Investigations into the roles of bacteroides fragilis thioredoxins during the oxidative stress response

Abstract

The Gram-negative Bacteroides fragilis is the most frequently isolated bacterium from anaerobic infections. Native to the human intestinal tract, it serves a symbiotic role breaking down complex polysaccharides and restricting the growth of potentially harmful organisms. However, if translocated into the peritoneal cavity, B. fragilis can induce abscess formation; a potentially life-threatening condition. Although an obligate anaerobe, this bacterium is capable of surviving aerobic environments for extended periods of time, and its role as an opportunistic pathogen depends on this capability. Aerobic survival relies on an oxidative stress response of genes activated either dependently or independently by the stress response regulator OxyR. An important subset of OxyR-independent genes are the thioredoxins (Trxs). B. fragilis contains an extensive catalog of six trx genes, and analyses indicated each is differentially regulated during oxidative conditions. Single and multiple trx deletions were produced to determine functional differences of the proteins. It was found that trxA was essential for growth while no other single trx deletion conferred anaerobic growth defects. The trxD gene was found to be induced by the thiol oxidant diamide and TrxD was shown to be protective during exposure to diamide as well. In a trxD mutant strain, diamide-induced expression of trxC, trxE, and trxF increased significantly, suggesting compensatory effects in the Trx system. TrxD and TrxE were determined to be the only two B. fragilis Trxs capable of reducing the aerobic ribonucleotide reductase (NrdAB) of E. col , suggesting specificity of Trx targets in B. fragilis. Further investigations into TrxD determined the trxD promoter region and transcriptional start site as well as demonstrating TrxD to be the Trx primarily responsible for the reduction of specific oxidative stress-induced proteins including AsnB (asparaginase II) and Tps (thiol peroxidase scavengase). These data provide initial insight into both the specific and overlapping functions of Trxs in B. fragilis.