Role of the Iron Storage Ferritins Dps and DpsL during the Prolonged Oxidative Stress Response of Bacteroides fragilis
Betteken, Michael I.
Bacteroides fragilis is a Gram negative anaerobe and member of the human intestinal tract microbiome. B. fragilis serves many beneficial roles within the intestinal tract; however, its translocation to the peritoneal cavity and the blood stream can result in peritonitis, intra-abdominal abscess formation, bacteremia and sepsis. We have shown that B. fragilis mediates both acute and Prolonged Oxidative Stress (POST) responses both in vitro and in vivo. This report characterizes the role and the genetic regulation of the iron storage proteins Dps and DpsL during the POST response. To test sensitivity to oxidative stress during the POST response a disk diffusion assay was developed using tert-butyl hydroperoxide (tBOOH). When the assay plates received aerobic exposure for three hours there was no zone of growth inhibition, whereas those kept under anaerobic conditions were highly sensitive to tBOOH. These results demonstrated an oxygen induced resistance to tBOOH that was mediated by prolonged aerobic exposure. To determine a mechanism for this POST induced resistance to tBOOH, a series of oxidative stress mutants were assayed. Only the Δdps mutant was sensitive to tBOOH after aerobic exposure indicating that Dps mediated the POST phenotype. Because of the similarities to Dps, the recently characterized DpsL (bfr) was tested for a role in the POST response. The Δbfr mutant demonstrated resistance to tBOOH after aerobic exposure similar to wildtype (WT); however, when a double Δdps Δbfr mutant was generated it demonstrated sensitivity to tBOOH that was greater than the Δdps mutant indicating that both Dps and DpsL play a role in the resistance phenotype. To explore the role that Dps and DpsL play in the survival of B. fragilis during infection, animal experiments were performed in the rat abscess model. Interestingly only the double Δdps Δbfr mutant was attenuated in this model whereas neither of the single mutants showed a defect in competition experiments with WT. This indicated that both Dps and DpsL play a role in survival during infection. To investigate genetic regulation during the POST response, it was essential to identify the second regulator of dps expression. It was previously shown that OxyR is a strong inducer of dps expression during acute oxidative stress; however, the ΔoxyR mutant was resistant to tBOOH after prolonged aerobic exposure similar to WT. This indicated that there was a second regulator of dps expression during the POST response. A known POST regulator, SigOF was investigated to determine if it played a role in this response. Similar to ΔoxyR the ΔsigOF mutant was resistant to tBOOH. Interestingly though a double ΔsigOF ΔoxyR mutant was sensitive to tBOOH in the POST assay and dps expression was reduced as shown by qRTPCR. These results strongly suggest that SigOF is the regulator responsible for dps expression during the POST response.
Betteken, Michael I.. (December 2015). Role of the Iron Storage Ferritins Dps and DpsL during the Prolonged Oxidative Stress Response of Bacteroides fragilis (Doctoral Dissertation, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/5116.)
Betteken, Michael I.. Role of the Iron Storage Ferritins Dps and DpsL during the Prolonged Oxidative Stress Response of Bacteroides fragilis. Doctoral Dissertation. East Carolina University, December 2015. The Scholarship. http://hdl.handle.net/10342/5116. June 19, 2018.
Betteken, Michael I., “Role of the Iron Storage Ferritins Dps and DpsL during the Prolonged Oxidative Stress Response of Bacteroides fragilis” (Doctoral Dissertation., East Carolina University, December 2015).
Betteken, Michael I.. Role of the Iron Storage Ferritins Dps and DpsL during the Prolonged Oxidative Stress Response of Bacteroides fragilis [Doctoral Dissertation]. Greenville, NC: East Carolina University; December 2015.
East Carolina University