Thioredoxin reductase is essential for thiol/disulfide redox control and oxidative stress survival of the anaerobe Bacteroides fragilis

Abstract

Results of this study showed that the anaerobic, opportunistic pathogen Bacteroides fragilis lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs. Analysis of the genome sequence revealed six Trx orthologs and an absence of genes required for synthesis of glutathione and glutaredoxins. In addition, it was shown that the thioredoxin reductase (TrxB)/Trx system is the major or sole redox system for thiol/disulfide cellular homeostasis in this anaerobic bacterium. Expression of the B. fragilis trxB gene was induced following treatment with diamide or H(2)O(2) or exposure to oxygen. This inducible trxB expression was OxyR independent. Northern blot hybridization analysis showed that the trxB mRNA was cotranscribed with lolA as a bicistronic transcript or was present as a monocistronic transcript that was also highly induced under the same conditions. The role of LolA, a prokaryotic periplasmic lipoprotein-specific molecular chaperone in the thiol/disulfide redox system, is unknown. A trxB deletion mutant was more sensitive to the effects of diamide and oxygen than the parent strain. In addition, the trxB mutant was unable to grow in culture media without addition of a reductant. Furthermore, the trxB mutant was not able to induce intraabdominal abscess formation in a mouse model, whereas the parent strain was. Taken together, these data strongly suggest that TrxB/Trx is the major, if not the sole, thiol/disulfide redox system in this anaerobe required for survival and abscess formation in a peritoneal cavity infection model.

FIG. 1.

Multiple alignment of the deduced amino acid sequences of B. fragilis (Bf) Trx orthologs (TrxA through TrxG plus TrxX) with E. coli (Ec) TrxA and TrxC amino acid sequences. The conserved Trx fold containing the CXXC redox active site is indicted by thick lines above and below the amino acid sequence region. A consensus of at least 50% identical amino acid residues is indicated by a black background. Conserved amino acid substitutions are indicated by a gray background. The predicted conserved prokaryotic membrane lipoprotein precursor signal peptide (15) present in TrxC is indicated by a thin line under the amino acid sequence. Alignment of peptide sequences was performed using the GCG programs pileup and box with the peptide scoring matrix default data file blosum62.cmp for comparison of amino acid substitutions.

FIG. 2.

Growth of B. fragilis parent strain 638R and the ΔtrxB mutant in BHIS (A) and in SDM (B). •, parent strain grown in medium containing cysteine; ○, parent strain grown in medium without cysteine; ▪, trxB mutant grown in medium containing cysteine; □, trxB mutant grown in medium without cysteine; ▴, trxB mutant grown in medium without cysteine but with DTT added at the time point indicated. O.D_{550 nm}, optical density at 550 nm.

FIG. 3.

(A) Northern hybridization analysis of total RNA of B. fragilis 638R (wt), IB298 (ΔoxyR), and IB263 constitutively activating OxyR [oxyR(Con)]. Cells were grown to mid-logarithmic phase in BHIS without addition of cysteine and then exposed to oxygen. Cultures were treated with H₂O₂ or exposed to oxygen as indicated in the text. The probe was a trxB internal gene fragment. The approximate sizes of the transcripts are indicated. DTT was used as a control reductant. (B) Diagram of lolA and trxB genetic organization and structure. The large arrows indicate the open reading frames and their direction of transcription. The arrows below the map indicate the lengths of the transcripts and their orientation. The bent arrow indicates the putative promoter predicted from data presented in Fig. 5. The lolA trxB intergenic nucleotide sequence region containing a predicted stem-loop structure is also shown. The lolA stop codon (UAG) and the trxB translational start codon (ATG) are underlined. The cleavage nucleotide regions of the bicistronic lolA trxB mRNA determined by the RNase protection assay are indicated by arrows.

FIG. 4.

Autoradiograph of Northern hybridization of total RNA from mid-log-phase B. fragilis 638R grown in BHIS without addition of cysteine. Cultures were treated with diamide at the concentrations indicated at the top. The probe was a trxB internal gene fragment. The approximate sizes of the transcripts are indicated. DTT was added to one culture as a control to show that addition of a reducing agent to the medium did not lead to induction of trxB.

FIG. 5.

Autoradiograph following primer extension analysis of the lolA trxB mRNA transcription initiation regions. Total RNA extracted from the wild-type strain under different stress conditions was used in the extension reactions. Cultures were treated by adding 50 μM H₂O₂, by exposure to atmospheric air for 1 h, or by adding 100 μM diamide. An untreated anaerobic control culture was also included. A DNA sequencing ladder generated with the same primer that was used for the primer extension reactions is shown on the left. The arrows indicate the initial +1 adenine for the P1 promoter and the +1 thymine for the P2 promoter. (B) Nucleotide sequence of the lolA trxB regulatory region. Based on a B. fragilis consensus (4), the predicted −10 and −35 promoter regions for the transcription initiation sites P1 and P2 are indicated by bold type and underlined with solid and dashed lines, respectively. The bent arrows indicate the initial +1 adenine nucleotide and the +1 thymine nucleotide for the P1 and P2 transcription start sites, respectively, at positions −26 and −8 relative to the translation start codon. The first 26 codons for lolA are shown.

FIG. 6.

Disk inhibition assay testing the sensitivity of B. fragilis strains to diamide in the absence of oxygen (bars with thick stripes) and in the presence of oxygen (bars with thin stripes) and to hydrogen peroxide (open bars). The cultures were grown overnight in BHIS, and 0.1 ml was plated on BHI agar without added cysteine. Ten microliters of a 2 M diamide solution or 0.88 M hydrogen peroxide was applied to a 6-mm filter paper placed in the center of each plate. Plates were incubated anaerobically overnight at 37°C. Duplicate plates with the diamide solution were exposed to air for 6 h at 37°C before they were placed in the anaerobic chamber and incubated as described above. Zones of growth inhibition (in millimeters) were measured. The error bars indicate the standard deviations of the means from three independent determinations. wt, wild type.

FIG. 7.

Growth of B. fragilis strains in soft agar tubes in the presence oxygen. A 0.1-ml aliquot of bacteria grown overnight in BHIS was mixed with 5 ml BHIS without cysteine containing 0.4% agar at 45°C under anaerobic conditions. After solidification of the agar, cultures were placed outside the anaerobic chamber and incubated aerobically at 37°C for 24 h. The clearance zone was measured from the top edge of the agar down to the growth line in the column. The arrows indicate the interface between bacterial growth and the zone of growth inhibition. NG, no growth.