Nine undergraduate students presented in the 2017 Undergraduate Microbiology Research poster session on April 20, 2017, they include the following:

Aissa Kergna (Okeoma lab)
Evan Lamb (McCarter lab)
Gabriela Lozano Flores (McCarter lab)
Juan P. Munoz (Maury lab)
Shondra Olson (Roller lab)
Vishal Perera (Okeoma lab)
Breanna Riesberg-Kramer (Klingelhutz lab)
Samantha Ryken (Roller lab)
Carly Twigg (Roller lab)

Congratulations to undergraduate student Juan P. Munoz, he is pictured with Professor Wendy Maury.  Munoz is the winner of the Allen J. Markovetz Award for best poster presentation at this year's undergraduate poster session. He was awarded the Stinski undergraduate research fellowship for the 2016-2017 academic year. Munoz graduates in May and plans to attend dental school. 

Read the following selected research descriptions from the participants.

Breanna Kramer-Riesberg 

Ebola Virus Infection of Skin as a Mechanism of Transmission   Breanna Kramer-Riesberg1, Francoise Gourrounc1, Marek Sliwinski2, Olena Shtanko3, Kai Rogers1, Jake Dillard1, Robert Davey3, Wendy Maury1, Aloysius Klingelhutz1   1Department of Microbiology, University of Iowa, Iowa City, IA 52242; 2Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614; 3Texas Biomedical Research Institute, San Antonio, TX 78245

The recent Ebola virus (EBOV) outbreak provided evidence for skin being a route of transmission of the virus. Yet, the skin cell populations that are susceptible to and support EBOV replication are unknown. We hypothesize that EBOV infects skin cells and that this is important for transmission and/or pathogenesis of the virus. In collaboration with a BSL4 facility, it was demonstrated that EBOV-GFP virus (identical to wild-type virus except with expression of GFP) productively infected human skin explants. To begin to address what specific cell types in skin support EBOV, we are using a BSL2 replication competent recombinant virus, EBOV GP-rVSV, to infect different skin cell types that are found in the different layers of skin. We found that human adult skin keratinocytes (epidermis) were readily infectable by EBOV GP-rVSV with notable differences in susceptbility depending on the culture media used. We also tested human subcutaneous undifferentiated preadipocytes and differentiated adipocytes (hypodermis) for infection by EBOV GP-rVSV. Only the differentiated adipocytes were readily infectable by the virus, suggesting subcutaneous adipocytes may be targets for EBOV infection. To begin to address how the virus is getting into susceptible cells, we are currently assessing what known EBOV receptors are expressed on these cells. We are also using receptor inhibitors, such as phosphotidylserine, and receptor antibodies to identify what receptors are being utilized by EBOV. These studies will be important for determining how EBOV is transmitted through skin and could lead to better strategies to stop the spread of EBOV.

Evan Lamb

Bacteria, like all other organisms, must seek out preferred conditions to survive. Therefore, many bacteria have developed means of motility to travel from one place to another, and a system of chemotaxis to sense and move towards or away from compounds. Vibrio parahaemolyticus, a marine bacterium and human pathogen, engages in socially directed surface motility known as ‘swarming’. This process is regulated by a cell-cell signaling molecule (the S signal). The S signal has been shown to modulate swarming gene expression, and we hypothesize it also directs swarming behavior by serving as a chemoattractant. Candidate chemoreceptors for the S signal were identified through bioinformatics and knock-out mutant strains were constructed. The mutants were then characterized with respect to swarming behavior (time of onset, rate of colony expansion, and competition races with wild type). The chemoreceptor mutants outcompeted the wild type (WT) strain in swarming competition assays. Ectopic expression of the chemoreceptors repressed swarming in mutants and WT strains. Expression of a control, unrelated chemoreceptor did not affect swarming. These results suggest that the S signal can serve as a social recruitment molecule. Future work will further quantify chemotaxis towards the S signal in WT and mutant strains and examine the role of S signaling during surface colonization.

Gabriela Lozano Flores

Peptidoglycan transmits the surface sensing signal to induce swarming in bacteria    Gabriela Lozano, Mahliyah Adkins-Threats, Maria Liana Morabe, and Linda McCarter  University of Iowa, Microbiology Department

The ability to adapt and grow on surfaces is an essential survival mechanism for many bacteria. When the Gram-negative marine bacterium Vibrio parahaemolyticus senses a surface it differentiates into a swarmer cell, which is capable of moving over surfaces. The polar flagellum, which is used for swimming motility, also serves as the surface sensor and interference with polar flagellar rotation results in induction of the swarmer cell program and production of a second surface-adapted motility system. Stators act as force generators for flagellar rotation. Since the stators interact with peptidoglycan, we hypothesize that peptidoglycan transmits the surface sensing signal detected by the polar flagellum. To investigate the role of peptidoglycan in this pathway, full gene and transpeptidase domain deletion mutations were introduced into mrcA, which encodes a bifunctional peptidoglycan synthetic enzyme with transglycosylase and transpeptidase activities. The resulting mrcA mutants were characterized and complemented. The mutants were sensitive to cell stressors such as bile and deoxycholate, consistent with making impaired peptidoglycan. On surfaces, the mutants failed to induce the swarmer cell program of gene expression, although their health was not generally impaired since they were capable of expressing capsule genes similar to wild type. Thus, alterations in peptidoglycan biosynthesis affect surface sensing signal transmission. Future work will explore the role of peptidoglycan in this signal transduction pathway and examine downstream signaling partners.

Shondra Olson

Herpes Simplex virus (HSV) is capable of spreading from an infected cell directly to an uninfected adjacent cell through cell-to-cell spread. UL51 has been identified as an important factor in cell-to-cell spread. Because of an overlap between the 5’ end of UL51 and the promoter of UL52, no complete deletion of UL51 has been made, however, cell-to-cell spread defects have been seen in partial deletions of UL51 and UL51 point mutants. UL51 partial deletions have also been observed to exhibit an accelerated cytopathic effect (CPE). An important part of cell-to-cell spread is the trafficking of newly formed virions to cell-to-cell junctions. We hypothesized that the accelerated rounding up of infected cells causes there to be fewer connections between adjacent cells earlier than in a wild-type infection, leading to decreased cell-to-cell spread in UL51 mutants. In order to assess if there is a relation between the degree of CPE and cell-to-cell spread efficiency, we developed an assay that allowed us to quantitate CPE by measuring empty space around cells. The assays were carried out using Δ73-244 and Δ167-244 UL51 partial deletions and UL51 point mutants. We found that UL51 partial deletions showed significantly greater areas of open space when compared to wild-type HSV, as did some UL51 point mutants. These results, when compared with plaque-sized assays of the same UL51 mutations, suggest that the accelerated CPE could be the cause of the reduced cell-to-cell spread observed.

Samantha Ryken

Characterization of Herpes Simplex Virus 1 UL51 Protein Self-interaction and Its Role in Cell-to-Cell Spread    Samantha Ryken and Richard Roller

Following reactivation from latency herpesviruses utilize the conserved ability of cell-to-cell spread to evade the host immune response and mediate disease and viral shedding. The UL51 protein of herpes simplex virus 1 is a conserved tegument protein that functions in virus assembly in the cytoplasm. Partial deletions of UL51 are associated with strong cell-to-cell spread defects. Since UL51 does not function as an enzyme its role in cell-to-cell spread is likely dependent on specific interactions with other proteins. It has been shown to form complexes with UL7, gE and itself. However, it is unknown whether the self-interaction is a direct interaction or an interaction mediated by another component. In order to determine whether the UL51 self-interaction requires other viral proteins, we are establishing a co-immunoprecipitation assay using plasmid expression of differentially tagged UL51 proteins. We expect that determining the mechanism of interaction that UL51 has with itself will allow mapping of regions important for that interaction and determination of the significance of the self-interactions for cell-to-cell spread.

Carly Twigg

Herpesviruses replicate, express, and package their genomes in the cell nucleus. They must move DNA-containing capsids from the nucleus to the cytoplasm to complete assembly and then exit the cell. They do this by a process called nuclear egress in which capsids become enveloped at the inner nuclear membrane and then de-envelope at the outer nuclear membrane. This process is accomplished by a complex of two conserved proteins, pUL34 and pUL31. Mutation of a charge cluster, CL13, changes arginine residues in UL34 to alanines. The CL13 mutation causes a severe growth defect that is correlated with promiscuous budding of the inner nuclear membrane. The CL13 growth phenotype can be suppressed by two classes of mutations. Mutations in the first class occur in the gene encoding pUL31, and mutations in the second class occur in another as yet unmapped gene that may encode a negative regulator of budding. Two mutants in the second class were found to largely suppress the CL13 growth phenotype. The genomes of these two suppressors and the parental virus were sequenced to identify suppressor-specific changes. They were found to contain one common SNP – a frameshift in UL35. One of these suppressor viruses also forms plaques significantly larger than the parent and this is correlated with a SNP in the intergenic region between US8 and US9.