Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe Bacteroides fragilis

Results of this study showed that the anaerobic, opportunisticpathogen Bacteroides fragilis lacks the glutathione/glutaredoxinredox system and possesses an extensive number of putative thioredoxin(Trx) orthologs. Analysis of the genome sequence revealed sixTrx orthologs and an absence of genes required for synthesisof glutathione and glutaredoxins. In addition, it was shownthat the thioredoxin reductase (TrxB)/Trx system is the majoror sole redox system for thiol/disulfide cellular homeostasisin this anaerobic bacterium. Expression of the B. fragilis trxBgene was induced following treatment with diamide or H₂O₂ orexposure to oxygen. This inducible trxB expression was OxyRindependent. Northern blot hybridization analysis showed thatthe trxB mRNA was cotranscribed with lolA as a bicistronic transcriptor was present as a monocistronic transcript that was also highlyinduced under the same conditions. The role of LolA, a prokaryoticperiplasmic lipoprotein-specific molecular chaperone in thethiol/disulfide redox system, is unknown. A trxB deletion mutantwas more sensitive to the effects of diamide and oxygen thanthe parent strain. In addition, the trxB mutant was unable togrow in culture media without addition of a reductant. Furthermore,the trxB mutant was not able to induce intraabdominal abscessformation in a mouse model, whereas the parent strain was. Takentogether, these data strongly suggest that TrxB/Trx is the major,if not the sole, thiol/disulfide redox system in this anaeroberequired for survival and abscess formation in a peritonealcavity infection model.

INTRODUCTION

Top

INTRODUCTION

Adaptation of anaerobic bacteria to aerobic environments hastaken different courses in different families of anaerobes.Some bacteria did not adapt and remained sensitive to aerobicenvironments. The bacteria that adapted had to cope with theproblem of thiol oxidation and developed different mechanismsto deal with the presence of oxygen (10). One of the latterbacteria is Bacteroides fragilis, a human intestinal obligateanaerobe which is the anaerobe most frequently isolated fromhuman infections. B. fragilis possesses a complex oxidativestress response mechanism which is required to maintain extendedaerotolerance. A set of at least 28 proteins is synthesizedin response to treatment with hydrogen peroxide or oxygen exposure.Some of these proteins, such as KatB, Dps, and AhpCF, have beenidentified (16, 37, 39, 40), but the mechanism that confersaerotolerance in this anaerobic bacterium still remains to beelucidated. In the presence of low, nanomolar concentrationsof oxygen, B. fragilis can maintain nearly normal growth andmetabolic activity by consuming oxygen (3). This activity isnot sufficient to allow growth under fully aerobic conditions,in which a wide range of cellular functions are inhibited (19).One of the cellular functions sensitive to oxygen is the thiol/disulfideredox balance. Thiols are oxidized within minutes followingexposure to air, and the oxidation of thiols produces reducedoxygen species that can be highly toxic (10). Iron-sulfur proteinsalso are very reactive, and it has been shown that the iron-sulfurclusters present in many metabolic enzymes of Bacteroides spp.are vulnerable to oxygen exposure and that this affects theiraerotolerance response (19, 32).

The intracellular environment is characterized by reducing conditionsgenerally due to the presence of small proteins with redox-activecysteine residues, including the thioredoxins (Trxs), the glutaredoxins(Grxs), the monocysteine tripeptide glutathione (GSH), and otherlow-molecular-weight thiols (10, 17). Trxs and Grxs are smallubiquitous thiol:disulfide oxidoreductases containing the two-cysteineredox motif CXXC. Trx contains this motif as WCGPC, while mostGrxs contain the CPYC motif (31, 55). Trxs and Grxs have beenimplicated in a variety of physiological processes and biologicalpathways. In addition, they play a role in defense against oxidativestress, either by reducing protein disulfide bonds producedby various oxidants or by scavenging reactive oxygen species.Trxs and Trx reductases (TrxB) are widespread in all phylogeneticbranches, including viruses, archaea, bacteria, protozoans,yeasts, plants, and animals (2), while GSH is absent in manybacteria, including some facultative gram-positive and anaerobicbacteria (9, 30, 47, 52).

The reduction of intracellular disulfides is mediated by NADPH,which operates together with flavin adenine dinucleotide-dependentTrxB to convert oxidized Trx to the free thiol form (2). Thiscellular redox mechanism is likely to be essential for adaptationof aerotolerant anaerobes to aerobic environments since theyrequire mechanisms to minimize the occurrence of thiol oxidationand to mitigate its consequences. TrxB/Trx systems have beenidentified in some anaerobe species, including Desulfovibriodesulfuricans (45), Clostridium pasteurianum (36), Porphyromonasgingivalis (23), amino acid-utilizing anaerobic bacteria (14),Clostridium acetobutylicum and Clostridium aminovalericum (22),and Chromatium vinosum (20).

Although Grx/GSH-related genes have not been found yet in thegenome sequences of strict anaerobes, a Grx homologue was acomponent of the AhpC peroxiredoxin reductase system in C. pasteurianum(36), and GSH has been found in crude extracts of Desulfovibriogigas (11). In this regard, analysis of B. fragilis genome sequences(6) revealed the presence of a Trx reductase and six Trx homologues,but no Grx, ${gamma}$ -glutamyl-L-cysteine synthetase, or GSH synthetasegenes were found. The large number of Trx orthologs in B. fragilisseems to be unusual compared to the smaller number of Trxs describedin other anaerobes (14, 20, 22, 23, 36, 45).

B. fragilis clinical isolates are some of the most aerotolerantanaerobic bacteria (41, 51). Thus, we propose that the expandedTrx system is an important component of their arsenal againstoxidative stress in vivo and in vitro. B. fragilis is the anaerobeisolated most often from clinical infections, and thus it isable to survive in oxygenated host tissues and withstand theoxidative burst of the host immune response during the initialsteps of infection until appropriate anaerobic conditions thatallow bacterial proliferation are present (42, 43, 44, 46).These findings prompted us to elucidate the role of the TrxB/Trxsystem in the physiology and aerotolerance of B. fragilis invivo and in vitro. In this paper we present evidence showingthat the TrxB/Trx system is the major thiol/disulfide redoxsystem in B. fragilis and is essential for bacterial survivaland abscess formation in an intraabdominal infection model.

MATERIALS AND METHODS

Top

MATERIALS AND METHODS

Bacterial strains and growth conditions. B. fragilis strains used in this study are listed in Table 1.Strains were grown anaerobically in brain heart infusion (BHI)broth supplemented with hemin, cysteine, and NaHCO₃ (BHIS) forroutine cultures. Rifamycin (20 µg/ml), 100 µg/mlgentamicin, 5 µg/ml tetracycline, 10 µg/ml erythromycin,and 25 µg/ml cefoxitin were added to the media when required.Semidefined medium (SDM) was prepared by adding 0.5 g/litertryptone to a modified chemically defined medium of Varel andBryant (53). The modified medium composition was as follows:KH₂PO₄, 1.15 g/liter; NH₄SO₄, 0.4 g/liter; NaCl, 0.9 g/liter;L-methionine, 75 mg/liter; MgCl₂·6H₂O, 20 mg/liter; CaCl₂·2H₂O,6.6 mg/liter; MnCl₂·4H₂O, 1 mg/liter; CoCl₂·6H₂O,1 mg/liter; resazurin, 1 mg/liter; L-cysteine, 500 mg/liter;hemin, 5 mg/liter; and glucose, 5 g/liter. Twenty millilitersof 10% NaHCO₃ was added per liter of medium, and the final pHwas 7.2. B. fragilis requires a reduced sulfur source to supportgrowth (53), which was tryptone when L-cysteine was omitted.

View this table:
[in this window]
[in a new window]

TABLE 1. Strains and plasmids used in this study

Construction of trxB deletion mutant. Briefly, a 2.1-kb chromosome fragment containing the trxB regionwas amplified by PCR using oligonucleotides containing modificationsto create restriction sites for SphI and BamHI and then clonedinto pUC19 to create pFD846. Next, an internal 60-bp BglII/ClaIfragment from the trxB gene was deleted and replaced with a2.1-kb BamHI/NarI cefoxitin (cfxA) resistance gene to constructa ${Delta}$ trxB::cfxA deletion in a new plasmid, pFD846/ ${Delta}$ trxB::cfxA. Thena 4.5-kb SphI/SstI fragment from pFD846/ ${Delta}$ trxB::cfxA was clonedinto the suicide vector pFD516 (50) to create pFD874. This plasmidwas mobilized from Escherichia coli DH10B into B. fragilis 638Rby triparental mating, and exconjugants were selected on BHIScontaining 20 µg/ml rifamycin, 100 µg/ml gentamicin,and 25 µg/ml cefoxitin. Sensitivity to erythromycin wasdetermined, and Southern blotting was performed to confirm thedouble-crossover allele exchange to create a trxB mutant designatedIB370.

A ${Delta}$ trxB ${Delta}$ oxyR mutant was constructed by mobilizing suicide vectorpFD754 (containing the ${Delta}$ oxyR::tetQ deletion [39]) into B. fragilisIB370 as described above. Exconjugants were selected on BHIScontaining 20 µg/ml rifamycin, 100 µg/ml gentamicin,5 µg/ml tetracycline, and 25 µg/ml cefoxitin. Sensitivityto either tetracycline/cefoxitin or erythromycin was used toidentify recombinants that were tetracycline and cefoxitin resistantand erythromycin sensitive. Strain IB372 was selected for furtherstudy.

A plasmid constitutively expressing trxB was constructed byPCR amplification of a 1,285-bp promoterless trxB gene containing21 bp upstream of the ATG start codon and 255 bp downstreamof the stop codon. The promoterless trxB gene fragment was clonedinto the BamHI site of the Bacteroides-E. coli shuttle expressionvector pFD340 (49) in the same orientation as the IS4351 promoter.The new construct, pFD892, was mobilized into B. fragilis IB370as described above. Transconjugants were selected on BHIS containing20 µg/ml rifamycin, 100 µg/ml gentamicin, and 10µg/ml erythromycin.

RNA extraction, Northern blot hybridization, and primer extension. For oxidative stress experiments, cultures were grown in BHISto an A₅₅₀ of 0.3 and treated with 50 µM H₂O₂ for 5 minprior to total RNA extraction. To induce oxygen stress, cultureswere split in half; one half was incubated anaerobically, andthe other half was shaken aerobically at 250 rpm using a volume/flaskratio of 1/5 as previously described (38). For diamide treatment,mid-log-phase cultures were treated with different concentrationsof diamide as indicated below. Total RNA extraction and Northernblot analysis of mRNA were carried out as previously described(38). Internal fragments of trxB and lolA were used as specificprobes when required. In a densitometry analysis the intensityof the autoradiograph was normalized to the relative intensityof total 23S and 16S rRNA detected on the ethidium bromide-stainedagarose gel to correct for any loading differences.

Primer extension analysis was performed with total RNA obtainedfrom mid-log-phase cells of B. fragilis 638R grown anaerobicallyand then subjected to oxidative stress conditions as describedpreviously (39). A lolA-specific oligonucleotide and a trxB-specificoligonucleotide were labeled with [ ${alpha}$ -³²P]ATP and used as primersfor the reverse transcriptase reaction.

Inhibition of growth by oxygen in a soft agar tube culture. A 0.1-ml aliquot of an overnight culture of B. fragilis in BHISbroth without L-cysteine was mixed with 5 ml of BHIS withoutadded L-cysteine and resazurin containing 0.4% agar at 45°Cin screw cap tubes (100 by 13 mm) inside an anaerobic chamber.The tubes were capped with sterile cotton stoppers and incubatedaerobically at 37°C for 24 h. The area from the top of theagar down to the edge of visible bacterial growth was consideredthe zone of growth inhibition by oxygen and was measured.

GSH determination. The total GSH/oxidized glutathione (GSSG) content in cells wasdetermined with a cycling enzyme assay utilizing glutathionereductase as described by Howe et al. (18) and Fahey et al.(9), with some modifications. Overnight cultures of B. fragilis638R and E. coli HB101 (control strain) were grown in SDM underanaerobic conditions and used to inoculate fresh SDM cultures.Bacteria were grown to mid-log phase (optical density at 550nm, 0.45), and then cultures were split. One half of each culturewas incubated aerobically in a shaker incubator at 37°Cfor 1 and 24 h. The other half was incubated under anaerobicconditions for the same time periods. The amount of total GSHwas determined in mid-log- and stationary-phase cells (24 hof incubation). Cells were washed in phosphate-buffered saline(50 mM phosphate buffer [pH 7.4], 150 mM NaCl). Cell pelletswere extracted in boiling 80% ethanol, dried at 105°C, weighedin preweighed tubes, and stored at –70°C until theassay was performed. Dried pellets were suspended in 800 µlof 2% 5-sulfosalicylic acid. GSH contents were determined using100-µl samples, and immediately prior to the assay, sampleswere treated with 3.9 µl of a 1:4 dilution of triethanolamineto bring the pH to 7.0. The presence of GSSG was also determinedin 100-µl samples by addition of 3.9 µl of 2-vinylpyridineprior to addition of 3.9 µl of a 1:4 dilution of triethanolamine.Briefly, the enzymatic assay was carried out using a 50-µltreated sample and a standard mixed with 100 µl of recyclingreaction buffer containing 42.7 mM sodium phosphate (pH 7.5),472 µM EDTA, 226 µM 5,5'-dithiobis(nitrobenzoicacid), 313 µM NADPH, and 20 U of glutathione reductase.Microplates were read at 405 nm every 45 s using a fluorescence/absorbanceplate reader (FL600; Bio-Tek). Standard curves were used todetermine the GSH/GSSG contents of the samples expressed inmicromoles per gram (dry weight).

Mouse model of abscess formation. The experimental model for intraabdominal infection describedpreviously was used, with minor modifications (7). B. fragilis638R and the isogenic trxB mutant IB370 used for challenge weregrown overnight in BHIS, concentrated, and then frozen untilthey were needed. The frozen cultures were titrated, and thenjust prior to use they were diluted to obtain the desired challengedose. C57BL male mice that were 6 to 8 weeks old were challengedvia intraperitoneal injection of 1 x 10⁶, 1 x 10⁷, or 1 x 10⁸cells in 0.2 ml (final volume) of BHI broth containing dilutedsterile rat cecal contents. The mice were sacrificed 6 dayslater and examined for intraabdominal abscesses. The presenceof one or more abscesses was considered a positive result. Animalsinoculated with broth plus sterile cecal contents alone withoutbacterial challenge did not form abscesses.

RESULTS

Top

RESULTS

Nucleotide sequence analysis of the TrxB/Trx system. The availability of the B. fragilis 638R genome sequence (6;http://www.sanger.ac.uk/Projects/B_fragilis/) allowed us tosearch for the presence of the Trx motif sequence. It was foundthat B. fragilis possesses an extensive Trx system consistingof a TrxB, six Trx orthologs, and more than 14 other proteinsthat were larger than Trxs but had the Trx-like protein foldand the CXXC motif (data not shown). The six Trx orthologs containthe redox-active center CXXC motif common to Trxs (8, 17), andfour of these orthologs, TrxA, TrxC, TrxE, and TrxF, containthe characteristic motif WCGPC (Fig. 1). TrxC also containsan N-terminal conserved prokaryotic membrane lipoprotein precursorsignal peptide (15). The TrxD active site contains WCVYC, whileTrxG has a WCPHC motif. In addition, another protein with homologyto the Trx fold motif family, tentatively designated TrxX, lacksthe reactive redox cysteine residues which were replaced byserine and tyrosine (WSPHY). When B. fragilis Trx homologueswere compared to other microbial Trxs using a progressive multiplealignment and parsimony analysis, it was found that except forTrxA, all B. fragilis Trx homologues were clustered together(data not shown). TrxA, however, grouped in a cluster togetherwith Flavobacterium, Corynebacterium, and Clostridium Trx homologues.

View larger version (75K):
[in this window]
[in a new window]

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.

The B. fragilis 638R trxB open reading frame contains a 948-bpnucleotide sequence. The deduced amino acid sequence is a 315-amino-acidprotein homologous to other bacterial Trx reductases. When B.fragilis 638R TrxB was compared to other TrxBs, it showed thehighest homology to Bacteroides thetaiotaomicron VPI-5482 TrxB(90% identity and 96% similarity) and P. gingivalis TrxB (84%identity and 80.5% similarity). Strong homology was also foundto TrxBs of E. coli (52.2% identity and 62.3% similarity), Haemophilusinfluenzae (51.4 identity and 61.5% similarity), and Mycobacteriumtuberculosis (49% identity and 59.3% similarity). In addition,the pyridine nucleotide disulfide oxidoreductase class II activesite is conserved in B. fragilis TrxB (data not shown). Thephylogenetic relationship between B. fragilis TrxB and 26 otherTrxB homologues from animals, plants, bacteria, and yeasts wasdetermined from a progressive multiple alignment of the aminoacid sequences, followed by parsimony analysis. This comparisonshowed that B. fragilis TrxB groups with other members of theunique Cytophaga-Flexibacter-Bacteroides phylum (see Fig. S1in the supplemental material).

GSH determination. During analysis of the B. fragilis TrxB/Trx system, we noticedthat there were no Grxs or any of the genes required for GSHbiosynthesis ( ${gamma}$ -glutamylcysteinyl synthetase and glutathionesynthase) in the genome. This suggested that the TrxB/Trx systemmight be the major or sole intracellular thiol/disulfide redoxsystem in this anaerobe. Consistent with this was the findingthat no GSH/GSSG was found in crude extracts of B. fragilisfrom mid-log-phase or stationary-phase cultures (limit of detection,5 pmol in 50 µl of sample or standard). Cultures exposedto aerobic conditions also lacked detectable GSH/GSSG. In contrast,GSH/GSSG was present in the crude extracts of E. coli controlcultures; 22.2 and 7.05 µmol/g (dry weight) were foundin logarithmic- and stationary-phase aerobic cultures, respectively,and in anaerobic cultures the levels were 2.81 and 4.96 µmol/g(dry weight), respectively. These values were comparable tolevels previously described for E. coli under aerobic and anaerobicconditions (9).

Effect of free thiol on growth of a trxB mutant. A trxB mutant was constructed to determine if another mechanismwas present that could maintain reduced cellular thiols andcompensate for loss of the TrxB/Trx system. The availabilityof thiol compounds in the media greatly affected growth of the ${Delta}$ trxB mutant in rich BHIS media and SDM. The ${Delta}$ trxB mutant wasunable to maintain growth in the absence of an exogenous freethiol source in SDM, but addition of cysteine or dithiothreitol(DTT) restored the ability of the trxB strain to grow (Fig.2A and 2B). The growth defect in rich media was less pronounced,probably because of the greater abundance of reduced sulfursources compared to SDM. Taken together, these findings suggestthat in B. fragilis the TrxB/Trx system is the major or solesystem for mediating the intracellular thiol/disulfide equilibrium;otherwise, the ${Delta}$ trxB mutant would not have been so dependenton added thiol for growth.

View larger version (14K):
[in this window]
[in a new window]

FIG. 2. Growth of B. fragilis parent strain 638R and the ${Delta}$ trxB mutant in BHIS (A) and in SDM (B). •, parent strain grown in medium containing cysteine; ${circ}$ , parent strain grown in medium without cysteine; ${blacksquare}$ , trxB mutant grown in medium containing cysteine; ${square}$ , trxB mutant grown in medium without cysteine; ${blacktriangleup}$ , 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.

Regulation of trxB expression by oxidative stress and diamide. The trxB gene was the second gene of what appeared to be a two-geneoperon that also encoded the lipoprotein chaperone, LolA. Theexpression of trxB following exposure to oxygen, to hydrogenperoxide, and to different concentrations of the thiol-specificoxidant diamide was examined by Northern hybridization. Theautoradiographs revealed that trxB mRNA was expressed as anapproximately 0.96-kb monocistronic transcript and as a lessabundant 1.8-kb bicistronic transcript containing the upstreamgene lolA (Fig. 3A). Expression of trxB monocistronic mRNA wasinduced approximately fivefold by both hydrogen peroxide andoxygen compared to anaerobic controls (Fig. 3A). Deletion ofoxyR did not significantly affect the expression of trxB followingoxidative stress, but the basal levels of the trxB transcriptwere slightly higher (twofold) anaerobically in the oxyR backgroundthan in the parent strain. trxB also was induced when cultureswere treated with diamide in a dose-dependent manner, indicatingthat trxB was expressed in response to a thiol/disulfide redoximbalance. Maximum induction of trxB mRNA (approximately sevenfold)was observed with 100 µM diamide compared to untreatedcontrols (Fig. 4). In addition, diamide also induced a 1.8-kblolA trxB bicistronic mRNA, confirming that lolA is cotranscribedwith trxB in B. fragilis following thiol redox stress. WhenNorthern blots were probed with an internal lolA gene fragment,the results confirmed that there was up-regulation of the 1.8-kblolA trxB bicistronic transcript (Fig. 3B) (unpublished data).

View larger version (40K):
[in this window]
[in a new window]

FIG. 3. (A) Northern hybridization analysis of total RNA of B. fragilis 638R (wt), IB298 ( ${Delta}$ oxyR), 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.

View larger version (54K):
[in this window]
[in a new window]

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.

Primer extension analysis of lolA revealed two transcriptioninitiation sites, P1 and P2 (Fig. 5). P1 is a constitutive promoterwhose transcription starts at an adenine nucleotide 26 basesupstream of the translation start codon. In contrast, P2 wasinduced upon exposure to oxidative stress and diamide treatment.P2 transcription starts at a thymine nucleotide 8 bases upstreamof the translation start side. A lolA transcriptional fusionwith the promoterless ß-xylosidase reporter gene confirmedthat the lolA promoter was induced by oxidative stress (datanot shown). In contrast, a trxB transcriptional fusion containingthe lolA-trxB intergenic region extending 238 bases upstreamfrom the trxB translational start site revealed that there wasno transcriptional initiation directly upstream of trxB (datanot shown). Because trxB was also detected as a single mRNAin a Northern blot analysis (Fig. 4A), we performed an RNaseprotection assay to determine whether there might have beenposttranscriptional modification of the lolA trxB bicistronicmRNA. The RNase protection assay used to map the lolA trxB intergenicregion showed that two 5' end protected sites of the trxB mRNAmapped at nucleotide positions –36/–35 and –21/–20with respect to the trxB translational start site (Fig. 3B anddata not shown).

View larger version (45K):
[in this window]
[in a new window]

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.

Sensitivity to diamide and peroxide. Diamide sensitivity mimics thiol oxidation due to oxygen exposurein anaerobic bacteria (29); therefore, sensitivity to diamidewas used to determine whether TrxB plays a role in the thiol/disulfidehomeostasis. As shown in Fig. 6, the trxB mutant was more sensitiveto diamide in disk diffusion assays than the parent strain andan oxyR mutant. When the trxB mutant was complemented with pFD892,the diamide sensitivity was nearly identical to that of theparent stain. Although the oxyR mutation alone had only a smalleffect on diamide sensitivity, the oxyR trxB double mutant showedgreatly increased sensitivity to diamide compared to the singlemutants, especially on the plates exposed to oxygen. This suggeststhat growth inhibition by diamide in both aerobic and anaerobicconditions was likely due to an imbalance in the cellular thiol/disulfidehomeostasis and that there might be a link between thiol redoxand peroxide stress responses in this anaerobic bacterium. Inthis regard, the trxB mutant strain was somewhat more sensitiveto hydrogen peroxide stress than the wild type, and this sensitivitywas additive with oxyR since the oxyR trxB double mutant wasinhibited more by hydrogen peroxide than either of the singlemutants (Fig. 6).

View larger version (36K):
[in this window]
[in a new window]

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.

Sensitivity to oxygen exposure in soft agar tubes. The effect of oxygen on the growth of the trxB strain was examinedin BHIS soft agar tubes incubated in air without added cysteine.Clearance of bacterial growth from the top of a soft agar tubeshowed that the trxB mutant was slightly more sensitive to oxygendiffusion into the agar than the oxyR or parent strain (Fig.7). The parent and oxyR strains had clear zones to 0.4 cm, whilethe trxB strain was inhibited by oxygen down to 0.9 cm intothe agar medium. Interestingly, the oxyR trxB double mutantstrain did not even grow into the bottom of the soft agar (depth,5.5 cm). Addition of cysteine to the medium was not able toovercome the growth inhibition of the double mutant in thisassay. The growth of the oxyR trxB double mutant under anaerobicconditions was similar to that of the parent strain (data notshown).

View larger version (98K):
[in this window]
[in a new window]

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.

Abscess formation. When B. fragilis leaves the anaerobic intestinal environmentto colonize more oxygenated tissues, such as the peritonealcavity, it encounters a hostile environment (46). It is presumedthat protection from oxidative stress is important for survivalin the initial stages of an extraintestinal infection (39, 40).Thus, the ability of a trxB strain to form intraabdominal abscessesin a mouse model was investigated. Compared to the parent strain,the trxB mutant was not able to form abscesses at all infectiondoses (Table 2). In contrast, the parent strain formed abscessesin some animals at every infection dose tested from 10⁶ to 10⁸CFU. Challenge with sterile cecal contents alone was used acontrol, and no abscesses were observed.

View this table:
[in this window]
[in a new window]

TABLE 2. Ability of B. fragilis strains to form intraabdominal abscesses in a mouse model

DISCUSSION

Top

DISCUSSION

In this study we demonstrated that the anaerobic opportunisticpathogen B. fragilis lacks the GSH/Grx system and possessesan extensive number of putative Trx orthologs. In addition,we showed that the TrxB/Trx system is apparently the major orsole system for the thiol/disulfide cellular equilibrium inthis bacterium. While both the GSH/Grx and TrxB/Trx systemsare generally present in facultative gram-negative bacteria,the lack of GSH/Grx in Bacteroides is consistent with reportsshowing the absence of detectable GSH in other anaerobic bacteria(9). The TrxB/Trx system seems to be the major component forthiol/disulfide balance in anaerobes, as well as in gram-positiveorganisms such as Bacillus subtilis, Staphylococcus aureus,and Lactobacillus lactis (47, 52, 54). TrxB homologues are essentialfor growth in both B. subtilis and S. aureus (47, 52) but notin L. lactis, where externally provided GSH or a reducing agentcan partially restore growth in a trxB1 mutant (54). Growthof the B. fragilis trxB mutant in SDM could be restored onlyin the presence of reducing agents, such as L-cysteine or DTT,while the growth deficiency in rich media was less pronounced.This finding could have been due to the presence of free GSHin the media or just the greater overall reducing potentialof rich media. Overall, these findings indicate that TrxB isthe major component in the reduction of oxidized Trxs in B.fragilis since the presence of an alternative thiol-reducingsystem was not evidently present to compensate for the lackof a functional TrxB.

The induction of trxB mRNA by atmospheric oxygen and by thespecific thiol oxidant diamide indicates that TrxB is an importantcomponent of the B. fragilis response to oxidative stress andcellular thiol/disulfide redox imbalance. These findings correlatewith the fact that TrxB is required for maximal growth duringoxidative stress in vitro (Fig. 6 and 7) and is essential forsurvival in vivo in an adverse aerobic environment (Table 2).The oxidative stress regulator of trxB expression was not identifiedin this work, but OxyR did not appear to be directly involved.The OxyR regulon, however, worked together with TrxB to providemaximum protection, as indicated by the additive effect thatthe trxB oxyR double mutations had on the sensitivity of cellsto oxidative stress in vitro. The lolA trxB operon appearedto be regulated at the transcriptional and posttranscriptionallevels. At the transcriptional level the lolA trxB constitutiveand stress-induced promoter regions (P1 and P2, respectively)do not have highly conserved consensus motifs with the previouslydescribed –10 and –35 sequence consensus in B. fragilis(4). This suggests that other regulatory promoter recognitionmechanisms are involved in lolA trxB mRNA expression. Posttranscriptionalregulation of polycistronic mRNA in B. fragilis has not beenpreviously described and represents a new mechanism involvedin oxidative and redox stress response regulation in this aerotolerantanaerobe. This regulation of the lolA trxB operon was unusualin that there was a greater abundance of trxB-specific mRNAthan of the 1.8-kb bicistronic message. This was likely to bedue to an increase in trxB mRNA stability following posttranscriptionalmodification of the lolA trxB mRNA since lolA trxB mRNA hada half-life of less than 1 min, while trxB single mRNA had ahalf-life for decay of around 25 min in the absence of oxygen(unpublished data).

The physiological roles of the Trxs in B. fragilis have notbeen established yet, but the observation that there are sixTrxs suggests that there is a wide range of functions perhapssimilar to those in photosynthetic cyanobacteria and plants,where Trxs have numerous roles (12, 24, 25, 35). Recent studieshave shown that the Trx system is involved in the oxidativestress, metal homeostasis, and stationary-phase responses inother anaerobes (16, 22, 45) and in glycine metabolism in gram-positiveanaerobic bacteria (1, 14 ). B. fragilis has a Trx peroxidasethat is linked to survival during exposure to organic peroxides(16), and it is likely that the Trxs are involved in a varietyof normal and stress response activities.

A classic role of Trx is as the hydrogen donor for class I ribonucleotidereductases (RRases) which are oxygen dependent. In a previousstudy we showed that B. fragilis possesses an oxygen-inducibleclass Ia RRase, NrdAB, which is important for survival duringoxygen exposure because it allows cells to resume growth underanaerobic conditions after aerobic exposure (48). This is consistentwith the fact that B. fragilis can resume growth and proliferateat the site of infection after the establishment of anaerobicconditions (42, 43, 44, 46). In many facultative bacteria, thereare three major classes of RRases which vary in sensitivityand in the requirement for oxygen. Class Ia RRases are usedfor growth in the presence of oxygen, and class III RRases areused for growth in the absence of oxygen (21). The class IIRRase is not affected by oxygen and can function independentof the presence of oxygen. Only class Ia (NrdAB) and class III(NrdDG) RRases are present in B. fragilis (48). Recently, ithas been shown that in E. coli, which possesses all three classesof RRases, the class Ia enzyme, NrdAB, can be reduced by eitherTrx 1 or 2 or by glutaredoxin 1 (13). In this regard, we thinkthat the TrxB/Trx system is likely to be the only reductivesystem linked to NrdAB induced in B. fragilis for anaerobicrecovery following oxygen exposure or oxidative stress. Thisconclusion is based on our findings showing the lack of an alternativeto the TrxB/Trx system.

The complexity of the thiol redox balance in B. fragilis isattributed not only to the presence of an extended number ofTrx homologues but also to the presence of at least 13 additionalthiol-disulfide oxidoreductase-like proteins with the CXXC motifand the Trx fold motif (data not shown). These proteins mayplay many roles in oxidation or reduction pathways in the cytoplasmicmembrane or periplasmis space, similar to the Dsb proteins inE. coli (31). The observation that the gene encoding LolA, aprokaryotic periplasmic lipoprotein-specific molecular chaperone(27, 28), is cotranscribed with trxB in an operon regulatedby oxidative and thiol redox stress supports the idea that someof these proteins are localized in the periplasm and membraneof this anaerobe. Further studies should help us to clarifyhow B. fragilis TrxB, the Trxs, and thiol disulfide oxidoreductase-likeproteins are employed to reduce different cytoplasm and periplasmsubstrates in an anaerobic organism.

The ability of B. fragilis to rapidly respond to an oxidativestress or a redox insult has been associated with its high aerotolerancecompared to that of other anaerobic bacteria (29, 37). Evenamong different strains of B. fragilis the clinical isolateshave been shown to be more aerotolerant than normal flora isolates(41, 51), and this characteristic has been assumed to be essentialfor their ability to survive in the more oxygenated host tissues,such as the peritoneal cavity, when they leave the anaerobicenvironment of the colon (37, 43). In this study, we showedthat the B. fragilis trxB deletion mutant strain completelylost the ability to form intraabdominal abscesses in a mousemodel. Recent reports have shown that Trxs and Trx reductaseplay an important role in the virulence and survival of pathogenicbacteria, yeasts, and protozoans (5, 26, 56) by contributingto the oxidative stress defense against toxic host cell-generatedoxygen-derived radicals. In this regard, we show here that TrxBcontributes to the survival of B. fragilis in the intraabdominalcavity, where the oxygen tension reaches 6% (33). One of theimportant roles of TrxB in the TrxB/Trx system during infectionmay be in the synthesis of deoxyribonucleotides in the presenceof oxygen, or TrxB may contribute to cellular protection byreduction of the Trx peroxidase (16, 48). In addition, animalstudies indicate that in the absence of functional TrxB, B.fragilis is unable to utilize host reduced thiol compounds assources for thiol reducing power to overcome the growth deficiencyin vivo. Further studies of this complex TrxB/Trx system shouldhelp clarify its role during normal growth in the colon andduring adaptation in more aerobic extraintestinal sites.

ACKNOWLEDGMENTS

We thank Richard A. Franklin for assistance with the GSH/GSSGcycling enzyme assay utilizing glutathione reductase.

This work was supported in part by Public Health Service grantAI40588 to C.J.S. and by Public Health Service grant AI068659to E.R.R.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology & Immunology, East Carolina University Brody School of Medicine, 600 Moye Blvd., Greenville, NC 27834. Phone: (252) 744-2700. Fax: (252) 744-3104. E-mail: smithcha{at}ecu.edu

Published ahead of print on 14 September 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Shire Human Genetic Therapies, 700 Main St.,Cambridge, MA 02139.

REFERENCES

Top