Microbiology Commons^™

Vaccination Against Onchocerca Volvulus Induces Igg-Mediated Protective Immunity Dependent On Neutrophils And Complement, Nathan M. Ryan, Jessica A. Hess, Mohini Nakhale, Annabel A. Ferguson, William Stump, Sara Belko, Rachel Monane, Robert S. Pugliese, Nikolai Petrovsky, Benjamin L. Makepeace, Sean A. Gray, Darrick Carter, Sara Lustigman, David Abraham

Department of Microbiology and Immunology Faculty Papers

Onchocerciasis remains a significant cause of morbidity and economic loss in sub-Saharan Africa. Despite the existence of effective therapeutics, a prophylactic vaccine targeting the etiologic agent, Onchocerca volvulus, is needed to control ongoing disease and transmission. Mice were vaccinated against O. volvulus with a fusion of the recombinant antigens Ov-103 and Ov-RAL-2 (Ov-FUS-1) with Advax-CpG adjuvant. Immunized mice developed protective immunity with the killing of third-stage larvae (L3) within 36 h of challenge infection. IgG from immunized mice passively transferred protective immunity to naïve mice, indicating that antigen-specific IgG mediated parasite elimination. Neutrophils were the most abundant subset of immune …

Analyzing Efficiency Of A Lentiviral Shrna Knockdown System In Human Enteroids Using Western Blot And Flow Cytometry, Adam P Wilson, Karni S Moshal, Addison P Franca, Sasirekha Ramani, Randle Gallucci, Hala Chaaban, Kathryn Y Burge

Faculty, Staff and Students Publications

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

Characterization Of Host Cell Specific Interactions During Spotted Fever Group Rickettsiae Infection, Emerson Towey

Graduate Theses, Dissertations, and Problem Reports (ETD)

Vector-borne diseases have been increasing over the past few years due to changes in climate and disturbances in the vectors’ natural environment. To mitigate the risks of these diseases, it is necessary to understand pathogen biology and how it interacts with a host to cause disease. Here we focus our attention on the tick-borne pathogen, Rickettsia rickettsii. R. rickettsii is a member of the spotted fever group (SFG) Rickettsiae and is the etiological agent of Rocky Mountain spotted fever (RMSF), a fatal human disease that is lacking an effective vaccine. Members of the bacterial genus Rickettsia are obligate intracellular endoparasites …

Multiple Rsv Strains Infecting Hep-2 And A549 Cells Reveal Cell Line-Dependent Differences In Resistance To Rsv Infection, Anubama Rajan, Felipe-Andres Piedra, Letisha Aideyan, Trevor Mcbride, Matthew Robertson, Hannah L. Johnson, Gina Marie Aloisio, David Henke, Cristian Coarfa, Fabio Stossi, Vipin Kumar Menon, Harshavardhan Doddapaneni, Donna Marie Muzny, Sara Joan Javornik Cregeen, Kristi Louise Hoffman, Joseph Petrosino, Richard A. Gibbs, Vasanthi Avadhanula, Pedro A. Piedra

Research Symposium

Background: Respiratory syncytial virus (RSV) is the major viral driver of a global pediatric respiratory disease burden disproportionately borne by the poor¹. Thus, RSV, like SARS-CoV-2, combines with congenital and environmental and host-history-dependent factors to create a spectrum of disease with greatest severity most frequently occurring in those least able to procure treatment.

Methods: Here we apply whole genome sequencing and a suite of other molecular biological techniques to survey host-virus dynamics in infections of two distinct cell lines (HEp2 and A549) with four strains representative of known RSV genetic diversity.

Results: We observed non-gradient patterns of RSV …

Modeling Nonsegmented Negative-Strand Rna Virus (Nnsv) Transcription With Ejective Polymerase Collisions And Biased Diffusion, Felipe-Andres Piedra

Research Symposium

Background: The textbook model of NNSV transcription predicts a gene expression gradient. However, multiple studies show non-gradient gene expression patterns or data inconsistent with a simple gradient. Regarding the latter, several studies show a dramatic decrease in gene expression over the last two genes of the respiratory syncytial virus (RSV) genome (a highly studied NNSV). The textbook model cannot explain these phenomena.

Methods: Computational models of RSV and vesicular stomatitis virus (VSV – another highly studied NNSV) transcription were written in the Python programming language using the Scientific Python Development Environment. The model code is freely available on GitHub: …

Overexpressing Two Helicobacter Pylori Small Rnas From A Bacterial Pathogenicity-Related Chromosomal Region To Investigate Their Regulation Of Virulence Genes, Roxanne N. Mcpeck, Olivia F. Morgan, Andrea R. Castillo Phd

2023 Symposium

The bacterial pathogen Helicobacter pylori infects the stomachs of approximately 50% of humanity, causing symptomatic disease (e.g., stomach ulcers, gastric cancer, and MALT lymphoma) in 10-15% of the infected. Colonizing the acidic, inhospitable stomach requires H. pylori to tightly regulate gene expression despite lacking many common bacterial genetic regulatory elements. The pathogen may compensate by using abundant non-protein-coding small RNAs (sRNAs) to regulate gene expression, including of infection-intensifying virulence genes. Additionally, severe disease and cancer correlate with infection by H. pylori strains that contain a nonessential chromosomal region, the cytotoxin-associated gene pathogenicity island (cagPAI). This encodes powerful virulence …

Protocol To Identify The Core Gene Supported By An Essential Gene In E. Coli Bacteria Using A Genome-Wide Suppressor Screen, Isao Masuda, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

We describe here a genome-wide screening approach to identify the most critical core reaction among a network of many that are supported by an essential gene to establish cell viability. We describe steps for maintenance plasmid construction, knockout cell construction, and phenotype validation. We then detail isolation of suppressors, whole-genome sequencing analysis, and reconstruction of CRISPR mutants. We focus on E. coli trmD, which encodes an essential methyl transferase that synthesizes m1G37 on the 3'-side of the tRNA anticodon. For complete details on the use and execution of this protocol, please refer to Masuda et al. (2022).

Proposing An Rna Interference (Rnai)-Based Treatment For Human Immunodeficiency Virus (Hiv) By Analyzing The Post-Transcriptional Gene Targeting Of Sars-Cov-2, Hepatitis C Virus, And A549 Lung Cancer Cells, Arjun Jagdeesh

Undergraduate Research Posters

Human Immunodeficiency Virus (HIV) is a retrovirus that infects CD4+ T cell lymphocytes in humans, leading to the development of Acquired Immunodeficiency Syndrome (AIDS) if left untreated. While current treatment methods, including antiretroviral combination treatments, effectively limit HIV replication, HIV can evade these treatments due to its high mutation rate. Long-term antiretroviral treatment can also be toxic to patients, meaning patients would benefit from a new mechanism of HIV treatment. RNA interference (RNAi) is an antiviral pathway found in mammals, plants, and insects that involves a small-interfering RNA that is incorporated into a protein complex called the RNA-induced Silencing Complex …

Understanding The Molecular Mechanism Of Ilv1 Stress Sensitivity In Yeast By Examination Of Differential Patterns Of Gene Expression Between Wild-Type And Ilv1 Knockout Strains Of Saccharomyces Cerevisiae, Emily Schuster

Honors Theses

As arguably the most widely used industrial eukaryote, Saccharomyces cerevisiae has become one of the many model organisms used to study molecular genetics. A recent unpublished study at the University of Tennessee - Chattanooga showed that yeast cells that had the ILV1 gene knocked out had reduced survivability under stress conditions. This study then focused on the role of ILV1 in the stress response of cells by measuring the expression levels of 11 candidate genes that are all involved in some critical pathway in the yeast to see how gene expression was influenced as a result of the loss of …

Factors Affecting The Antibacterial Activity Of Cefiderocol On Klebsiella Pneumoniae With Diverse Characteristics, Lana Jamal Rashed Daoud

Theses

Antimicrobial resistance (AMR) is a growing serious threat that poses a burden on the healthcare system. Spread of AMR bacteria limits treatment options; thus, new antimicrobial agents are required. Cefiderocol (CFDC) is a novel siderophore cephalosporin, approved by the FDA in 2019 for use in treating infections caused by multi-drug resistant (MDR) pathogens; but it is not yet in clinical use in the UAE. CFDC is equipped with stabilizers against β-lactamases as well as a siderophore side chain that allows it to penetrate the outer membrane of bacteria through the iron uptake pathways. The aim of this study is to …

Periodic Spatial Disturbances Alter The Expression Of Quorum Sensing Virulence Factors In Pseudomonas Aeruginosa, Laura García-Diéguez

All HCAS Student Capstones, Theses, and Dissertations

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe acute and chronic illnesses. Current antibiotic-based approaches fail to effectively treat P. aeruginosa infections due to the effectiveness and robustness of the quorum sensing signaling system (QS). Pathogenic bacteria, such as P. aeruginosa, employ this population density-dependent communication mechanism to confer antimicrobial resistance, propagate infection, and coordinate the expression of virulence factors, through the production and detection of autoinducing signaling molecules (AI). As such, there is a growing interest in developing novel non-antibiotic-based techniques to attenuate the pathogenicity of P. aeruginosa by disrupting the functionality of its QS system. Previous …

Mapk-Mediated Transcription Factor Gatad Contributes To Cry1ac Resistance In Diamondback Moth By Reducing Pxmalp Expression, Le Guo, Zhouqiang Cheng, Jianying Qin, Dan Sun, Shaoli Wang, Qingjun Wu, Neil Crickmore, Xuguo Zhou, Alejandra Bravo, Mario Soberón, Zhaojiang Guo, Youjun Zhang

Entomology Faculty Publications

The benefits of biopesticides and transgenic crops based on the insecticidal Cry-toxins from Bacillus thuringiensis (Bt) are considerably threatened by insect resistance evolution, thus, deciphering the molecular mechanisms underlying insect resistance to Bt products is of great significance to their sustainable utilization. Previously, we have demonstrated that the down-regulation of PxmALP in a strain of Plutella xylostella (L.) highly resistant to the Bt Cry1Ac toxin was due to a hormone-activated MAPK signaling pathway and contributed to the resistance phenotype. However, the underlying transcriptional regulatory mechanism remains enigmatic. Here, we report that the PxGATAd transcription factor (TF) is responsible for the …

Assesment Of Antibiotic Resistant Gene Expression In Clinical Isolates Of Pseudomonas Aeruginosa, Dustin Esmond

Biology Theses

Increasing prevalence of nosocomial infections by antimicrobial resistant pathogens resulting in higher mortality rates and financial burden is of great concern. Pseudomonas aeruginosa represents one of six highly virulent “ESKAPE” pathogens that exhibit considerable intrinsic drug resistance as well as mechanisms for acquiring further resistance. As many of these mechanisms are regulated through gene expression, we sought to identify regulatory strategies and patterns at play in 23 clinical isolates collected from Baku, Azerbaijan and Tyler, Texas, USA. Real-time quantitative polymerase chain reaction was performed on six gene targets implicated in resistance and contrasted with antibiotic phenotypes. We found AmpC cephalosporinase …

Lipoxin A 4 (Lxa 4 ) Promotes Reduction And Antibiotic Efficacy Against Pseudomonas Aeruginosa Biofilm, Julianne M. Thornton, Jean Walker, Prem Yugandhar Kadiyam Sundarasivarao, Bernd Spur, Ana Rodriguez, Kingsley Yin

Rowan-Virtua Research Day

Pseudomonas aeruginosa (P aeruginosa) is an opportunistic bacterium commonly found in wound infections and airways of cystic fibrosis patients P aeruginosa readily forms biofilms which can reduce the efficacy of antibiotics used to eradicate the pathogen We have previously shown that a Specialized Pro resolving Mediator ( Lipoxin A 4 (LxA 4 is a quorum sensing inhibitor which can reduce P aeruginosa virulence In this study, we examined the direct actions of LxA 4 and RvD 2 on P aeruginosa biofilm formation and virulence gene expression The influence of LxA 4 on antibiotic efficacy and the combined effects on biofilm …

The Role Of Med31 And Med12 In Directing Adipogenesis Of Human Adult-Derived Mesenchymal Stem Cells, Joseph Straub

Master's Theses

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multisubunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules: head, middle, tail, and kinase. In our introduction, we show the state of the field of Mediator study with a focus on the critical kinase module. In the following chapters, we used siRNA knockdowns to investigate the roles of the highly-conserved core subunit MED31 and the kinase module …

Disruption Of Cellular P-Bodies During An Adenovirus Infection​, La`Quita Randolph, Kevin Neubrecht, Kasey A. Karen

Graduate Research Showcase

Adenovirus has made major contributions in medicine by serving as a model DNA virus to study other viruses, such as human papillomavirus (HPV). Adenoviruses are a diverse family of nonenveloped, double-stranded DNA viruses that are ubiquitous to animals and humans. There are over 67 serotypes of human adenoviruses that can cause a variety of illnesses including, gastroenteritis, conjunctivitis and respiratory infections. Adenovirus can cause these infections by invading host cells and producing an environment that is favorable for viral replication. During the early phases of infection, adenovirus expresses various viral proteins such as E4 11k, which has multiple functions. One …

The Role Of Conserved Poly(A) Sequences In The 3'Utr Of Zika Virus Gene Expression, Kristen Kaytes

Legacy Theses & Dissertations (2009 - 2024)

Following its discovery in 1947, Zika virus garnered little attention in the global stage until 2015, when a rapidly spreading epidemic brought the virus, and its devastating clinical manifestations to light. Zika virus pathologies include Congenital Zika Syndrome, a known causative of microcephaly, as well as spontaneous miscarriages and the development of Guillain Barré syndrome. Despite recent advances in research, the molecular biology of Zika virus is largely unknown.

Novel Small Rnas Expressed By Bartonella Bacilliformis Under Multiple Conditions Reveal Potential Mechanisms For Persistence In The Sand Fly Vector And Human Host, Shaun Wachter, Linda D. Hicks, Rahul Raghavan, Michael F. Minnick

Biology Faculty Publications and Presentations

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We …

Environmental Influences On Expression Of Virulence- And Survival-Associated Genes And Epigenetic Modifications Of Dna In Vibrio Vulnificus, James W. Conrad

USF Tampa Graduate Theses and Dissertations

Vibrio vulnificus is an autochthonous inhabitant of warm, brackish coastal waters and is an opportunistic pathogen that causes the highest mortality of all seafood-related illnesses (1). These fatal infections are generally caused by the clinically-associated vcgC/16S rRNA type B genotypes within the biotype I group (2–5). However, the reasons for the heightened infectiousness of the clinically-associated strains over environmentally-associated ones remains elusive, as no unique clinically-associated virulence genes have been identified through genomic sequencing or other strategies. DNA methylation may contribute to regulation of virulence by affecting gene transcription, and was investigated in the highly virulent V. vulnificus strain CMCP6. …

Direct Binding Of Tfeα Opens Dna Binding Cleft Of Rna Polymeras, Sung-Hoon Jun, Jaekyung Hyun, Hoyoung Kim, Michael S. Bartlett, Hyun-Soo Cho, Katsuhiko S. Murakami

Biology Faculty Publications and Presentations

Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAPTFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix …

A Mini-Tn5-Derived Transposon With Reportable And Selectable Markers Enables Rapid Generation And Screening Of Insertional Mutants In Gram-Negative Bacteria, Eric S. Nazareno, Bimala Acharya, C. Korsi Dumenyo

Agricultural and Environmental Sciences Faculty Research

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The …

Molecular Characterisation Of Hsp70 Gene Expression, Histopathology And Immune System Of Snakehead Fish, Channa Striata, Under Temperature And Bacterial Stress, Eid Elhussein Ihlam Ibrahim

Student Works (2020-2029)

Snakeheads are air-breathing freshwater fishes containing two genera, Channa (Asia) and Parachanna (Africa). The Asian genus is dominated by Channa striata which is found in southern and Southeastern parts of the continent. In this research, environmental and bacteria stressor to Channa striata were investigated. HSP70 gene was studied as an environmental stress gene (ChHSP70). Results showed a cDNA sequence of the gene to be (~918bp). It was also shown the gene is highly similar to that of other fish species. Constructing a phylogenetic tree of the gene across vertebrate species revealed six main clusters; same class remained conserved by showing …

Gene Expression Analysis Of Effectors Involved In Early Interaction Of Banana And Fusarium Oxysporum F. Sp. Cubense, Nee Kiew Poon

Student Works (2020-2029)

Banana is a popular but disease-laden fruit crop. It is very susceptible to various phytopathogens such as viruses, bacteria, fungi and nematodes. A particularly infectious and economically important fungal disease in banana is Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). The disease has been plaguing the banana industry since late 19th century. Despite the early discovery of this phytopathogen of banana, little is known on its the mechanism of pathogenicity. In other plants, fungal effectors have been found to function in manipulating plant mechanisms during infection and many have been identified and characterized. In this study, the …

Neurodegenerative Modeling: Tau Protein, Degradative Pathways, And Gene Expression Profiling Of Human Ipsc-Derived Neural Precursors And Differentiated 3-D Neural Sphere Versus 2-D Monolayer Cultures, Kyle H. Anthoney

Cal Poly Humboldt theses and projects

Human induced pluripotent stem cells offer a model for human brain development and disease by differentiation into brain organoids; however, current neural culture systems lack the microenvironment, neuronal circuits and connectivity, vascular circulation, and immune system that exist in vivo. After differentiation and development of neuronal and non-neuronal cell types within two formats of cell cultures, we can visualize and recapitulate in vivo protein accumulation, gene expression, and degradative processes such as autophagy. Using RNA extraction, purification methods and reverse transcription I compared traditional monolayer cultures and novel 3-D neural sphere cultures via gene expression analysis. This analysis indicated …

Rhodococcus Opacus Pd630 Genetic Tool Development To Enable The Conversion Of Biomass, Drew Michael Delorenzo

McKelvey School of Engineering Graduate Student Theses & Dissertations

The discovery of fossil fuels facilitated a new era in human history and allowed many firsts, such as the mass production of goods, the ability to travel and communicate long distances, the formation of population dense cities, and unprecedented improvements in quality of life. Alternative sources of energy and chemicals are needed, however, as hydrocarbon reserves continue to deplete and the effects of burning fossils on the planet become better understood. Lignocellulosic biomass is the most abundant raw material in the world and a viable alternative to petroleum-derived products. The pre-treatment of lignocellulose (e.g., thermocatalytic depolymerization, enzymatic hydrolysis, pyrolysis, etc.) …

The Role Of Multidrug Resistance Regulators Mara, Soxs, Rob And Rama In Regulating Virulence Traits In Salmonella Enterica, Srinivas Thota

Dissertations

Enteric pathogens sense numerous signals specific to the anatomical location in the intestine and integrate them with the complex regulatory networks to temporally and spatially regulate their virulence genes. MarA, SoxS, Rob and RamA are homologous transcription factors that belong to AraC family of proteins in Salmonella enterica that primarily were thought to be involved in rendering antibiotic resistance to bacteria by up regulating efflux pumps and down regulating outer membrane porins. The fact that these transcription factors respond to the same intestinal compounds that regulate virulence genes in Salmonella motivated us to look for other roles of these transcription …

Characterization Of Early Biofilm Formation And Physiology In Neisseria Gonorrhoeae, Kelly Eckenrode

Dissertations, Theses, and Capstone Projects

Many bacteria rely on the dynamics of their extracellular appendages to perform important tasks, like motility and biofilm formation. Interestingly, these dynamics have been linked to physiological responses in some pathogenic bacteria; therefore, it is important to understand more about the role of physical forces in bacteria. I used the causative agent of the human disease gonorrhea, Neisseria gonorrhoeae, as a model system to study the role of physical force on early biofilm formation. The advantage of this system is that cell-cell interactions are controlled by extracellular filaments called type IV pili (tfp). Tfp is composed of monomers that give …

Post-Translational Modifications And Functional Studies Of Dksa In Escherichia Coli, Andrew Charles Isidoridy

Legacy Theses & Dissertations (2009 - 2024)

DksA is a bacterial gene regulator that functions synergistically with the stress alarmone ppGpp to mediate the stringent response. DksA also functions independently of ppGpp to regulate transcription of a number of genes. DksA function is dependent on its binding affinity to RNA polymerase and requires specific interactions between RNAP and catalytic amino acids located on the coiled coil tip, D74 and A76. While much of the previous work on DksA has focused on understanding the mechanisms of action and the numerous gene targets for transcriptional regulation, little is known about the mechanisms by which DksA expression and function may …

Transcriptional Regulation Of Dksa In E. Coli, Daniel Thomas Woods

Legacy Theses & Dissertations (2009 - 2024)

DksA is a global transcription factor that binds RNAP directly to regulate the expression of many genes and operons, including ribosomal RNA, in a ppGpp-dependent or ppGpp–independent manner. It is also involved in facilitating the process of DNA replication by removing stalled transcription elongation complexes that could block the progress of the replication fork. In addition, DksA is important for colonization, establishment of biofilms, and pathogenesis. In order to sustain these various functions, an adequate level of cellular DksA is required. This work tested the hypothesis that the E. coli dksA is substantially regulated at the level of transcription. Using …

Gesture: An Online Hand-Drawing Tool For Gene Expression Pattern Search, Chunyan Wang, Yiqing Xu, Xuelin Wang, Li Zhang, Suyun Wei, Qiaolin Ye, Youxiang Zhu, Hengfu Yin, Manoj Nainwal, Luis Tanon-Reyes, Feng Cheng, Tongming Yin, Ning Ye

Molecular Biosciences Faculty Publications

Gene expression profiling data provide useful information for the investigation of biological function and process. However, identifying a specific expression pattern from extensive time series gene expression data is not an easy task. Clustering, a popular method, is often used to classify similar expression genes, however, genes with a ‘desirable’ or ‘user-defined’ pattern cannot be efficiently detected by clustering methods. To address these limitations, we developed an online tool called GEsture. Users can draw, or graph a curve using a mouse instead of inputting abstract parameters of clustering methods. GEsture explores genes showing similar, opposite and time-delay expression patterns with …