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

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.  Ph.D

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

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.

Thioredoxins in Redox Maintenance and Survival during Oxidative Stress of Bacteroides fragilis▿ †

The anaerobe Bacteroides fragilis is a gram-negative, opportunistic pathogen that is highly aerotolerant and can persist in aerobic environments for extended periods. In this study, the six B. fragilis thioredoxins (Trxs) were investigated to determine their role during oxidative stress. Phylogenetic analyses of Trx protein sequences indicated that four of the six Trxs (TrxA, TrxC, TrxD, and TrxF) belong to the M-type Trx class but were associated with two different M-type lineages. TrxE and TrxG were most closely associated to Y-type Trxs found primarily in cyanobacteria. Single and multiple trx gene deletions were generated to determine functional differences between the Trxs. The trxA gene was essential, but no anaerobic growth defects were observed for any other single trx deletion or for the ΔtrxC ΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxG quintuple mutant. Regulation of the trx genes was linked to the oxidative stress response, and all were induced by aerobic conditions. The ΔtrxC ΔtrxE ΔtrxF ΔtrxG and the ΔtrxC ΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxG multiple deletion strains were impaired during growth in oxidized media, but single trx gene mutants did not have a phenotype in this assay. TrxD was protective during exposure to the thiol oxidant diamide, and expression of trxD was induced by diamide. Diamide-induced expression of trxC, trxE, and trxF increased significantly in a trxD mutant strain, suggesting that there is some capacity for compensation in this complex Trx system. These data provide insight into the role of individual Trxs in the B. fragilis oxidative stress response