VIRTUAL STUDENT SCHOLARS SYMPOSIUM 2020 PRESENTATIONS

Microbiology and Molecular Biology - Poster Presentations

High School Division

Mutant variant of the Unc93B1 gene confers recognition of CpG-A by Toll-like receptor 9
Aimi Sekiguchi, Berkshire School

The innate immune system protects the body by recognizing pathogens and initiating immune responses. Toll-like-receptor 9 (TLR9) is a pathogen receptor localized in in the endolysosome, and it recognizes single-stranded DNA (ssDNA). Studies have shown that ssDNA sensing by TLR9 requires various molecules such as cathepsins, DNase2a, and Unc93B1, but the exact mechanism underlying TLR9 recognition of its ligands is not yet completely understood. Here I show that the Unc93B1 gene from plasmacytoid dendritic cells (pDCs) confers Ba/F3 cell responses to CpG-A, a ssDNA TLR9 ligand. Functional cloning of the cDNA library of pDCs, a CpG-A responsive cell line, has shown that different mutations and a splicing variant of Unc93B1 enhance TLR9 responses to CpG-A. Although further work is necessary to identify the independent functions of each mutant and variant, the comparative studies of TLR9 activation in Ba/F3 cells expressing wt Unc93B1 and mutant Unc93B1 splicing variants suggest previously unknown roles of Unc93B1 in DNA recognition by TLR9.

Undergraduate Division

Investigation of the Role of the Rna Binding Protein CsrA in the Emerging Pathogen Escherichia Albertii
April E Pivonka, Saint Joseph's University

Attaching and effacing (A/E) pathogens are one of the main causes of severe infantile diarrhea and death in developing countries. A/E bacteria induce these effects by means of a type 3 secretion system (T3SS) encoded by the pathogenicity island, locus of enterocyte effacement (LEE). The T3SS connects the bacterial cytosol to the host and enables effectors to be dispatched in the infected host. One such effector is Tir. Tir is integrated in the membrane of the infected host where it functions as a receptor for the protein intimin, located on the outer bacterial membrane. Other effectors destroy intestinal microvilli, diminishing the ability of intestinal cells to absorb water and nutrients, leading to diarrhea. Examples of A/E bacteria include enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Escherichia albertii. E. albertii was first isolated from a diarrheic infant in Bangladesh in 1991. However, the bacterium continues to be mistyped as EPEC or EHEC. Thus, little is known about the bacterium’s virulence. This highlights the necessity of identifying as many unique traits of E. albertii as possible for correct classification.
Previously our lab demonstrated the RNA-binding protein CsrA posttranscriptionally controls gene expression from the LEE in EPEC. CsrA affects mRNA stability, transcriptional elongation, and translation. This study was undertaken to investigate the role of CsrA in regulation of the LEE in E. albertii. First, the csrA gene was deleted in E. albertii using a recombineering-based protocol developed in our lab. Using Western Blotting, we examined the levels of the LEE-encoded Tir protein in the mutant. Our results revealed a decrease in the abundance of Tir in the mutant bacteria, suggesting that crsA is required for its synthesis. We also examined the role of csrA in glycogen biosynthesis. Glycogen production was increased in the csrA mutants compared to the wild type E. albertii, demonstrating that csrA plays a role in suppressing glycogen synthesis. Future experiments are aimed at elucidating the molecular mechanism by which CsrA regulates the LEE and controls glycogen biosynthesis in E. albertii. Our results represent the first report to implicate CsrA in virulence and metabolism in E. albertii - a bacterium that is severely understudied, although an increasing number of reports suggest the bacterium could be a cause of several outbreaks which were incorrectly attributed to other A/E pathogens. These results are critical for developing clinical measures to counteract this emerging pathogen.
References 1. Bhatt S, Edwards AN, Nguyen HT, Merlin D, Romeo T, Kalman D. The RNA binding protein CsrA is a pleiotropic regulator of the locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli. Infect Immun. 2009;77(9):3552–3568. doi:10.1128/IAI.00418-09 2. Egan M, Ramirez J, Xander C, Upreti C, Bhatt S. Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii. Biol Proced Online. 2016;18:3. Published 2016 Feb 3. doi:10.1186/s12575-015-0032-8 3. Romeo T, Gong M, Liu MY, Brun-Zinkernagel AM. Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties. J Bacteriol. 1993;175(15):4744–4755. doi:10.1128/jb.175.15.4744-4755.1993

The RNA Chaperone ProQ Regulates Virulence and Motility in Enteropathogenic Escherichia coli
Emily E Costello, Saint Joseph's University

Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that forms A/E lesions on the surface of infected intestinal cells and destroys their microvilli, decreasing the ability of these cells to absorb nutrients and water, which leads to diarrhea. The locus of enterocyte effacement (LEE) encodes the proteins for a type 3-secretion system (T3SS). The T3SS allows for a conduit to form between the bacterium and the host cell so the bacterial cell can secrete the translocated intimin receptor (Tir) into the host cell. Tir then associates with intimin on the bacterial cell which allows for A/E lesion formation.

Thus, understanding the regulation of the T3SS is important for developing any effective clinical approaches to counteract the bacterium. Previously, our lab showed that the RNA chaperone protein Hfq modulates the virulence of EPEC. This study was undertaken to investigate the role of another cryptic RNA chaperone, ProQ, in regulation of the LEE. ProQ enhances the binding of small regulatory RNAs (sRNAs) to their target mRNAs which can affect transcriptional elongation, mRNA stability, and/or translation. We engineered a proQ deletion strain of EPEC by lambda red recombineering and compared its LEE gene expression profile and motility with respect to the unmutagenized wild type parent strain. Our results suggest that deletion of proQ leads to an increase in the synthesis of Tir. Thus, ProQ appears to be a negative regulator of the LEE. Conversely, the proQ mutant exhibited decreased motility, suggesting that ProQ is required for bacterial motility. To the best of our knowledge, this is the first study to implicate ProQ in motility and virulence of EPEC.

Future studies will be aimed at investigating the mechanism by which ProQ regulates the LEE and motility. Our study will enable us to explore the complexity of the riboregulatory landscape in EPEC. Moreover, our results will also aid in the development of effective therapies to combat infections by A/E pathogens.

Distinct Roles for Xenopus laevis AHR Paralogs in Cell Cycle Regulation
Ali L Fox, Kenyon College

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates toxicity of dioxin-like compounds. In the absence of xenobiotics, AHR also plays roles in development and physiology. These include promotion of cell cycle progression through the G1/S checkpoint. The frog Xenopus laevis has two AHR paralogs, AHR1α and AHR1β . We recently generated mutant derivatives of the XLK-WG cell line, each lacking a functional version of either AHR1α or AHR1β, and showed that the two receptors mediate distinct transcriptional responses to specific agonists. This study tested the hypothesis that each frog AHR plays a distinctive role in the regulation of cell cycle progression. Using the water-soluble formazan (WST-8) method, we found that AHR1α ^-/-cells displayed significantly lower levels of proliferation than XLK-WG and AHR1β ^-/- . Cell cycle distribution of each line was determined using propidium iodide staining and flow cytometry. AHR1β ^-/- and wild type lines exhibited similar distributions of cells in the various phases. By comparison, AHR1α^-/- cells had diminished S and G2/M phase populations, consistent with attenuated G1/S checkpoint progression. This may indicate a distinctive role for AHR1α in cell cycle regulation. Additionally, a discrete sub-G1 population was unique to AHR1α ^-/- cells. This population did not represent cellular debris or apoptotic cells, and the implications of this sub-G1 phase is an area of ongoing investigation. Most vertebrates have multiple AHRs. This study contributes to understanding each receptor and how their expression affects interpretation of data from non-mammalian toxicological models. [NIH: R15 ES011130]

Unraveling Analgesics Metabolizing Bioprospects Using Functional Metagenomics
Cristian Sanlatte, University of Puerto Rico Mayagüez

Over the counter drugs have become one of the most reliable and fastest solutions to relieve common muscle pain and inflammations. For example, drugs such as ibuprofen (IBU) and paracetamol (PA) are highly used within the pharmacological industry, hospitals, and the common household. However, its overuse has placed it as an environmental threat to aquatic environments. Furthermore, the presence of these compounds have an adverse effect on the aquatic biodiversity evidenced by cases of oxidative stress damage. Therefore, as a means of removing these contaminants, this research aims to search for bioprospects capable of metabolizing the active compound of IBU or PA using culture independent techniques. Centralia, Guajataca reservoir and Guánica’s Dry Forest metagenomic libraries were screened by growing metagenomic clones in M9 medium with IBU or PA as its sole Carbon source. The plates were incubated at 37°C and candidate colonies were scored daily for 5 days. A total of twenty-eight potential analgesics metabolizing bioprospects (PAMB) were isolated. The growth of seven PAMB was reconfirmed using optical density in M9 liquid media supplemented with IBU or PA. From the PAMB screened, six candidates showed considerable growth under the conditions tested. These where evaluated through molecular analysis to confirm the presence of a fosmid. Current work is in progress to identify its metagenomic fragment and perform mutagenesis experiments that validate the candidate’s ability to use the analgesics as its carbon source. Considering that IBU and PA are currently emerging contaminants, these PAMB represent an alternative method towards bioremediating these contaminants.

Graduate Division

Using Recombination-Dependent Lethal Mutations to Stabilize Reporter Flaviviruses
Coleman Baker, University of Texas Medical Branch

Members of the Flavivirus genus cause widespread disease; and despite concerted efforts, medical countermeasures are insufficient. Infectious reporter viruses are invaluable tools in furthering these efforts. However, the stability of reporter flaviviruses has been challenged by viral RNA recombination, leading to deletion of the engineered reporter gene during viral replication. The instability of reporter viruses has limited their application to research and countermeasure development. Thus, new approaches to overcome the instability of reporter flaviviruses are critically needed to advance the flavivirus field. To create a stable flavivirus bearing a full-length luciferase gene, we engineered mutations that are rendered virus-lethal upon recombination. Flaviviruses require a threshold of positive charges in the N-terminus of the capsid for infectious virus to be produced. Using this information, we engineered charge-reversing mutations in the capsid portion upstream of the reporter gene. Upon recombination, these changes become part of the full capsid, where they are lethal for viral particle formation. Thus, only non-recombined reporter virus propagates. We tested this strategy using ZIKV bearing NanoLuc and passaged both virus with and without capsid mutations. The recombination-dependent lethal mutations succeeded in stabilizing the NanoLuc ZIKV through ten passages, while WT reporter virus showed early instability at five passages. These results, assayed by RT-PCR, were corroborated by sequencing, focus forming assay, and luciferase assay. This method was also adapted to a NanoLuc YF17D virus with similar successful results. This strategy overcomes the shortfalls of traditional reporter flaviviruses, opening new doors to better disease diagnosis, drug screening, and trans-gene delivery.

VIRTUAL STUDENT SCHOLARS SYMPOSIUM 2020 PRESENTATIONS

Microbiology and Molecular Biology - Poster Presentations

High School Division

Undergraduate Division

Graduate Division

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