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.

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.

cis-Encoded Small RNAs, a Conserved Mechanism for Repression of Polysaccharide Utilization in Bacteroides.

UNLABELLED: Bacteroides is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host and a potential role in a wide range of disease syndromes. The predominance of this genus is due in large part to expansion of paralogous gene clusters, termed polysaccharide utilization loci (PULs), dedicated to the uptake and catabolism of host-derived and dietary polysaccharides. The nutritive value and availability of polysaccharides in the gut vary greatly; thus, their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that induce PUL expression in response to the presence of specific glycan substrates. However, the existence of additional regulatory mechanisms has been predicted to explain phenomena such as hierarchical control and catabolite repression. In this report, a previously unknown layer of regulatory control was discovered in Bacteroides fragilis Exploratory transcriptome sequencing (RNA-seq) analysis revealed the presence of cis-encoded antisense small RNAs (sRNAs) associated with 15 (30%) of the B. fragilis PULs. A model system using the Don (degradation of N-glycans) PUL showed that the donS sRNA negatively regulated Don expression at the transcriptional level, resulting in a decrease in N-glycan utilization. Additional studies performed with other Bacteroides species indicated that this regulatory mechanism is highly conserved and, interestingly, that the regulated PULs appear to be closely linked to the utilization of host-derived glycans rather than dietary plant polysaccharides. The findings described here demonstrate a global control mechanism underlying known PUL regulatory circuits and provide insight into regulation of Bacteroides physiology. IMPORTANCE: The human gut is colonized by a dense microbiota which is essential to the health and normal development of the host. A key to gut homeostasis is the preservation of a stable, diverse microbiota. Bacteroides is a dominant genus in the gut, and the ability of Bacteroides species to efficiently compete for a wide range of glycan energy sources is a crucial advantage for colonization. Glycan utilization is mediated by a large number of polysaccharide utilization loci (PULs) which are regulated by substrate induction. In this report, a novel family of antisense sRNAs is described whose members repress gene expression in a distinct subset of PULs. This repression downregulates PUL expression in the presence of energy sources that are more readily utilized such as glucose, thereby allowing efficient glycan utilization.

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.

Targeted antisense radiotherapy and dose fractionation using a (177)Lu-labeled anti-bcl-2 peptide nucleic acid-peptide conjugate.

INTRODUCTION: The overall goal of these studies was to test the hypothesis that simultaneous down-regulation of a tumor survival gene and delivery of internally emitted cytotoxic radiation will be more effective than either treatment modality alone. The objectives were to evaluate the therapeutic efficacy of a (177)Lu-labeled anti-bcl-2-PNA-Tyr(3)-octreotate antisense conjugate in a mouse model bearing human non-Hodgkin's lymphoma (NHL) tumor xenografts and to optimize targeted antisense radiotherapy by dose fractionation. METHODS: In the initial therapy studies, tumor-bearing mice were given saline, nonradioactive DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate, (177)Lu-DOTA-Tyr(3)-octreotate, (177)Lu-DOTA-PNA-peptide alone, or (177)Lu-DOTA-PNA-peptide followed by a chase dose of nonradioactive PNA-peptide. The MTD of (177)Lu-DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate was then determined. Subsequently single dose MTD and four weekly fractionated doses were directly compared, followed by histopathologic evaluation. RESULTS: Antisense radiotherapy using 4.44 MBq of the (177)Lu-DOTA-PNA-peptide followed by nonradioactive PNA-peptide was significantly more effective than other low dose treatment regimens. A dose of 18.5 MBq of (177)Lu-DOTA-PNA-peptide was determined to be the approximate maximum tolerated dose (MTD). The median times to progression to a 1cm(3) tumor volume were 32 and 49 days for single dose MTD and fractionated dose (4 × 4.63 MBq) groups, respectively. Histopathology revealed metastases in the single dose groups, but not in the dose fractionation group. CONCLUSIONS: Targeted antisense radiotherapy using (177)Lu-DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate and DOTA-PNA-peptide conjugate effectively inhibited tumor progression in a mouse model of NHL. Furthermore, a dose fractionation regimen had a significant advantage over a single high dose, in terms of tumor growth inhibition and prevention of metastasis. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Down-regulating bcl-2, an anti-apoptotic proto-oncogene, is a mechanism to reverse chemotherapy resistance or failure in humans with NHL. We have developed a (177)Lu-DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate conjugate for targeted antisense radiotherapy, in which down-regulation of bcl-2 and delivery of cytotoxic radiation occur simultaneously. Our previous studies have shown highly specific inhibition of bcl-2 protein, additive in vitro cytotoxic effects on human lymphoma cells, and favorable biodistribution and dosimetric properties. Lutetium-177 targeted antisense radiotherapy demonstrates a significant advantage over conventional (177)Lu-peptide receptor radionuclide therapy in a mouse model of NHL. Our preclinical studies identified an effective combination of antisense and radionuclide therapy, with the goal of future clinical trials in patients.

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.

Bombesin peptide conjugated gold nanocages internalize via clathrin mediated endocytosis.

The nature of interaction and mechanism of internalization of receptor-avid peptide nanoparticles with cells is not yet completely understood. This article describes the cellular internalization mechanism and intracellular trafficking of peptide conjugated receptor targeted porous Gold nanocages (AuNCs) in cancer cells. We synthesized and characterized a library of AuNCs conjugated with bombesin (BBN) peptide. Evidence of selective affinity of AuNC-BBN toward gastrin releasing peptide receptors (GRPR) was obtained using radiolabeled competitive cell binding assay. Endocytic mechanism was investigated using cell inhibitor studies and monitored using optical and transmission electron microscopy (TEM). Results show AuNC-BBN uptake in PC3 cells is mediated by clathrin mediated endocytosis (CME). Indeed, in the presence of CME inhibitors, AuNC-BBN uptake in cells is reduced up to 84%. TEM images further confirm CME characteristic clathrin coated pits and lysosomal release of AuNCs. These results demonstrate that peptide ligands conjugated to the surface of nanoparticles maintain their target specificity. This bolsters the case for peptide robustness and its persisting functionality in intracellular vehicular delivery systems.

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 recA operon: A novel stress response gene cluster in Bacteroides fragilis.

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections.

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.

The extracytoplasmic function sigma factor EcfO protects Bacteroides fragilis against oxidative stress.

The anaerobe Bacteroides fragilis is a highly aerotolerant, opportunistic pathogen that is an important component of the human intestinal microbiota. Aerotolerance has been linked to a robust oxidative stress response, which in turn is necessary for maximal virulence in a mouse intra-abdominal abscess model. During oxidative stress, there is a dynamic change in gene expression that encompasses a third of the genome, but there is a paucity of information on factors that control this response. A large number of transcription regulators, including about 14 extracytoplasmic function (ECF) sigma factors, are affected by oxidative stress, and one of these, EcfO, was used as a model of ECF sigma factor activity during stress. Genetic and biochemical experiments showed that EcfO was located in an operon with a structurally unique anti-sigma factor, Reo. Cells deleted for EcfO were impaired during exposure to oxygen or other forms of oxidative stress, whereas reo mutants were more resistant to stress. Protein-protein interaction experiments demonstrated that Reo directly interacts with and regulates the activity of EcfO. Expression microarray and chromatin affinity precipitation assays were used to identify target genes regulated by EcfO, and an EcfO recognition sequence was identified. The results revealed that EcfO controls a regulon of novel lipoproteins whose distribution in nature is restricted to members of the Bacteroidetes phylum.

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.

Nuclear targeting with cell-specific multifunctional tricarbonyl M(I) (M is Re, (99m)Tc) complexes: synthesis, characterization, and cell studies.

Auger-emitting radionuclides such as (99m)Tc have been the focus of recent studies aiming at finding more selective therapeutic approaches. To explore the potential usefulness of (99m)Tc as an Auger emitter, we have synthesized and biologically evaluated novel multifunctional structures comprising (1) a pyrazolyl-diamine framework bearing a set of donor atoms to stabilize the [M(CO)(3)](+) (M is Re, (99m)Tc) core; (2) a DNA intercalating moiety of the acridine orange type to ensure close proximity of the radionuclide to DNA and to follow the internalization and subcellular trafficking of the compounds by confocal fluorescence microscopy; and (3) a bombesin (BBN) analogue of the type X-BBN[7-14] (where X is SGS, GGG) to provide specificity towards cells expressing the gastrin releasing peptide receptor (GRPr). Of the evaluated (99m)Tc complexes, Tc ( 3 ) containing the GGG-BBN[7-14] peptide showed the highest cellular internalization in GRPr-positive PC3 human prostate tumor cells, presenting a remarkably high nuclear uptake in the same cell line. Live-cell confocal imaging microscopy studies with the congener Re complex, Re ( 3 ), showed a considerable accumulation of fluorescence in the nucleus, with kinetics of uptake similar to that exhibited by Tc ( 3 ). Together, these data show that the acridine orange intercalator and the metal fragment are colocalized in the nucleus, which indicates that they remain connected despite the lysosomal degradation of Tc ( 3 )/Re ( 3 ). These compounds are the first examples of (99m)Tc bioconjugates that combine specific cell targeting with nuclear internalization, a crucial issue to explore use of (99m)Tc in Auger therapy.

Genetic and functional analyses of the mob operon on conjugative transposon CTn341 from Bacteroides spp.

Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Radioactive gold nanoparticles in cancer therapy: therapeutic efficacy studies of GA-198AuNP nanoconstruct in prostate tumor-bearing mice.

Biocompatibility studies and cancer therapeutic applications of nanoparticulate beta-emitting gold-198 (198Au; beta(max) = 0.96 MeV; half-life of 2.7 days) are described. Gum arabic glycoprotein (GA)-functionalized gold nanoparticles (AuNPs) possess optimum sizes (12-18 nm core diameter and 85 nm hydrodynamic diameter) to target individual tumor cells and penetrate through tumor vasculature and pores. We report the results of detailed in vivo therapeutic investigations demonstrating the high tumor affinity of GA-198AuNPs in severely compromised immunodeficient (SCID) mice bearing human prostate tumor xenografts. Intratumoral administration of a single dose of beta-emitting GA-198AuNPs (70 Gy) resulted in clinically significant tumor regression and effective control in the growth of prostate tumors over 30 days. Three weeks after administration of GA-198AuNPs, tumor volumes for the treated animals were 82% smaller as compared with tumor volume of control group. The treatment group showed only transitory weight loss in sharp contrast to the tumor-bearing control group, which underwent substantial weight loss. Pharmacokinetic studies have provided unequivocal evidence for the optimum retention of therapeutic payload of GA-198AuNPs within the tumor site throughout the treatment regimen with minimal or no leakage of radioactivity to various nontarget organs. The measurements of white and red blood cells, platelets, and lymphocytes within the treatment group resembled those of the normal SCID mice, thus providing further evidence on the therapeutic efficacy and concomitant in vivo tolerance and nontoxic features of GA-198AuNPs. FROM THE CLINICAL EDITOR: In this study, the biocompatibility and cancer therapeutic applications of glycoprotein (GA) functionalized gold nanoparticles containing b-emitting Au-198 are described in SCID mice bearing human prostate tumor xenografts. The findings of significant therapeutic efficacy, good in vivo tolerance and non-toxic features make these particles ideal candidates for future human applications.

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.

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.

Laboratory maintenance and cultivation of bacteroides species.

Bacteroides species are Gram-negative, obligate anaerobic bacteria. They are the predominant indigenous bacterial species in the human intestinal tract, where they play an important role in the normal physiology of the host, but they can also be significant opportunistic pathogens. The fact that these are obligate anaerobes is the prevailing feature that affects the methodology used for their cultivation. Several techniques are described for anaerobic culturing; the anaerobic glove box and anaerobic jars are the two techniques most adaptable to a range of research needs. Straightforward methods are presented for propagation on solid media and in broth cultures, and for the long-term storage and maintenance of stock cultures. The Bacteroides species are saccharolytic, which is the second feature of their physiology that impacts cultivation methodology. Several flexible media formulations, including a defined minimal media, are provided that allow the researcher to choose the carbon source best suited for his or her work.

The Bacteroides fragilis transcriptome response to oxygen and H2O2: the role of OxyR and its effect on survival and virulence.

The intestinal anaerobic symbiont, Bacteroides fragilis, is highly aerotolerant and resistant to H(2)O(2). Analysis of the transcriptome showed that expression of 45% of the genome was significantly affected by oxidative stress. The gene expression patterns suggested that exposure to oxidative stress induced an acute response to rapidly minimize the immediate effects of reactive oxygen species, then upon extended exposure a broad metabolic response was induced. This metabolic response induced genes encoding enzymes that can supply reducing power for detoxification and restore energy-generating capacity. An integral aspect of the metabolic response was downregulation of genes related to translation and biosynthesis which correlated with decreased growth and entry into a stationary phase-like growth state. Examination of oxyR mutants showed that they were impaired for the acute response and they induced the expanded metabolic response with only minimal exposure to stress. The oxyR mutants were more sensitive to oxidants in vitro and in vivo they were attenuated in an intra-abdominal abscess infection model. Aerotolerance and resistance to oxidative stress are physiological adaptations of B. fragilis to its environment that enhance survival in extra-intestinal sites and promote opportunistic infections.