Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

Analysis of Six tonB Gene Homologs in Bacteroides fragilis Revealed That tonB3 is Essential for Survival in Experimental Intestinal Colonization and Intra-Abdominal Infection.

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp), and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB's role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro. Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1, ΔtonB2, ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain, which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1, ΔtonB2, ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo. The genetic organization of tonB1, tonB2, tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space-in contrast to an oxidative environment in aerobes-it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

Characterization of the oxidative stress response regulatory network in Bacteroides fragilis: An interaction between BmoR and OxyR regulons promotes abscess formation in a model of intra-abdominal infection.

OBJECTIVES: Bacteroides fragilis is an anaerobic bacterium that is commonly found in the human gut microbiota and an opportunistic pathogen in extra-intestinal infections. B. fragilis displays a robust response to oxidative stress which allows for survival in oxygenated tissues such as the peritoneal cavity and lead to the formation of abscesses. In this study, we investigated the synergy of the oxidative stress response regulators OxyR and BmoR in the ability of B. fragilis to resist oxidative damage and to survive in extra-intestinal infection. METHODS: A ΔbmoR ΔoxyR double mutant B. fragilis strain was constructed, and its oxidative stress response was compared to parental and single mutant strains in phenotypical assays and gene expression analysis. The pathogenic potential in an in vivo mouse model of abscess formation was also evaluated. RESULTS: Expression analysis showed a coordinated control of thioredoxin C (trxC) gene expression by BmoR and OxyR during oxygen exposure, with upregulation of trxC in the bmoR mutant strain (4.9-fold increase), downregulation in the oxyR mutant (2.5-fold decrease), and an intermediate level of deregulation (2-fold increase) in the double mutant strain compared to the parent strain. Expression analysis during oxidative stress conditions also showed that BmoR is a major repressor of the CoA-disulfide reductase gene (upregulated 47-fold in the bmoR mutant) while OxyR plays a minor repression role in this gene (upregulated 2.5-fold in the oxyR mutant). Exposure to atmospheric oxygen for up to 72 h revealed that the deletion of bmoR alone had no significant effect in in vitro survival phenotype assays, though it partially abolishes the OxyR sensitivity phenotype in the bmoR/oxyR double mutant strain compared to oxyR mutant. In vivo assays showed that bmoR and oxyR mutants were significantly impaired in the formation and development of abscesses compared to the parent strain in an experimental intra-abdominal infection mouse model. CONCLUSION: Although the full extent of genes whose expression are modulated by BmoR and OxyR is yet to be defined, we present evidence that these regulators have overlapping functions in B. fragilis response to oxidative stress and ability to form abscess in extra-intestinal tissues.

The uroS and yifB Genes Conserved among Tetrapyrrole Synthesizing-Deficient Bacteroidales Are Involved in Bacteroides fragilis Heme Assimilation and Survival in Experimental Intra-abdominal Infection and Intestinal Colonization.

The human intestinal anaerobic commensal and opportunistic pathogen Bacteroides fragilis does not synthesize the tetrapyrrole protoporphyrin IX in order to form heme that is required for growth stimulation and survival in vivo Consequently, B. fragilis acquires essential heme from host tissues during extraintestinal infection. The absence of several genes necessary for de novo heme biosynthesis is a common characteristic of many anaerobic bacteria; however, the uroS gene, encoding a uroporphyrinogen III synthase for an early step of heme biosynthesis, is conserved among the heme-requiring Bacteroidales that inhabit the mammalian gastrointestinal tract. In this study, we show that the ability of B. fragilis to utilize heme or protoporphyrin IX for growth was greatly reduced in a ΔuroS mutant. This growth defect appears to be linked to the suppression of reverse chelatase and ferrochelatase activities in the absence of uroS In addition, this ΔuroS suppressive effect was enhanced by the deletion of the yifB gene, which encodes an Mg2+-chelatase protein belonging to the ATPases associated with various cellular activities (AAA+) superfamily of proteins. Furthermore, the ΔuroS mutant and the ΔuroS ΔyifB double mutant had a severe survival defect compared to the parent strain in competitive infection assays using animal models of intra-abdominal infection and intestinal colonization. This shows that the presence of the uroS and yifB genes in B. fragilis seems to be linked to pathophysiological and nutritional competitive fitness for survival in host tissues. Genetic complementation studies and enzyme kinetics assays indicate that B. fragilis UroS is functionally different from canonical bacterial UroS proteins. Taken together, these findings show that heme assimilation and metabolism in the anaerobe B. fragilis have diverged from those of aerobic and facultative anaerobic pathogenic bacteria.

Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections.

Bacteroides fragilis is the most common anaerobe isolated from clinical infections, and in this report we demonstrate a characteristic of the species that is critical to their success as an opportunistic pathogen. Among the Bacteroides spp. in the gut, B. fragilis has the unique ability of efficiently harvesting complex N-linked glycans from the glycoproteins common to serum and serous fluid. This activity is mediated by an outer membrane protein complex designated as Don. Using the abundant serum glycoprotein transferrin as a model, it has been shown that B. fragilis alone can rapidly and efficiently deglycosylate this protein in vitro and that transferrin glycans can provide the sole source of carbon and energy for growth in defined media. We then showed that transferrin deglycosylation occurs in vivo when B. fragilis is propagated in the rat tissue cage model of extraintestinal growth, and that this ability provides a competitive advantage in vivo over strains lacking the don locus.

Dps and DpsL Mediate Survival In Vitro and In Vivo during the Prolonged Oxidative Stress Response in Bacteroides fragilis.

UNLABELLED: Bacteroides fragilis is a Gram-negative anaerobe and member of the human intestinal tract microbiome, where it plays many beneficial roles. However, translocation of the organism to the peritoneal cavity can lead to peritonitis, intra-abdominal abscess formation, bacteremia, and sepsis. During translocation, B. fragilis is exposed to increased oxidative stress from the oxygenated tissues of the peritoneal cavity and the immune response. In order to survive, B. fragilis mounts a robust oxidative stress response consisting of an acute and a prolonged oxidative stress (POST) response. This report demonstrates that the ability to induce high levels of resistance to tert-butyl hydroperoxide (tBOOH) after extended exposure to air can be linked to the POST response. Disk diffusion assays comparing the wild type to a Δdps mutant and a Δdps Δbfr mutant showed greater sensitivity of the mutants to tBOOH after exposure to air, suggesting that Dps and DpsL play a role in the resistance phenotype. Complementation studies with dps or bfr (encoding DpsL) restored tBOOH resistance, suggesting a role for both of these ferritin-family proteins in the response. Additionally, cultures treated with the iron chelator dipyridyl were not killed by tBOOH, indicating Dps and DpsL function by sequestering iron to prevent cellular damage. An in vivo animal model showed that the Δdps Δbfr mutant was attenuated, indicating that management of iron is important for survival within the abscess. Together, these data demonstrate a role for Dps and DpsL in the POST response which mediates survival in vitro and in vivo. IMPORTANCE: B. fragilis is the anaerobe most frequently isolated from extraintestinal opportunistic infections, but there is a paucity of information about the factors that allow this organism to survive outside its normal intestinal environment. This report demonstrates that the iron storage proteins Dps and DpsL protect against oxidative stress and that they contribute to survival both in vitro and in vivo. Additionally, this work demonstrates an important role for the POST response in B. fragilis survival and provides insight into the complex regulation of this response.

Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis.

BACKGROUND: Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear. METHODS: A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole. RESULTS: Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo. CONCLUSIONS: Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.

The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis.

Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis cannot be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis.

Ferritin-like family proteins in the anaerobe Bacteroides fragilis: when an oxygen storm is coming, take your iron to the shelter.

Bacteroides are gram-negative anaerobes and one of the most abundant members the lower GI tract microflora where they play an important role in normal intestinal physiology. Disruption of this commensal relationship has a great impact on human health and disease. Bacteroides spp. are significant opportunistic pathogens causing infections when the mucosal barrier integrity is disrupted following predisposing conditions such as GI surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma and inflammatory bowel diseases. B. fragilis accounts for 60-90 % of all anaerobic infections despite being a minor component of the genus (<1 % of the flora). Clinical strains of B. fragilis are among the most aerotolerant anaerobes. When shifted from anaerobic to aerobic conditions B. fragilis responds to oxidative stress by inducing the expression of an extensive set of genes involved in protection against oxygen derived radicals and iron homeostasis. In Bacteroides, little is known about the metal/oxidative stress interactions and the mobilization of intra-cellular non-heme iron during the oxidative stress response has been largely overlooked. Here we present an overview of the work carried out to demonstrate that both oxygen-detoxifying enzymes and iron-storage proteins are essential for B. fragilis to survive an adverse oxygen-rich environment. Some species of Bacteroides have acquired multiple homologues of the iron storage and detoxifying ferritin-like proteins but some species contain none. The proteins found in Bacteroides are classical mammalian H-type non-heme ferritin (FtnA), non-specific DNA binding and starvation protein (Dps) and the newly characterized bacterial Dps-Like miniferritin protein. The full contribution of ferritin-like proteins to pathophysiology of commensal and opportunistic pathogen Bacteroides spp. still remains to be elucidated.

Bile salt hydrolase in non-enterotoxigenic Bacteroides potentiates colorectal cancer.

Bile salt hydrolase (BSH) in Bacteroides is considered a potential drug target for obesity-related metabolic diseases, but its involvement in colon tumorigenesis has not been explored. BSH-expressing Bacteroides is found at high abundance in the stools of colorectal cancer (CRC) patients  with overweight and in the feces of a high-fat diet (HFD)-induced CRC mouse model. Colonization of B. fragilis 638R, a strain with low BSH activity, overexpressing a recombinant bsh gene from B. fragilis NCTC9343 strain, results in increased unconjugated bile acids in the colon and accelerated progression of CRC under HFD treatment. In the presence of high BSH activity, the resultant elevation of unconjugated deoxycholic acid and lithocholic acid activates the G-protein-coupled bile acid receptor, resulting in increased ß-catenin-regulated chemokine (C-C motif) ligand 28 (CCL28) expression in colon tumors. Activation of the ß-catenin/CCL28 axis leads to elevated intra-tumoral immunosuppressive CD25+FOXP3+ Treg cells. Blockade of the ß-catenin/CCL28 axis releases the immunosuppression to enhance the intra-tumoral anti-tumor response, which decreases CRC progression under HFD treatment. Pharmacological inhibition of BSH reduces HFD-accelerated CRC progression, coincident with suppression of the ß-catenin/CCL28 pathway. These findings provide insights into the pro-carcinogenetic role of Bacteroides in obesity-related CRC progression and characterize BSH as a potential target for CRC prevention and treatment.

Characterization of the Bacteroides fragilis bfr gene product identifies a bacterial DPS-like protein and suggests evolutionary links in the ferritin superfamily.

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O(2) or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer.

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 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.

Bacteroides fragilis requires the ferrous-iron transporter FeoAB and the CobN-like proteins BtuS1 and BtuS2 for assimilation of iron released from heme.

The intestinal commensal and opportunistic anaerobic pathogen Bacteroides fragilis has an essential requirement for both heme and free iron to support growth in extraintestinal infections. In the absence of free iron, B. fragilis can utilize heme as the sole source of iron. However, the mechanisms to remove iron from heme are not completely understood. In this study, we show that the inner membrane ferrous iron transporter ∆feoAB mutant strain is no longer able to grow with heme as the sole source of iron. Genetic complementation with the feoAB gene operon completely restored growth. Our data indicate that iron is removed from heme in the periplasmic space, and the released iron is transported by the FeoAB system. Interestingly, when B. fragilis utilizes iron from heme, it releases heme-derived porphyrins by a dechelatase activity which is upregulated under low iron conditions. This is supported by the findings showing that formation of heme-derived porphyrins in the ∆feoAB mutant and the parent strain increased 30-fold and fivefold (respectively) under low iron conditions compared to iron replete conditions. Moreover, the btuS1 btuS2 double-mutant strain (lacking the predicted periplasmic, membrane anchored CobN-like proteins) also showed growth defect with heme as the sole source of iron, suggesting that BtuS1 and BtuS2 are involved in heme-iron assimilation. Though the dechelatase mechanism remains uncharacterized, assays performed in bacterial crude extracts show that BtuS1 and BtuS2 affect the regulation of the dechelatase-specific activities in an iron-dependent manner. These findings suggest that the mechanism to extract iron from heme in Bacteroides requires a group of proteins, which spans the periplasmic space to make iron available for cellular functions.

Deletion of BmoR affects the expression of genes related to thiol/disulfide balance in Bacteroides fragilis.

Bacteroides fragilis, an opportunistic pathogen and commensal bacterium in the gut, is one the most aerotolerant species among strict anaerobes. However, the mechanisms that control gene regulation in response to oxidative stress are not completely understood. In this study, we show that the MarR type regulator, BmoR, regulates the expression of genes involved in the homeostasis of intracellular redox state. Transcriptome analysis showed that absence of BmoR leads to altered expression in total of 167 genes. Sixteen of these genes had a 2-fold or greater change in their expression. Most of these genes are related to LPS biosynthesis and carbohydrates metabolism, but there was a significant increase in the expression of genes related to the redox balance inside the cell. A pyridine nucleotide-disulfide oxidoreductase located directly upstream of bmoR was shown to be repressed by direct binding of BmoR to the promoter region. The expression of two other genes, coding for a thiosulphate:quinone-oxidoreductase and a thioredoxin, are indirectly affected by bmoR mutation during oxygen exposure. Phenotypic assays showed that BmoR is important to maintain the thiol/disulfide balance in the cell, confirming its relevance to B. fragilis response to oxidative stress.

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, 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.

Anaerobic utilization of Fe(III)-xenosiderophores among Bacteroides species and the distinct assimilation of Fe(III)-ferrichrome by Bacteroides fragilis within the genus.

In this study, we show that Bacteroides species utilize Fe(III)-xenosiderophores as the only source of exogenous iron to support growth under iron-limiting conditions in vitro anaerobically. Bacteroides fragilis was the only species able to utilize Fe(III)-ferrichrome while Bacteroides vulgatus ATCC 8482 and Bacteroides thetaiotaomicron VPI 5482 were able to utilize both Fe(III)-enterobactin and Fe(III)-salmochelin S4 as the only source of iron in a dose-dependent manner. We have investigated the way B. fragilis assimilates Fe(III)-ferrichrome as initial model to understand the utilization of xenosiderophores in anaerobes. B. fragilis contains two outer membrane TonB-dependent transporters (TBDTs), FchA1 and FchA2, which are homologues to Escherichia coli ferrichrome transporter FhuA. The disruption of fchA1 gene had only partial growth defect on Fe(III)-ferrichrome while the fchA2 mutant had no growth defect compared to the parent strain. The genetic complementation of fchA1 gene restored growth to parent strain levels indicating that it plays a role in Fe(III)-ferrichrome assimilation though we cannot rule out some functional overlap in transport systems as B. fragilis contains abundant TBDTs whose functions are yet not understood. However, the growth of B. fragilis on Fe(III)-ferrichrome was abolished in a feoAB mutant indicating that Fe(III)-ferrichrome transported into the periplasmic space was reduced in the periplasm releasing ferrous iron prior to transport through the FeoAB transport system. Moreover, the release of iron from the ferrichrome may be linked to the thiol redox system as the trxB deletion mutant was also unable to grow in the presence of Fe(III)-ferrichrome. The genetic complementation of feoAB and trxB mutants completely restored growth on Fe(III)-ferrichrome. Taken together, these findings show that Bacteroides species have developed mechanisms to utilize ferric iron bound to xenosiderophores under anaerobic growth conditions though the regulation and role in the biology of Bacteroides in the anaerobic intestinal environment remain to be understood.

Genetic analysis of an important oxidative stress locus in the anaerobe Bacteroides fragilis.

The obligate anaerobe, Bacteroides fragilis, is a highly aerotolerant intestinal tract organism that has evolved a complex oxidative stress response (OSR). The redox regulator OxyR controls several OSR genes (katB, dps, and ahpC), but there is little else known about other genes it regulates. To identify additional genes in the OxyR regulon, two-dimensional gel electrophoresis was used to isolate proteins from a mutant that constitutively expresses genes in the regulon. The 28,500 Da protein thioredoxin peroxidase (Tpx) was identified. Two additional genes induced during oxidative stress were identified adjacent to tpx, a putative RNA-binding protein (rbpA) and a cytochrome-c peroxidase (ccp). Transcriptional analyses showed that tpx and rbpA were transcribed as monocistronic mRNA species or as a bicistronic operon. Transcription of tpx was induced by exposure to air or H(2)O(2) from an OxyR-dependent promoter and to a lesser extent from a second OxyR-independent promoter. Expression of the rbpA gene during oxidative stress was regulated by the OxyR-dependent tpx promoter resulting in the bicistronic tpx/rbp mRNA. The ccp gene was expressed only as a monocistronic message and induction was only observed after exposure to H(2)O(2) in an OxyR-independent manner. Disruption of the tpx operon or ccp resulted in sensitivity to the organic peroxides cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP) but not to H(2)O(2). This work brings the total of oxyR-controlled genes in B. fragilis to five and suggests the existence of a second peroxide response regulator that controls ccp expression.

Expression of Bacteroides fragilis hemolysins in vivo and role of HlyBA in an intra-abdominal infection model.

Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis "ping-pong" ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after 4 days and 12-fold after 8 days postinfection. Expression of hlyC mRNA increased sixfold after 1 day, 45-fold after 4 days, and 16-fold after 8 days postinfection, respectively. The hlyD and hlyE mRNAs were induced approximately 40-fold and 30-fold, respectively, after 4-days postinfection. The hlyF expression increased approximately threefold after 4-days postinfection. hlyG was induced approximately fivefold after 4 and 8 days postinfection. The hlyIII mRNA levels had a steady increase of approximately four-, eight-, and 12-fold following 1, 4, and 8 days postinfection, respectively. These findings suggest that B. fragilis hemolysins are induced and differentially regulated in vivo. Both parent and hlyBA mutant strains reached levels of approximately 3-8 × 10(9) cfu/mL after 1 day postinfection. However, the hlyBA mutant strain lost 2 logs in viable cell counts compared with the parent strain after 8 days postinfection. This is the first study showing HlyBA is a virulence factor which plays a role in B. fragilis survival in an intra-abdominal abscess model.

Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis.

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies.

The interaction of Bacteroides fragilis with components of the human fibrinolytic system.

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis.