2012 Summer Scholar Participants-HHMI Undergraduate Program - Carnegie Mellon University

2012 HHMI Summer Scholar Participants

Abigail Simmons

Abigail Simmons, Carnegie Mellon University

Mentor: Aaron Mitchell

Role of Superoxide Dismutases in Adherence and Biofilm Formation in Candida albicans

Candida albicans is a major fungal pathogen of humans. Infections often result from biofilm formation on tissue and implanted medical devices. Thus, understanding the genetic mechanisms that underlie biofilm formation may have impact on human health. Sod4, Sod5, and Sod6 are all Cu-Zn dependent superoxide dismutases found on the cell surface of C. albicans. Previous work from our lab indicates that, in addition to Sod5's well characterized role in breaking down superoxide, it may also play a role in adherence. Zfu2 is a transcription factor and deletion mutants of this gene are defective in adherence and biofilm formation; one of the only cell surface proteins that is downregulated in the zfu2 deletion mutant is Sod5.  In addition, overexpression of SOD5 improves adherence of a zfu2 deletion mutant.  Thus we hypothesized that a SOD5 deletion mutant would be deficient in adherence and biofilm formation. We also hypothesized that SOD4 and SOD6, which are close homologs to SOD5, may also play a role in adherence. To test this hypothesis I first constructed SOD4, SOD5 and SOD6 homozygous deletion mutants. I then characterized the mutants using BioFlux adherence assay, which tests cell adherence to a silicone substrate, as well as additional phenotypic plates. To our surprise none of the deletion mutants tested showed an adherence defect. Instead the mutants trended toward increased adherence, though the increases did not reach statistical significance. If Sod4/5/6 are indeed adhesins, then perhaps a compensatory mechanism in each deletion mutant causes other adhesin genes, such as the other SOD genes, to be upregulated.  Alternatively, SOD5 overexpression may have an indirect effect on cell wall properties that improves adherence in the zfu2 mutant.  We will use gene expression assays and create double or triple SOD mutants to distinguish between these hypotheses.

Naomi Gunawardena

Naomi Gunawardena, Carnegie Mellon University

Mentor: Aaron Mitchell

Screening of Various Candida albicans Mutants for Stress Phenotypes

Candida albicans is the most prevalent fungal pathogen of humans. C. albicans encounters salt, oxidative, and cell wall stresses in the human body and its ability to respond to these stressors allows it to grow, form biofilms, and infect its host. Thus, there is great value in understanding genes that are responsible for Candida albicans’ stress response and virulence. This project involved screening a collection of Candida albicans mutants for sensitivity to various stresses in order to identify genes that may be involved in C. albicans’ ability to adapt. 283 different C. albicans mutant strains were spotted on plates containing NaCl, H2O2, and Congo red (a cell wall inhibitor) with relevant controls. Over 40 mutants were found to be sensitive to cell wall stress, 6 sensitive to salt stress, and 12 sensitive to oxidative stress. 8 mutants were sensitive to multiple stresses. A number of mutants were also found to be resistant to the stresses, while others did not show a stress response at all. An mrr1-/- mutant displayed sensitivity to cell wall stress and salt stress. Based on previously published microarray data, we hypothesize that MRRI is a positive regulator of PGA31, which is responsible for the cell wall stress response, and of OSM2, which is responsible for the salt stress response. The data obtained will be put together into a file that others may use as a starting point for learning about uncharacterized genes. Any genes of interest identified may be explored further through other phenotype assays, such as those for cell-substrate adherence and biofilm formation. This will allow us to begin putting together pathways that regulate the Candida albicans stress response. In terms of medical relevance, these identified genes may be possible targets for antifungal therapies in the future.
Benjamin Kuo

Benjamin Kuo, Carnegie Mellon University

Mentor: John Woolford

Investigation of the Recruitment of Has1 in the Yeast Ribosome Assembly Pathway

Ribosome biogenesis is a highly regulated, step-wise pathway, which requires ~200 assembly factors that help fold and process rRNA, and facilitate the binding of ~80 ribosomal proteins to rRNA. Has1, a DEAD-box RNA helicase, functions in assembly of 60S ribosomal subunits, more specifically, in the removal of the ITS1 spacer RNA from the 27SA3 pre-rRNA to form the 27SBS processing intermediate. Hence, it is of interest to determine the protein targets and cofactors of Has1 to further understand its role in 60S ribosome assembly. To identify potential cofactors, we began by asking how Has1 is recruited into pre-ribosomes. We did this by purifying pre-ribosomes from four different yeast strains that are conditional for expression of four different proteins, rpL8, Nop7, rpL17, and Nog2. Previous results in the lab showed that these four proteins are consecutively recruited to pre-ribosomes. Hence, these proteins were depleted and the presence of Has1 in pre-ribosomes was probed by western blotting. The results indicate that Has1 is absent from pre-ribosomes when L8 or Nop7 are depleted, but it is present when L17 or Nog2 are depleted. Thus, Has1 is recruited into pre-ribosomes immediately after Nop7. In fact, there are 5 other assembly factors that are recruited with Nop7 in an interdependent fashion. We hypothesize that one of these six assembly factors is directly interacting with Has1 to recruit it into pre-ribosomes. We have found that two of these assembly factors, Nop15 and Rlp7, show two-hybrid positive interactions with Has1. Furthermore, truncations of the carboxyl terminal extension of Has1 show that this C-terminal tail domain of Has1 is necessary for the two-hybrid interactions and also for recruitment of Has1 into pre-ribosomes. To follow this up, we would like to pinpoint the amino acids in the tail domain of Has1 that are important for its interactions with Nop15 and Rlp7 to determine how specific interactions facilitate recruitment of Has1 into pre-ribosomes. Therefore, we have elucidated the timing of Has1 recruitment into pre-ribosomes, and would like to further characterize the molecular mechanism of recruitment in the future.

Wendy Li

Wendy Li, Carnegie Mellon University

Mentor: Chien Ho

Analysis of the Interaction between Macrophages and T-cells Labeled with a Dual Modality Contrast Agent

Currently, the labeling and tracking of cells in-vivo by magnetic resonance imaging (MRI) has great potential for diagnosing and determining treatment for disease diseases, such as graft rejection, cancer, cellular therapy, etc. By tracking the accumulation of labeled immune cells at the site of organ rejection, we have shown that cardiac rejection can be monitored through MRI. IOPC-NH2, a contrast agent, is a promising particle for tracking T-cells in-vivo by MRI because it labels T-cells with over 90% efficiency. In order to apply this new methodology to monitoring cardiac rejection, however, the interactions between immune cells involved in cardiac rejection (e.g. T-cells and macrophages) following T-cell labeling with IOPC-NH2 must be examined.  Previously, it was shown through confocal microscopy and flow cytometry that labeling of normal T-cells with IOPC-NH2 particles does not affect cell-cell interactions or change the expression of cell surface markers.  These results are a promising indication that T-cells can be labeled in-vitro with IOPC-NH2 particles without affecting the normal function of macrophages and T-cells. However, it is now important to examine the effects of IOPC-NH2 labeling on activated immune cells from cardiac allograft recipients. Immune cells were obtained from the spleens of normal rats and graft recipients. Cell surface marker populations on T-cells and macrophages were determined through flow cytometry. It was found that labeling activated T-cells with IOPC-NH2 had no significant effects on the populations of cell surface markers on T-cells or macrophages. These results further confirm that labeling T-cells with IOPC-NH2 does not cause changes in immune cell function.

Semawit Gebrehiwot

Semawit Gebrehiwot, Carnegie Mellon University

Mentor: Adam Linstedt

Site-directed Mutagenesis of Shiga Toxin

Shiga toxin is a toxin mainly produced by the Shigatoxigenic group of Escherichia coli (STEC). It has been associated with outbreaks all over the world and spreads through food and water contamination. Shiga acts by invading cells through endocytosis and evading degradation by trafficking with GPP130 through the Golgi. It then travels through the endoplasmic reticulum and the cytoplasm where it hinders protein synthesis by destroying the 28S subunit of the ribosome. It has recently been observed that manganese causes GPP130 degradation, which in turn causes the degradation of Shiga. Therefore it has been theorized that manganese could have possible benefits as a form of treatment. Another possibility is that blocking the GPP130 and Shiga interaction will give a similar result. Shiga toxin has five B subunits that form a homopentamer which participate in the trafficking of the Shiga toxin and an A subunit that participates in the destruction of ribosomes. The goal of this project was to mutate the B subunit of Shiga toxin so that it loses the ability to bind GPP130. In order to create the mutated Shiga, I have studied proposed binding models to predict possible blocking mutations, mutated the B subunit cDNA using the quickchange method, purified the mutated versions of the expressed B subunit through nickel affinity chromatography and performed a binding assay to determine which mutations were successful. By accomplishing this goal we have gained a better understanding of Shiga toxin and possible forms of treatment.