Thioredoxins in redox maintenance and survival during oxidative stress of Bacteroides fragilis

Abstract

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 DeltatrxC DeltatrxD::cfxA DeltatrxE DeltatrxF DeltatrxG quintuple mutant. Regulation of the trx genes was linked to the oxidative stress response, and all were induced by aerobic conditions. The DeltatrxC DeltatrxE DeltatrxF DeltatrxG and the DeltatrxC DeltatrxD::cfxA DeltatrxE DeltatrxF DeltatrxG 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.

FIG. 1.

Phylogenetic comparison of 27 Trx proteins from diverse sources. ClustalW was used to align protein sequences for 27 Trx proteins. The unrooted bootstrap consensus tree was inferred using the minimum-evolution method with 500 bootstrap replicates (41). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Phylogenetic analyses were conducted in MEGA4 (50). The following sequences with accession numbers were used: Burkholderia, YP_333769; human, NP_003320; E. coli TrxA, AAA67582; Porph TrxC, P. gingivalis AAQ65495; Syn M, Synechococcus ZP_01124485; Anabaena Y, ABA23368; Flavobacterium, CAL43878; Nostoc Y, NP_485933; Syn Y, Synechocystis NP_442168; Campylobacter, YP_178167; Helicobacter, NP_223481; Porph TrxA, P. gingivalis NP_904389; Streptomyces, CAB72414; TrxA, YP_210347; TrxC, YP_212311; TrxD, YP_211860; TrxE, YP_212629; TrxF, YP_212630; TrxG, YP_210941; Arabidopsis H1, CAA78462; Arabidopsis F1, AAD35003; Arabidopsis X1, NP_564566; Syn X, Synechocystis NP_440611; Syn C, Synechocystis NP_439965; E. coli TrxC, NP_417077; Arabidopsis M1, AAF15948; Nostoc M, NP_485906.

FIG. 2.

Genetic loci of the six B. fragilis trx genes. The maps are drawn to scale; the dashed lines above the trx genes show the regions deleted in each trx mutant, and the black lines under the trx genes represent the approximate sizes of the mRNAs observed in Fig. 3. Genes: unk, unknown with no matches in database; dnaE, DNA polymerase III; fldA, flavodoxin; doxDA, thiosulfate quinone oxidoreductase; mauG, tryptophan tryptophylquinone synthesis; cztBC, heavy metal efflux pump; hel, DNA helicase; hyp-Ptase, hypothetical phosphatase; nfnB, oxygen-insensitive nitroreductase; rbr, rubrerythrin-like; per, peroxide response regulator homolog; hyp-reg, hypothetical DNA binding protein; spoU, SpoU-like RNA methylase; arsF, sulfatase.

FIG. 3.

Northern hybridization analysis of total RNA of B. fragilis strain 638R (wild type). RNA was isolated from cells grown to mid-logarithmic phase in BHIS and then treated as described in the text: 500 μM diamide (D), 50 μM hydrogen peroxide (P), exposed to air (O), or untreated (An). (A) Autoradiographs of blots hybridized to radiolabeled probes containing the entire open reading frame of each trx gene as indicated. The approximate sizes of the transcripts are shown. The apparent bands (*) at about 1.5 and 2.5 kb are a commonly observed compression artifact caused by the 16S and 23S rRNAs. (B) Fold increase of transcript levels under each condition compared to the anaerobic control based on densitometric values. Black bars, 500 μM diamide; white bars, 50 μM H₂O₂; hatched bars, aerobic exposure.

FIG. 4.

Growth analysis of B. fragilis trx and oxyR mutant strains in anaerobic and oxidized media. Strains were grown overnight in BHIS and then inoculated into either fully oxidized medium (Ox) or anaerobic medium (An). Growth was measured on a spectrophotometer at 550 nm. The results shown are the averages from triplicate observations in two growth experiments. Strains IB101 (wild type, ⧫), IB298 (ΔoxyR, ⋄), IB498 (ΔtrxC ΔtrxE ΔtrxF ΔtrxG ▪), IB499 (ΔtrxC ΔtrxE ΔtrxF ΔtrxG ΔoxyR::tetQ □), IB483 (ΔtrxC ΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxG ▴), and IB500 (ΔtrxC ΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxG ΔoxyR::tetQ ▵) were used.

FIG. 5.

Effect of each Trx on survival during oxidative stress. (A) Wild-type strain 638R was compared to trx mutant strains in diamide disk diffusion assays on BHIS plates with no added cysteine. (B) Strain 638R harboring the empty expression vector pFD340 was compared to 638R strains harboring pFD340 containing B. fragilis trx genes in diamide disk diffusion assays on defined minimal medium. The values are mean diameters of growth inhibition zones measured in three independent experiments performed in triplicate and are given in millimeters. The error bars indicate standard deviations. *, P < 0.01 compared to wild-type strain. Strains in panel A: wild type (WT), 638R; ΔtrxC, IB458; ΔtrxD, IB469; ΔtrxE, IB490; ΔtrxF, IB491; ΔtrxG, IB471; and ΔtrxEF, IB492.

FIG. 6.

Rescue of strain IB483 diamide sensitivity phenotype by plasmid ptrxD. Diamide disk diffusion assays were used to compare sensitivities of the wild-type strain 638R, the quadruple trx mutant strain IB498, the quintuple trx mutant strain IB483, and IB483 expressing trxD on plasmid ptrxD. Black bars represent plates placed directly into an anaerobic incubator after plating, and open bars represent plates placed in an aerobic incubator for 6 h prior to being placed into the anaerobic incubator. The values are mean diameters of growth inhibition from three independent experiments performed in triplicate, and are given in millimeters. The error bars indicate standard deviations. *, P < 0.001 compared to wild-type strain.

FIG. 7.

Transcriptional analysis of trx genes. The parental strain (wild type [WT], B. fragilis strain 638R) and the isogenic trxD mutant (ΔtrxD, strain IB469) were exposed for 5 min to 500 μM, 100 μM, and 50 μM diamide or maintained under standard anaerobic conditions (0 μM control). For each condition, RNA was isolated and real-time RT-PCR was performed in triplicate. The sigma-54 modulation protein gene was used as a standard, and the results are expressed as fold induction relative to levels under the control condition. The values are means of fold induction, compared to the 0 μM control, from two independent experiments. The error bars indicate standard deviations.