Discipline: Biological Sciences
Karien Dixon - Tougaloo College
Co-Author(s): Percus Z. Mody and Justin A. Thornton, Mississippi State University, MS Jason W. Rosch, St. Jude Children’s Research Hospital, TN
Streptococcus pneumoniae (pneumococcus) is a bacteria responsible for invasive and noninvasive diseases such as pneumonia, meningitis, and otitis media. This pathogen produces hydrogen peroxide via pyruvate oxidase (ΔSpxB), which is known to be a virulence factor. Pneumococcal hydrogen peroxide is known to act as a messenger, setting in motion intracellular signals, sequentially, activating and upregulating certain genes. In this study, we quantitate changes in stress gene expressions to wild type or ΔSpxB-negative strains. We hypothesized that global gene expression in lung epithelial cells would be affected by exposure to hydrogen peroxide produced by ΔSpxB. We exposed A549 cells to either media alone, a wild type strain, or an isogenic mutant lacking pyruvate oxidase for 30 minutes or 1 hour at 37 Cº. Total RNA was then isolated and used for microarray analysis, and results were verified by qRT-PCR. Beta actin served as an internal housekeeping gene for qRT-PCR. The microarray identified several genes that were differentially expressed in response to production of hydrogen peroxide. The genes FOS, IL-8, N4RA2, and EGR-1 were found to be up-regulated. Quantitative PCR verified that the A549 cells that were exposed to the wild type strain had greater gene expression than those cells exposed to the strain lacking pyruvate oxidase at both 30 min and 1hr postexposure. The effect of peroxide on gene expression was timedependent with greater fold-changes seen at 1hr for both the wild-type and mutant strain. The fold changes for cells exposed to bacteria for 30 min were: Fos (10.7-fold vs 5.3-fold), IL-8 (1.3fold vs 1-fold), EGR1 (5.9-fold vs 2.9-fold), and NR4A2 (7.2-fold vs 4.2-fold) for T4R and ΔSpxB, respectively. The fold changes for cells exposed to bacteria for 1 hr were: Fos (17.2-fold vs 9fold), IL-8 (4.2-fold vs 2.9-fold), EGR1 (12.3-fold vs 6.4-fold), NR4A2 (14.4-fold vs 8.6-fold) for T4R and ΔSpxB, respectively. This study shows that pneumococcal hydrogen peroxide causes a greater expression of stress response genes than hydrogen peroxide negative strains. While it is known that production of hydrogen peroxide by pneumococcus is detrimental to the host, identifying processes involved in the response to this virulence factor is essential for future research.
Funder Acknowledgement(s): I would like to thank Dr. Justin Thornton and Dr. Janet Donaldson for help in the field. I would also like to thank Jessica E. Drakeford, Joseph Bryant, Lindsey Brown, and Dr. Bianca Garner for their help. Funding was provided by Mississippi State University’s College of Arts and Sciences and the Jackson Heart Study Program.
Faculty Advisor: Justin A. Thornton,