Smulian MD (Committee Member); William Miller PhD (Committee Member) Subjects: Microbiology

PhD, University of Cincinnati, 2008, Medicine : Cell and Molecular Biology

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (MΦ). Studies in human and murine MΦ have demonstrated that the intracellular growth of H. capsulatum yeasts is exquisitely sensitive to the availability of iron. As H. capsulatum produces hydroxymate siderophores, we sought to determine if siderophore production was required for intracellular survival in MΦ, and in a murine model of pulmonary histoplasmosis. UC7 SIDA silenced yeasts and SIDA knockout yeasts (UC8 ΔsidA) grew normally in rich medium, did not synthesize siderophores, and were unable to grow on apotransferrin-chelated medium. The intracellular growth of UC7 SIDA silenced and UC8 ΔsidA yeasts in MΦ was significantly decreased compared to wild type (WT) yeasts, but growth was restored to WT levels by the addition of exogenous iron. Compared to WT (G217B) yeasts, C57BL/6 mice infected with demonstrated significantly reduced growth in the lungs and spleens seven days after infection. Additionally, we sought to identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human MΦ. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron replete medium, but not on iron deficient media. On iron deficient medium, the growth of the vma1 mutant was restored in the presence of wild type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within MΦ was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28°C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostas (open full item for complete abstract)

Committee: Simon Newman PhD (Committee Chair); George Smulian MD (Committee Member); Wallace Ip PhD (Committee Member); Steve Kleene PhD (Committee Member); Gary Dean PhD (Committee Member) Subjects: Biology