The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production by Staphylococcus aureus.

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O-phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.

A Ferric-Peroxo Intermediate in the Oxidation of Heme by IsdI.

The canonical heme oxygenases (HOs) catalyze heme oxidation via a heme-bound hydroperoxo intermediate that is stabilized by a water cluster at the active site of the enzyme. In contrast, the hydrophobic active site of IsdI, a heme-degrading enzyme from Staphylococcus aureus, lacks a water cluster and is expected to oxidize heme by an alternative mechanism. Reaction of the IsdI-heme complex with either H2O2 or m-chloroperoxybenzoic acid fails to produce a specific oxidized heme iron intermediate, suggesting that ferric-hydroperoxo or ferryl derivatives of IsdI are not involved in the catalytic mechanism of this enzyme. IsdI lacks a proton-donating group in the distal heme pocket, so the possible involvement of a ferric-peroxo intermediate has been evaluated. Density functional theory (DFT) calculations indicate that heme oxidation involving a ferric-peroxo intermediate is energetically accessible, whereas the energy barrier for a reaction involving a ferric-hydroperoxo intermediate is too great in the absence of a proton donor. We propose that IsdI catalyzes heme oxidation through nucleophilic attack by the heme-bound peroxo species. This proposal is consistent with our previous demonstration by nuclear magnetic resonance spectroscopy that heme ruffling increases the susceptibility of the meso-carbon of heme to nucleophilic attack.

IruO is a reductase for heme degradation by IsdI and IsdG proteins in Staphylococcus aureus.

Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K(m) of 15 ± 4 µM, a k(cat) of 5.2 ± 0.7 min(-1), and a k(cat)/K(m) of 5.8 × 10(3) M(-1) s(-1). From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-ß-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.

Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis.

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

Transcriptional responses of Burkholderia cenocepacia to polymyxin B in isogenic strains with diverse polymyxin B resistance phenotypes.

BACKGROUND: Burkholderia cenocepacia is a Gram-negative opportunistic pathogen displaying high resistance to antimicrobial peptides and polymyxins. We identified mechanisms of resistance by analyzing transcriptional changes to polymyxin B treatment in three isogenic B. cenocepacia strains with diverse polymyxin B resistance phenotypes: the polymyxin B-resistant parental strain K56-2, a polymyxin B-sensitive K56-2 mutant strain with heptoseless lipopolysaccharide (LPS) (RSF34), and a derivative of RSF34 (RSF34 4000B) isolated through multiple rounds of selection in polymyxin B that despite having a heptoseless LPS is highly polymyxin B-resistant. RESULTS: A heptoseless LPS mutant of B. cenocepacia was passaged through multiple rounds of selection to regain high levels of polymyxin B-resistance. This process resulted in various phenotypic changes in the isolate that could contribute to polymyxin B resistance and are consistent with LPS-independent changes in the outer membrane. The transcriptional response of three B. cenocepacia strains to subinhibitory concentrations of polymyxin B was analyzed using microarray analysis and validated by quantitative Real Time-PCR. There were numerous baseline changes in expression between the three strains in the absence of polymyxin B. In both K56-2 and RSF34, similar transcriptional changes upon treatment with polymyxin B were found and included upregulation of various genes that may be involved in polymyxin B resistance and downregulation of genes required for the synthesis and operation of flagella. This last result was validated phenotypically as both swimming and swarming motility were impaired in the presence of polymyxin B. RSF34 4000B had altered the expression in a larger number of genes upon treatment with polymyxin B than either K56-2 or RSF34, but the relative fold-changes in expression were lower. CONCLUSIONS: It is possible to generate polymyxin B-resistant isolates from polymyxin B-sensitive mutant strains of B. cenocepacia, likely due to the multifactorial nature of polymyxin B resistance of this bacterium. Microarray analysis showed that B. cenocepacia mounts multiple transcriptional responses following exposure to polymyxin B. Polymyxin B-regulated genes identified in this study may be required for polymyxin B resistance, which must be tested experimentally. Exposure to polymyxin B also decreases expression of flagellar genes resulting in reduced swimming and swarming motility.

A decade of Burkholderia cenocepacia virulence determinant research.

The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immunocompromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.

Iron Uptake Oxidoreductase (IruO) Uses a Flavin Adenine Dinucleotide Semiquinone Intermediate for Iron-Siderophore Reduction.

Many pathogenic bacteria including Staphylococcus aureus use iron-chelating siderophores to acquire iron. Iron uptake oxidoreductase (IruO), a flavin adenine dinucleotide (FAD)-containing nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase from S. aureus, functions as a reductase for IsdG and IsdI, two paralogous heme degrading enzymes. Also, the gene encoding for IruO was shown to be required for growth of S. aureus on hydroxamate siderophores as a sole iron source. Here, we show that IruO binds the hydroxamate-type siderophores desferrioxamine B and ferrichrome A with low micromolar affinity and in the presence of NADPH, Fe(II) was released. Steady-state kinetics of Fe(II) release provides kcat/Km values in the range of 600 to 7000 M-1 s-1 for these siderophores supporting a role for IruO as a siderophore reductase in iron utilization. Crystal structures of IruO were solved in two distinct conformational states mediated by the formation of an intramolecular disulfide bond. A putative siderophore binding site was identified adjacent to the FAD cofactor. This site is partly occluded in the oxidized IruO structure consistent with this form being less active than reduced IruO. This reduction in activity could have a physiological role to limit iron release under oxidative stress conditions. Visible spectroscopy of anaerobically reduced IruO showed that the reaction proceeds by a single electron transfer mechanism through an FAD semiquinone intermediate. From the data, a model for single electron siderophore reduction by IruO using NADPH is described.

Antibiotic Capture by Bacterial Lipocalins Uncovers an Extracellular Mechanism of Intrinsic Antibiotic Resistance.

The potential for microbes to overcome antibiotics of different classes before they reach bacterial cells is largely unexplored. Here we show that a soluble bacterial lipocalin produced by Burkholderia cenocepacia upon exposure to sublethal antibiotic concentrations increases resistance to diverse antibiotics in vitro and in vivo These phenotypes were recapitulated by heterologous expression in B. cenocepacia of lipocalin genes from Pseudomonas aeruginosa, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus Purified lipocalin bound different classes of bactericidal antibiotics and contributed to bacterial survival in vivo Experimental and X-ray crystal structure-guided computational studies revealed that lipocalins counteract antibiotic action by capturing antibiotics in the extracellular space. We also demonstrated that fat-soluble vitamins prevent antibiotic capture by binding bacterial lipocalin with higher affinity than antibiotics. Therefore, bacterial lipocalins contribute to antimicrobial resistance by capturing diverse antibiotics in the extracellular space at the site of infection, which can be counteracted by known vitamins.IMPORTANCE Current research on antibiotic action and resistance focuses on targeting essential functions within bacterial cells. We discovered a previously unrecognized mode of general bacterial antibiotic resistance operating in the extracellular space, which depends on bacterial protein molecules called lipocalins. These molecules are highly conserved in most bacteria and have the ability to capture different classes of antibiotics outside bacterial cells. We also discovered that liposoluble vitamins, such as vitamin E, overcome in vitro and in vivo antibiotic resistance mediated by bacterial lipocalins, providing an unexpected new alternative to combat resistance by using this vitamin or its derivatives as antibiotic adjuvants.

Identification of synergists that potentiate the action of polymyxin B against Burkholderia cenocepacia.

Burkholderia cenocepacia and other members of the Burkholderia cepacia complex (BCC) are highly multidrug-resistant bacteria that cause severe pulmonary infections in patients with cystic fibrosis. A screen of 2686 compounds derived from marine organisms identified molecules that could synergise with polymyxin B (PMB) to inhibit the growth of B. cenocepacia. At 1 µg/mL, five compounds synergised with PMB and inhibited the growth of B. cenocepacia by ≥70% compared with growth in PMB alone. Follow-up testing revealed that one compound from the screen, the aminocoumarin antibiotic novobiocin, synergised with PMB and colistin against tobramycin-resistant clinical isolates of B. cenocepacia and Burkholderia multivorans. In parallel, we show that novobiocin sensitivity is common among BCC species and that these bacteria are even more susceptible to an alternative aminocoumarin, clorobiocin, which also had an additive effect with PMB against B. cenocepacia. These studies support using aminocoumarin antibiotics to treat BCC infections and show that synergisers can be found to increase the efficacy of antimicrobial peptides and polymyxins against BCC bacteria.

Structural-functional studies of Burkholderia cenocepacia D-glycero-ß-D-manno-heptose 7-phosphate kinase (HldA) and characterization of inhibitors with antibiotic adjuvant and antivirulence properties.

As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-ß-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

Extreme antimicrobial Peptide and polymyxin B resistance in the genus burkholderia.

Cationic antimicrobial peptides and polymyxins are a group of naturally occurring antibiotics that can also possess immunomodulatory activities. They are considered a new source of antibiotics for treating infections by bacteria that are resistant to conventional antibiotics. Members of the genus Burkholderia, which includes various human pathogens, are inherently resistant to antimicrobial peptides. The resistance is several orders of magnitude higher than that of other Gram-negative bacteria such as Escherichia coli, Salmonella enterica, or Pseudomonas aeruginosa. This review summarizes our current understanding of antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. These bacteria possess major and minor resistance mechanisms that will be described in detail. Recent studies have revealed that many other emerging Gram-negative opportunistic pathogens may also be inherently resistant to antimicrobial peptides and polymyxins and we propose that Burkholderia sp. are a model system to investigate the molecular basis of the resistance in extremely resistant bacteria. Understanding resistance in these types of bacteria will be important if antimicrobial peptides come to be used regularly for the treatment of infections by susceptible bacteria because this may lead to increased resistance in the species that are currently susceptible and may also open up new niches for opportunistic pathogens with high inherent resistance.

Extreme antimicrobial peptide and polymyxin B resistance in the genus Burkholderia.

Cationic antimicrobial peptides and polymyxins are a group of naturally occurring antibiotics that can also possess immunomodulatory activities. They are considered a new source of antibiotics for treating infections by bacteria that are resistant to conventional antibiotics. Members of the genus Burkholderia, which includes various human pathogens, are inherently resistant to antimicrobial peptides. The resistance is several orders of magnitude higher than that of other Gram-negative bacteria such as Escherichia coli, Salmonella enterica, or Pseudomonas aeruginosa. This review summarizes our current understanding of antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. These bacteria possess major and minor resistance mechanisms that will be described in detail. Recent studies have revealed that many other emerging Gram-negative opportunistic pathogens may also be inherently resistant to antimicrobial peptides and polymyxins and we propose that Burkholderia sp. are a model system to investigate the molecular basis of the resistance in extremely resistant bacteria. Understanding resistance in these types of bacteria will be important if antimicrobial peptides come to be used regularly for the treatment of infections by susceptible bacteria because this may lead to increased resistance in the species that are currently susceptible and may also open up new niches for opportunistic pathogens with high inherent resistance.

A two-tier model of polymyxin B resistance in Burkholderia cenocepacia.

Burkholderia cenocepacia is an environmental bacterium causing serious human opportunistic infections and is extremely resistant to multiple antibiotics including antimicrobial peptides, such as polymyxin B (PmB). Extreme antibiotic resistance is attributed to outer membrane impermeability ('intrinsic' resistance). Previous work showed that production of full-length lipopolysaccharide (LPS) prevents surface binding of PmB. We hypothesized that two tiers of resistance mechanisms rendering different thresholds of PmB resistance exist in B. cenocepacia. To test this notion, candidate genes were mutated in two isogenic strains expressing full-length LPS or truncated LPS devoid of heptose ('heptoseless LPS') respectively. We uncovered various proteins required for PmB resistance only in the strain with heptoseless LPS. These proteins are not involved in preventing PmB binding to whole cells or permeabilization of the outer membrane. Our results support a two-tier model of PmB resistance in B. cenocepacia. One tier sets a very high threshold mediated by the LPS and the outer membrane permeability barrier. The second tier sets a lower threshold that may play a role in PmB resistance only when outer membrane permeability is compromised. This model may be of general applicability to understanding the high antimicrobial peptide resistance of environmental opportunistic pathogens.

Burkholderia cenocepacia O polysaccharide chain contributes to caspase-1-dependent IL-1beta production in macrophages.

Burkholderia cenocepacia infections in CF patients involve heightened inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory IL-1ß secretion is important in airway inflammation and tissue damage. However, little is known about this pathway in macrophages upon B. cenocepacia infection. We report here that murine macrophages infected with B. cenocepacia K56-2 produce proinflammatory cytokine IL-1ß in a TLR4 and caspase-1-mediated manner. We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to IL-1ß production and pyroptotic cell death. Furthermore, we showed that the malfunction of the CFTR channel augmented IL-1ß production upon B. cenocepacia infection of murine macrophages. Taken together, we identified eukaryotic and bacterial factors that contribute to inflammation during B. cenocepacia infection, which may aid in the design of novel approaches to control pulmonary inflammation.

Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia.

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugd(BCAL2946), ugd(BCAM0855) and ugd(BCAM2034), all of which were individually inactivated. Only inactivation of ugd(BCAL2946) resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugd(BCAL2946) or ugd(BCAM0855) but not ugd(BCAM2034) was expressed from plasmids. The growth of a conditional ugd(BCAL2946) mutant, created in the Deltaugd(BCAM0855) background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugd(BCAL2946) or ugd(BCAM0855) expressed in trans, but not by ugd(BCAM2034). Biochemical analysis of the purified, recombinant forms of Ugd(BCAL2946) and Ugd(BCAM0855) revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD(+). Purified Ugd(BCAM2034) showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugd(BCAL2946) was 5.4- and 135-fold greater than that of ugd(BCAM0855) and ugd(BCAM2034), respectively. Together, these data indicate that the combined activity of Ugd(BCAL2946) and Ugd(BCAM0855) is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugd(BCAL2946), is required for polymyxin B resistance.

A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability.

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo.

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis.

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-d-manno-octulosonic acid and three glycero-manno-heptose residues. The HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.

Reconstitution of O-specific lipopolysaccharide expression in Burkholderia cenocepacia strain J2315, which is associated with transmissible infections in patients with cystic fibrosis.

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.

Modulation of p210(BCR-ABL) activity in transduced primary human hematopoietic cells controls lineage programming.

Retroviral transduction of primary hematopoietic cells with human oncogenes provides a powerful approach to investigating the molecular mechanisms controlling the normal proliferation and differentiation of these cells. Here we show that primitive human CD34(+) cord blood cells, including multipotent as well as granulopoietic- and erythroid-restricted progenitors, can be efficiently transduced with a MSCV-BCR-ABL-IRES-GFP retrovirus, resulting in the sustained expression by their progeny of very high levels of tyrosine phosphorylated p210(BCR-ABL). Interestingly, even in the presence of growth factors that supported the exclusive production of granulopoietic cells from green fluorescent protein (GFP)-transduced control cells, BCR-ABL-transduced progenitor subpopulations generated large numbers of erythropoietin-independent terminally differentiating erythroid cells and reduced numbers of granulopoietic cells. Analyses of individual clones generated by single transduced cells in both semisolid and liquid cultures showed this BCR-ABL-induced erythroid differentiation response to be elicited at a high frequency from all types of transduced CD34(+) cells independent of their apparent prior lineage commitment status. Additional experiments showed that this erythroid differentiation response was largely prevented when the cells were transduced and maintained in the presence of the BCR-ABL-specific tyrosine kinase inhibitor, STI-571. These findings indicate that overexpression of BCR-ABL in primary human hematopoietic cells can activate an erythroid differentiation program in apparently granulopoietic-restricted cells through a BCR-ABL kinase-dependent mechanism, thus providing a new molecular tool for elucidating mechanisms underlying lineage fate determination in human hematopoietic cells and infidelity in human leukemia.