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Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board ‘anti-σ factor’ in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Urogenital Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection throughout the world. While progress has been made to better understand how type strains develop and respond to environmental stress in vitro, very few studies have examined how clinical isolates behave under similar conditions. Here, we examined the development and persistence phenotypes of several clinical isolates, to determine how similar they are to each other, and the type strain C. trachomatis D/UW-3/Cx. The type strain was shown to produce infectious progeny at a higher magnitude than each of the clinical isolates, in each of the six tested cell lines. All chlamydial strains produced the highest number of infectious progeny at 44 h post-infection in the McCoy B murine fibroblast cell line, yet showed higher levels of infectivity in the MCF-7 human epithelial cell line. The clinical isolates were shown to be more susceptible than the type strain to the effects of penicillin and iron deprivation persistence models in the MCF-7 cell line. While subtle differences between clinical isolates were observed throughout the experiments conducted, no significant differences were identified. This study reinforces the importance of examining clinical isolates when trying to relate in vitro data to clinical outcomes, as well as the importance of considering the adaptations many type strains have to being cultured in vitro.

© 2017 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists Background: We know very little about the microbiota inhabiting the upper female reproductive tract and how it impacts on fertility. Aims: This pilot study aimed to examine the vaginal, cervical and endometrial microbiota for women with a history of infertility compared to women with a history of fertility. Materials and methods: Using a retrospective case–control study design, women were recruited for collection of vaginal, cervical and endometrial samples. The microbiota composition was analysed by 16S ribosomal RNA (rRNA) gene amplification and endometrial expression of selected human genes by quantitative reverse transcription polymerase chain reaction. Results: Sixty-five specimens from the reproductive tract of 31 women were successfully analysed using 16S rRNA gene amplicon sequencing (16 controls and 15 cases). The dominant microbial community members were consistent in the vagina and cervix, and generally consistent with the endometrium although the relative proportions varied. We detected three major microbiota clusters that did not group by tissue location or case–control status. There was a trend that infertile women more often had Ureaplasma in the vagina and Gardnerella in the cervix. Testing for the expression of selected genes in the endometrium did not show evidence of correlation with case–control status, or with microbial community composition, although Tenascin-C expression correlated with a history of miscarriage. Conclusions: There is a need for further exploration of the endometrial microbiota, and how the microbiota members or profile interplays with fertility or assisted reproductive technologies.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-A resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCE Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

Devastating fires in Australia over 2019–20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (pChlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.

To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis., Highlights • We have constructed antibiotic-resistance cassettes and suicide vectors for use in Burkholderia and related species. • These vectors facilitate construction of mutants by gene disruption with antibiotic-resistance markers. • We have validated the utility of the vectors for marked genetic inactivation in members of the genus Burkholderia.

OrbS and PvdS are extracytoplasmic function (ECF) σ factors that regulate transcription of operons required for the biosynthesis of the siderophores ornibactin and pyoverdine in the Burkholderia cepacia complex and Pseudomonas spp., respectively. Here we show that promoter recognition by OrbS requires specific tetrameric −35 and −10 element sequences that are strikingly similar to those of the consensus PvdS-dependent promoter. However, whereas Pseudomonas aeruginosa PvdS can serve OrbS-dependent promoters, OrbS cannot utilize PvdS-dependent promoters. To identify features present at OrbS-dependent promoters that facilitate recognition by OrbS, we carried out a detailed analysis of the nucleotide sequence requirements for promoter recognition by both OrbS and PvdS. This revealed that DNA sequence features located outside the sigma binding elements are required for efficient promoter utilization by OrbS. In particular, the presence of an A-tract extending downstream from the −35 element at OrbS-dependent promoters was shown to be an important contributor to OrbS specificity. Our observations demonstrate that the nature of the spacer sequence can have a major impact on promoter recognition by some ECF σ factors through modulation of the local DNA architecture. IMPORTANCE ECF σ factors regulate subsets of bacterial genes in response to environmental stress signals by directing RNA polymerase to promoter sequences known as the −35 and −10 elements. In this work, we identify the −10 and −35 elements that are recognized by the ECF σ factor OrbS. Furthermore, we demonstrate that efficient promoter utilization by this σ factor also requires a polyadenine tract located downstream of the −35 region. We propose that the unique architecture of A-tract DNA imposes conformational features on the −35 element that facilitates efficient recognition by OrbS. Our results show that sequences located between the core promoter elements can make major contributions to promoter recognition by some ECF σ factors.

International audience; Burkholderia cenocepacia is an opportunistic bacterial pathogen that poses a significant threat to individuals with cystic fibrosis by provoking a strong inflammatory response within the lung. It possesses a type VI secretion system (T6SS), a secretory apparatus that can perforate the cellular membrane of other bacterial species and/or eukaryotic targets, to deliver an arsenal of effector proteins. The B. cenocepacia T6SS (T6SS-1) has been shown to be implicated in virulence in rats and contributes toward actin rearrangements and inflammasome activation in B. cenocepacia-infected mac-rophages. Here, we present bioinformatics evidence to suggest that T6SS-1 is the archetype T6SS in the Burkholderia genus. We show that B. cenocepacia T6SS-1 is active under normal laboratory growth conditions and displays antibacterial activity against other Gram-negative bacterial species. Moreover, B. cenocepacia T6SS-1 is not required for virulence in three eukaryotic infection models. Bioinformatics analysis identified several candidate T6SS-dependent effectors that may play a role in the antibacterial activity of B. cenocepacia T6SS-1. We conclude that B. cenocepacia T6SS-1 plays an important role in bacterial competition for this organism, and probably in all Burkholderia species that possess this system, thereby broadening the range of species that utilize the T6SS for this purpose.

TssA is a core component of the type VI secretion system, and phylogenetic analysis of TssA subunits from different species has suggested that these proteins fall into three distinct clades. Whilst representatives of two clades, TssA1 and TssA2, have been the subjects of investigation, no members of the third clade (TssA3) have been studied. Constructs of TssA from Burkholderia cenocepacia, a representative of clade 3, were expressed, purified and subjected to crystallization trials. Data were collected from crystals of constructs of the N-terminal and C-terminal domains. Analysis of the data from the crystals of these constructs and preliminary structure determination indicates that the C-terminal domain forms an assembly of 32 subunits in D 16 symmetry, whereas the N-terminal domain is not involved in subunit assocation.

Background: Bronchopulmonary dysplasia (BPD) is a commonly used outcome for randomized neonatal trials. Objectives: The aim of the present study was to determine whether a diagnosis of BPD or respiratory morbidity (RM1 or RM2) at 12 months corrected age better predicted subsequent RM in extremely low gestational age infants (23-28 weeks of gestation). Methods: Initial analysis was undertaken in a development cohort of 76 infants who underwent pulmonary function tests (PFTs) at 12 months corrected age. Parents completed infant respiratory diaries 2 weeks before the PFTs. Analysis was then undertaken in a validation cohort of 227 infants whose parents completed a 4-week respiratory diary when their infant was 12 months corrected age. BPD at 28 days (BPD28d) and 36 weeks post-menstrual age (BPD36w), RM1 (≥3 days and/or nights of cough, wheeze, and/or medicine use) and RM2 (≥4 days and/or nights of cough, wheeze, and/or respiratory medicine use) each week for 2 weeks at 12 months corrected age were assessed with regard to prediction of respiratory outcomes at 24 months documented by respiratory health questionnaires. Results: BPD28d and BPD36w were not significantly associated with any respiratory outcome. Areas under the receiver operating characteristic curves were significantly better for either definition of RM than BPD28d or BPD36w for all outcomes. Conclusions: RM documented by parental completed diaries at 12 months corrected age better predicted respiratory outcome at 24 months corrected age than BPD regardless of diagnostic criteria.

I was born in central London, but my parents moved to the eastern suburbs when I was still an infant. I was the eldest of four brothers and had a normal childhood. I was very keen on soccer from an...

Objectives To assess longitudinally small airway function in children born extremely prematurely and whether there was a correlation between airway function in infancy and at 11-14 years. Working hypotheses There would be tracking of airways obstruction and small airway function would deteriorate during childhood in those born extremely prematurely. Study design A longitudinal study. Patient-subject selection Thirty-five children with a mean gestational age of 26 weeks had lung function assessed at 1 year corrected and 11-14 years of age. Methodology Lung volumes were measured by helium gas dilution (FRCHe ) and plethysmography (FRCpleth ) and small airway function assessed by calculating the FRCHe :FRCpleth ratio. Airway function was assessed at 1 year corrected by measurement of airway resistance (Raw ) and at 11-14 years by assessment of Raw , forced expiratory flow from 75% of vital capacity (FEF75 ), and forced expiratory volume at one second (FEV1 ). Results At the first assessment, the children had a mean (SD) FRCHe :FRCpleth of 0.90 (0.13) and at the second, 0.83 (0.12) (P = 0.035). There was a significant 0.54% decrease (95%CI: -1.02%, -0.06%) in FRCHe :FRCpleth for increased age per year after adjusting for birth weight, gestational age, sex, and bronchopulmonary dysplasia (P = 0.027). There were significant correlations between Raw at the first assessment and Raw (P = 0.012), FEF75 (P = 0.034), and FEV1 (P = 0.04) at 11-14 years. Conclusions These results demonstrate in those born extremely prematurely there is tracking of airway function during childhood.

The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric β or β' subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric α-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the α C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any α-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.

The bacterial pathogen that finds itself, either by accident or design, within the human or animal host, senses and adapts to the prevailing conditions by modulating its gene expression on a global...

The Escherichia coli guaB promoter (P(guaB)) regulates transcription of two genes, guaB and guaA, that are required for the synthesis of guanosine 5'-monophosphate (GMP), a precursor for the synthesis of guanine nucleoside triphosphates. Transcription from P(guaB) increases as a function of increasing cellular growth rate, and this is referred to as growth rate-dependent control (GRDC). Here we investigated the role of the factor for inversion stimulation (FIS) in the regulation of this promoter. The results showed that there are three binding sites for FIS centred near positions -11, +8 and +29 relative to the guaB transcription start site. Binding of FIS to these sites results in repression of P(guaB) in vitro but not in vivo. Deletion of the fis gene results in increased P(guaB) activity in vivo, but GRDC of P(guaB) is maintained.

The Escherichia coli guaB promoter (P guaB ) regulates the transcription of two genes, guaB and guaA , that are required for de novo synthesis of GMP, a precursor for the synthesis of guanine nucleoside triphosphates. The activity of P guaB is subject to growth rate-dependent control (GRDC). Here we show that the A+T-rich sequence located between positions −59 and −38 relative to the guaB transcription start site stimulates transcription from P guaB ∼8- to 10-fold and, in common with other UP elements, requires the C-terminal domain of the RNA polymerase α subunit for activity. Like the rrnB P1 UP element, the P guaB UP element contains two independently acting subsites located at positions −59 to −47 and −46 to −38 and can stimulate transcription when placed upstream of the lacP1 promoter. We reveal a novel role for the P guaB UP element by demonstrating that it is required for GRDC. The involvement of the UP element in GRDC also requires the participation of sequences located at least 100 bp upstream of the guaB transcription start site. These sequences are required for down-regulation of P guaB activity at lower growth rates.

The Gram-negative proteobacterium Burkholderia pseudomallei can survive and multiply within a variety of eukaryotic cells, including macrophages. This property is believed to be important for its ability to cause the disease melioidosis in a wide range of animal species, including humans. To identify determinants that are important for the ability of B. pseudomallei to survive within macrophages, in vivo expression technology (IVET) was employed. Several putative macrophage-inducible genes were identified that are likely to contribute to the virulence of B. pseudomallei, including three genes (tssH-5, tssI-5 and tssM-5) located within the same type VI secretion system cluster (tss-5), mntH, encoding a natural resistance-associated macrophage protein (NRAMP)-like manganese ion transporter, and a haem acquisition gene, bhuT. The macrophage-inducibility of the tss-5 gene cluster was confirmed by reporter gene analysis. Construction of tssH-5 and bhuT null mutants indicated that expression of the tss-5 unit and the bhu operon were not required for intramacrophage survival. A further five tss units were identified within the B. pseudomallei genome that, together with tss-5, account for approximately 2.3 % of the total genome size. The presence of six type VI secretion systems in this organism is likely to be an important factor in making this bacterium such a versatile pathogen.

The bacteriophage lambda p(M) promoter is required for maintenance of the lambda prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the lambda repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of p(M). CI activates p(M) through cooperative binding to the O(R)1 and O(R)2 sites within the O(R) operator, with the O(R)2-bound CI dimer making contact with domain 4 of the RNA polymerase sigma subunit (sigma(4)). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of p(M). We also show that the location of alphaCTD at the p(M) promoter changes in the presence of CI. Thus, in the absence of CI, one alphaCTD is located on the DNA at position -44 relative to the transcription start site, whereas in the presence of CI, alphaCTD is located at position -54, between the CI-binding sites at O(R)1 and O(R)2. These results suggest that contacts between CI and both alphaCTD and sigma are required for efficient CI-dependent activation of p(M).

The Burkholderia cepacia complex (Bcc) is comprised of at least 10 closely related species of Gram-negative proteobacteria that are associated with infections in certain groups of immunocompromised individuals, particularly those with cystic fibrosis. Infections in humans tend to occur in the lungs, which present an iron-restricted environment to a prospective pathogen, and accordingly members of the Bcc appear to possess efficient mechanisms for iron capture. These bacteria specify up to four different types of siderophore (ornibactin, pyochelin, cepabactin and cepaciachelin) that employ the full repertoire of iron-binding groups present in most naturally occurring siderophores. Members of the Bcc are also capable of utilising some exogenous siderophores that they are not able to synthesise. In addition to siderophore-mediated mechanisms of iron uptake, the Bcc possess mechanisms for acquiring iron from haem and from ferritin. The Bcc therefore appear to be well-equipped for life in an iron-poor environment.

Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn 5 Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ , encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS , located upstream from orbI . The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) σ factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron-regulated σ 70 -dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF σ factor gene orbS . A mechanism is also proposed for the biosynthesis of ornibactin.

DNA vaccines have considerable potential for disease prophylaxis and therapy, but are generally poorly immunogenic. A number of means of enhancing immunogenicity have been assessed, including the co-expression of cytokines, the use of heterologous prime-boost regimes, and the addition of more conventional adjuvants. In this study we have assessed the effects on gp120 DNA immunogenicity of in-frame fusion of tumor necrosis factor alpha DNA to DNA encoding a large fragment of HIV gp120. The studies were performed using a DNA prime, protein boost regime and a heterologous boosting protein. Fusion of TNFα DNA enhanced Th1 related immune responses against both the priming and the boosting gp120. In-frame fusion of interferon gamma-encoding DNA at the 5′ end of the chimeric molecule, to create a tripartite fusion, had no additional effect on immunogenicity.

Nucleic acid vaccination has many potential advantages over traditional methods, but suffers from the fact that DNA vaccines tend to be relatively poorly immunogenic. Attempts to enhance DNA vaccine immunogenicity have included the addition of cytokine-encoding plasmids into the formulation, as well as the use of heterologous prime-boost regimes and the addition of conventional adjuvants, such as alum. We have previously shown that interferon gamma fusions have enhanced immunogenicity as recombinant protein vaccines. We have assessed here the immunogenicity of an interferon gamma-gp120 fusion delivered as a DNA vaccine, in the context of a prime-boost strategy and in the presence of absence of aluminium phosphate. Fusion of gp120 DNA to interferon gamma-encoding DNA resulted in strongly enhanced priming, especially of Th1 responses, including IgG2a responses to a protein boost.

The bacteriophage lambda CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the -35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) to demonstrate that one alphaCTD binds near position -41 at p(E), whilst the other alphaCTD binds further upstream. The alphaCTD bound near position -41 is oriented such that its 261 determinant is in close proximity to sigma(70). The location of alphaCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for alphaCTD at promoters where activators bind sites overlapping the -35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of alphaCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in alphaCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of alphaCTD at the three lysogenic promoters may be different.

The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn 5 Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

A role for the Escherichia coli RNA polymerase alpha subunit in transcription antitermination dependent on bacteriophage lambda N protein has been previously inferred from the isolation of rpoA mutants that alter the efficiency of this process. This report describes studies on the efficiency of N-dependent transcription antitermination in a strain containing the rpoA341 mutation, which interferes with this process. The effect of mutations in genes coding for different Nus factors and/or plasmids overexpressing nus genes on bacteriophage lambda development in an E. coli rpoA341 host was examined. In addition, the effect of overproduction of the N protein in these genetic backgrounds was assessed. Analogous bacterial strains were employed to measure the efficiency of the antitermination process using the lacZ reporter gene under control of the lambda p(R) promoter, and containing the phage nutR region and the t( R1) terminator between the promoter and lacZ. The experimental results suggest interactions between components of the N-antitermination complex, which have been established biochemically, as well as additional functional relationships within the complex. Furthermore, the results indicate that amino acid substitution in the alpha subunit C-terminal domain encoded by the rpoA341 mutation may specifically disrupt the function of the NusA and NusE proteins. During this analysis, it was also found that the E. coli nusA1 mutant exhibits a conditional lethal phenotype.

Arch Dis Child Fetal Neonatal Ed 2003;88:F329‐F332 Objective: To determine if chest radiograph appearance at 28 days or 36 weeks postmenstrual age (PMA) can predict recurrent wheeze or cough at follow up in prematurely born infants more effectively than readily available clinical data. Design: Chest radiographs of infants entered into the UKOS trial, who had had a chest radiograph at 28 days and 36 weeks PMA and completed six months of follow up, were assessed for the presence of fibrosis, interstitial shadows, cystic elements, and hyperinflation. At 6 months of corrected age, the occurrence and frequency of wheeze and cough since discharge were determined using a symptom questionnaire. Patients: A total of 185 infants with a median gestational age of 26 (range 23‐28) weeks. Results: Thirty seven infants wheezed more than once a week, compared with the rest of the cohort. These infants had significantly higher chest radiograph scores at 28 days (p = 0.020) and 36 weeks PMA (p = 0.005), with significantly higher scores at 28 days for fibrosis (p = 0.017) and at 36 weeks PMA for fibrosis (p = 0.001) and cystic elements (p = 0.0007). They had also been ventilated for longer (p = 0.013). Forty four infants coughed more than once a week; they did not differ significantly from the rest of the cohort. An abnormal chest radiograph score at 36 weeks PMA had the largest area under the receiver operator characteristic curve with regard to prediction of frequent wheeze. Conclusion: An abnormal chest radiograph appearance at 36 weeks PMA predicts frequent wheeze at follow up and appears to be a better predictor than readily available clinical data.

It was found that infection of Escherichia coli by bacteriophage lambda is inhibited in the presence of certain bile salts and carbohydrates when cells are in the "OFF" state for production of the phase-variable cell surface protein antigen 43 (Ag43). The inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption. Expression of the gene encoding Ag43 (agn43) from a plasmid or inactivation of the oxyR gene (encoding an activator of genes important for defence against oxidative stress) suppressed this inhibition. A mutation, rpoA341, in the gene encoding the alpha subunit of RNA polymerase also facilitated phage adsorption in the presence of bile salts and carbohydrates. The rpoA341 mutation promoted efficient production of Ag43 in a genetic background that would otherwise be in the "OFF" phase for expression of the agn43 gene. Analysis of a reporter gene fusion demonstrated that the promoter for the agn43 gene was more active in the rpoA341 mutant than in the otherwise isogenic rpoA(+) strain. The combined inhibitory action of bile salts and carbohydrates on phage adsorption and the abolition of this inhibition by production of Ag43 was not restricted to lambda, as a similar phenomenon was observed for the coliphages P1 and T4.

Plethysmographic measurement of airway resistance (R(aw)) has been determined by single-point analysis, usually at 50% of maximum inspiratory flow (MIF). Computer-assisted (best fit) analysis, however, allows R(aw) to be calculated by applying a regression line to any portion of the plethysmograph pressure-flow loop. We determined whether the results of best fit analysis using a computer program, sampling at 200 Hz, were influenced by the portion of the inspiratory loop analysed and if best fit or single-point analysis gave more reproducible results. Twenty infants of median gestational age 26 (range 24-28) weeks, were studied at a median age of 12 (12-14) months corrected for prematurity. R(aw) was calculated by best fit analysis between 0 and 33% MIF, 0 and 50% MIF and 0 and 67% MIF and single-point analysis at 50% of MIF. Similar mean R(aw) values were obtained by best fit analysis between 0 and 33% MIF (2.79 kPa/(l/s)) and 0 and 50% MIF (3.01 kPa/(l/s)) and single-point analysis at 50% MIF (2.86 kPa/(l/s)), but best fit analysis between 0 and 67% gave higher results (3.60 kPa/(l/s)), p < 0.0001. Within the linear portion of the inspiratory loop, the mean intrasubject coefficient of variation was lowest for best fit analysis between 0 and 50% MIF. Best fit computerized analysis between 0 and 50% MIF is recommended as the analysis of choice.

The transcription factor Fis activates the Escherichia coli rRNA promoters rrnB P1 and rrnE P1 by binding to sites centered at -71 and -72, respectively, and interacting with the C-terminal domain of the alpha subunit of RNA polymerase (RNAP alphaCTD). To understand the mechanism of activation by Fis at these promoters, we used oriented alpha-heterodimeric RNAPs and heterodimers of Fis to determine whether one or both subunits of alpha and Fis participate in the alphaCTD-Fis interaction. Our results imply that only one alphaCTD in the alpha dimer and only one activation-proficient subunit in the Fis dimer are required for activation by Fis. A library of alanine substitutions in alpha was used to identify the alphaCTD determinants required for Fis-dependent transcription at rrnB P1 and rrnE P1. We propose that the transcriptional activation region of the promoter-proximal subunit of the Fis dimer interacts with a determinant that includes E273 of one alphaCTD to activate transcription. We further suggest that the Fis contact to alphaCTD results in alphaCTD interactions with DNA that differ somewhat from those that occur at UP elements in the absence of Fis. The accompanying paper shows that the 273 determinant on alphaCTD is also targeted by Fis at the proP P2 promoter where the activator binds overlapping the -35 hexamer. Thus, similar Fis-alphaCTD interactions are used for activation of transcription when the activator is bound at very different positions on the DNA.

Introduction Ticagrelor is a more potent platelet inhibitor than clopidogrel but also has a more rapid offset of inhibitory effect. The optimal timing of discontinuation of ticagrelor prior to coronary artery bypass graft (CABG) surgery is unknown. In the ONSET/OFFSET study of patients with stable coronary artery disease, ticagrelor’s effects dissipated within 48–120 hours of discontinuation. However, there is evidence that the pharmacodynamics of antiplatelet therapy are significantly altered during an acute coronary syndrome (ACS) and the ONSET/OFFSET study results should not be extrapolated to ACS patients. Although the PLATO study suggested that it was safe to discontinue ticagrelor 48 hours prior to CABG, regulatory authorities recommended that ticagrelor should be discontinued 5 days prior to CABG. The PLATO study also showed that ticagrelor was associated with fewer deaths following pulmonary adverse events and sepsis compared to clopidogrel. It is unknown whether ticagrelor’s additional property of inhibiting cellular adenosine uptake might underlie this observation. The aim of this study was to characterise the offset of ticagrelor’s activity in inhibiting platelets and also in inhibiting cellular adenosine uptake in ACS patients. Methods Patients admitted with ACS, treated with ticagrelor and referred for CABG were recruited. Venous blood was drawn from patients at the following 6 timepoints: 2 hours (h), 24 hour, 48 hour, 72 hour, 96 hour, and 120 hour after the last dose of ticagrelor prior to CABG. Whole-blood aggregometry was carried out using the Multiplate analyzer with adenosine diphosphate 6.45 µM as agonist and the final value of area under the curve (AUC) (units, U) was recorded. Results were analysed using linear mixed models. A local protocol was referred to, where an AUC of >50U supports safe progression to surgery, while values 50U indicates need for re-testing 24–48 hours later. Inhibition of adenosine uptake was determined by measuring the adenosine plasma concentration using liquid chromatography. Results 13 patients were recruited, all of whom received ticagrelor prior to CABG surgery. At 96 hour post ticagrelor, the mean AUC was 82.8U, (95% confidence interval, 60.4, 105.2). Only at 96 hour was the lower limit of the confidence interval >50U. From the mixed model analysis, the comparison between 72 hour and 120 hour post ticagrelor showed a significant difference (p=0.007) while 96 hour and 120 hour showed no significant difference (p=1.000). Adenosine plasma concentration was measured in 7 patients who received ticagrelor and showed no significant difference in adenosine plasma concentration across all timepoints. Conclusion ACS patients might be safe to undergo CABG surgery 4 days after the cessation of ticagrelor. Ticagrelor might have no measurable effect on adenosine transport in ACS patients.

Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ≤ 0.01) and additionally 715j-pobA utilised transferrin (P ≤ 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ≤ 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection.

The recently identified bacterial type VI secretion system (T6SS) has rapidly become one of the most interesting areas of research in microbiology. In a relatively short period of time the relationship between the T6SS and the bacteriophage T4 tail and baseplate has been established. However, a number of questions concerning the T6SS remain the focus of a large number of researchers worldwide. Key questions that need to be addressed include how this system assembles in the cell envelope and the mechanism by which it translocates effector proteins across two membranes, the identification of such effectors and their function, how this secretion system contributes to virulence, interbacterial interactions and/or adaptation to the environment, and the evolutionary relationship between T6SS machine and bacteriophage T4. Focused on how the proteins constituting the secretion system interact, we recently identified a sub-complex of the T6SS comprised of four cell envelope proteins: the inner membrane-anchored TssL, TssM and TagL proteins and the outer membrane-associated TssJ lipoprotein. We further demonstrated that the TagL subunit carries a specific domain allowing anchorage of the secretion system to the peptidoglycan (PG) layer. Herein, we discuss these results, examine whether PG-binding motifs are found within other T6SS subunits and express hypotheses regarding the role of PG-binding motifs in type VI secretion.

Cytokines, including interferon (IFN)-gamma, can be effective immunologic adjuvants but often lack the potency of other, more reactogenic compounds. On the basis of the observation that attachment of IFN-gamma to antigen could further enhance its adjuvanticity, a chimeric protein involving IFN-gamma and gp120 of human immunodeficiency virus was produced, using varying lengths of amino acid linkers between the two moieties. All resultant fusion proteins appeared to be dimerized, but full IFN-gamma biological activity was present only with the longest, 34-aa linker. Immunization with the fusion protein gave rise to enhanced primary antibody responses to gp120, particularly of the IgG2a subclass. In addition, both T cell proliferation and IFN-gamma production in response to antigen were strongly enhanced by primary immunization with the fusion protein. IFN-gamma fused to antigen is a more potent adjuvant for Th1-like responses than is IFN-gamma mixed with antigen.

The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element. Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.

The ferric uptake regulator (Fur) functions as a transcription repressor of many genes in bacteria in response to iron, but the presence of a functional equivalent of this protein has not been demonstrated in Burkholderia cepacia. A segment of the Burkholderia pseudomallei fur gene was amplified using degenerate primers and used to identify chromosomal restriction fragments containing the entire fur genes of B. cepacia and B. pseudomallei. These fragments were cloned and sequenced, revealing the Fur protein of both species to be a polypeptide of 142 amino acids possessing a high degree of amino acid sequence identity to Fur of other members of the beta subclass of the Proteobacteria. Primer extension analysis demonstrated that transcription of B. cepacia fur originated from a single promoter located 36 bp upstream from the fur translation initiation codon. The Fur polypeptide of B. cepacia was shown to functionally substitute for Fur in an Escherichia coli fur mutant. Single copy fur-lacZ fusions were constructed and used to examine the regulation of B. cepacia fur. The B. cepacia fur promoter was not responsive to iron availability, the presence of hydrogen peroxide or the superoxide generator methyl viologen. In addition, fur expression was not significantly influenced by carbon source. Interestingly, the presence of the divergently transcribed omlA/smpA gene upstream of fur in some members of the gamma subclass of the Proteobacteria is retained in several genera within the beta taxon, including Burkholderia.

Respiratory syncytial virus (RSV) is a common cause of respiratory illness in young children, almost all will have been infected by the age of two years old. Very young infants, and those with underlying disease, are at risk of severe RSV disease, but even those who were previously healthy can suffer recurrent respiratory symptoms 9 to 10 years after their initial infection. The management of RSV infection is essentially supportive, thus prophylaxis offers the best hope of reducing the morbidity and mortality of RSV infection. There is no safe and effective RSV vaccine to use in those infants who are at highest risk from the infection. Immunoprophylaxis, however, has been shown to have benefits in randomised controlled trials. Standard immunoglobulin, however, is ineffective as its administration does not achieve an adequate titre of neutralising antibodies. RSV immunoglobulin (RSV-IGIV, RespiGam, Massachusetts Public Health Laboratories, Boston, MA), in contrast, contains high levels of RSV neutralising antibody and has been shown to significantly reduce hospitalisation in preterm infants with or without bronchopulmonary dysplasia (BPD). Its use is not recommended in infants with cyanotic congenital heart disease (CHD), as it was associated with an excess of adverse events. A humanised RSV monoclonal antibody (Palivizumab, MEDI-493, Synagis, MedImmune Inc, Gaithersburg, MD) also significantly reduces hospitalisation for RSV infection in high risk infants, but without serious side effects. The American Academy of Paediatrics has recommended that immunoprophylaxis should be considered for young children at high risk of severe RSV infection and that palivizumab is the preferred agent. Studies have suggested it is essential to carefully select patients for immunoprophylaxis, if its use is to be cost-effective.

Binding of CD40 by CD154 expressed on activated T cells is a pivotal event in T cell help to B cells, macrophages, and other antigen-presenting cells. Expression of CD154 by MHC mismatched cells, in contrast to expectations, strongly suppressed alloantibody responses against the cells. This was caused by a failure of priming of antibody responses by the CD154 expressing cells. We hypothesize that this lack of response against CD154 expressing cells may represent a mechanism that has evolved to prevent autoantibody responses being generated against the CD154 antigen itself, as B cells expressing antibody reactive with CD154 would probably escape deletion on binding antigen in the bone marrow due to rescue by the simultaneous ligation of CD40.

Background —Coronary artery bypass grafting improves survival in patients with >70% luminal diameter narrowing of the 3 major epicardial coronary arteries, particularly if there is involvement of the proximal portion of the left anterior descending (LAD) coronary artery. Measurement of coronary flow reserve can be used to identify functionally important luminal narrowing of the LAD artery. Although magnetic resonance imaging (MRI) has been used to visualize coronary arteries and to measure flow reserve noninvasively, the utility of MRI for detecting significant LAD stenoses is unknown. Methods and Results —Thirty subjects (23 men, 7 women, age 36 to 77 years) underwent MRI visualization of the left main and LAD coronary arteries as well as measurement of flow in the proximal, middle, or distal LAD both at rest and after intravenous adenosine (140 μg/kg per minute). Immediately thereafter, contrast coronary angiography and when feasible, intracoronary Doppler assessments of coronary flow reserve, were performed. There was a statistically significant correlation between MRI assessments of coronary flow reserve and (a) assessments of coronary arterial stenosis severity by quantitative coronary angiography and (b) invasive measurements of coronary flow reserve ( P 70% in the distal left main or proximal/middle LAD arteries was 100% and 83%, respectively. Conclusions —Noninvasive MRI measures of coronary flow reserve correlated well with similar measures obtained with the use of intracoronary Doppler flow wires and predicted significant coronary stenoses (>70%) with a high degree of sensitivity and specificity. MRI-based measurement of coronary flow reserve may prove useful for identification of patients likely to obtain a survival benefit from coronary artery bypass grafting.

Background: The α subunit of eubacterial RNA polymerase comprises an N-terminal assembly domain and a mobile C-terminal domain which provides an activation contact site for class I transcription activators. One particular C-terminal α mutant, rpoA341, impairs the response of Escherichia coli RNA polymerase to several activators, including MelR. Results: The in vivo properties of a set of C-terminally truncated α variants were investigated. Derivatives of 230 amino acids or longer were assembled into functional RNA polymerase. However, derivatives greater than 271 residues in length were sensitised to proteolysis near K271. Deletion of only 13 C-terminal amino acids impaired the response to CRP at a class I promoter whereas the complete removal of the α C-terminal domain did not prevent complementation of MelR-dependent PmelAB activity in the rpoA341 mutant. Conclusions: Our results refine the C-terminal limit of the α assembly domain to between residues 221 and 230. The 13 extreme C-terminal amino acids are exposed in the holoenzyme and participate in the protection of an otherwise proteolytically sensitive bond near K271. Their presence is also essential for transcription activation at class I CRP-dependent promoters. The rpoA341-mediated substitution, K271E, does not define an activation contact site for MelR.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ∼44 bp of the DNA sequence preceding and/or overlapping the predicted −35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR “recognition motifs” at different responsive promoters appears to be limited.