132 resultados para Subtelomeric Deletion


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Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic resistant bacterium Burkholderia cenocepacia protects less resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sub-lethal concentrations of PmB and other bactericidal antibiotics induce reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS such as superoxide ion and hydrogen peroxide was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sub-lethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation, and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study exposes BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance, and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

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Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles (BcCVs) that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low-molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the BCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since BCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in B. multivorans confers to this bacterium a similar phagosomal maturation delay as found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity.

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Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C35 extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria.whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria.

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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.

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We present a fully-distributed self-healing algorithm DEX, that maintains a constant degree expander network in a dynamic setting. To the best of our knowledge, our algorithm provides the first efficient distributed construction of expanders - whose expansion properties hold deterministically - that works even under an all-powerful adaptive adversary that controls the dynamic changes to the network (the adversary has unlimited computational power and knowledge of the entire network state, can decide which nodes join and leave and at what time, and knows the past random choices made by the algorithm). Previous distributed expander constructions typically provide only probabilistic guarantees on the network expansion which rapidly degrade in a dynamic setting, in particular, the expansion properties can degrade even more rapidly under adversarial insertions and deletions. Our algorithm provides efficient maintenance and incurs a low overhead per insertion/deletion by an adaptive adversary: only O(log n) rounds and O(log n) messages are needed with high probability (n is the number of nodes currently in the network). The algorithm requires only a constant number of topology changes. Moreover, our algorithm allows for an efficient implementation and maintenance of a distributed hash table (DHT) on top of DEX, with only a constant additional overhead. Our results are a step towards implementing efficient self-healing networks that have guaranteed properties (constant bounded degree and expansion) despite dynamic changes.

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UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host.

IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

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Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined P ADJ = 1.15 × 10(-6), OR 1.332 (1.187-1.496)]. This association was characterized by a highly significant sex difference (P diff = 0.0032) in that it was clearly confined to females with genome-wide significance [P ADJ = 2.62 × 10(-8), OR 1.541 (1.324-1.796); males: P ADJ = 0.382, OR 1.084 (0.905-1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMSaveD pathogenesis.

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Purpose: Suppressor of cytokine signalling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 critically controls STAT3 activation, cytokine signalling and inflammatory gene expression in macrophages and microglia. In this study, we investigated the role of SOCS3/STAT3 in myeloid cells in the initiation and progression of diabetic retinopathy (DR). 
Methods: Mice with a conditional deletion of SOCS3 in myeloid cells (LysMCre-SOCS3 fl/fl) and C57BL/6J (as control) were rendered diabetic by a low-dose multiple intraperitoneal injections of Stroptozocine. Diabetes related retinal changes, including leukostasis, acellular capilliaries, and microglial activation were assessed at different stages of disease. Bone marrow derived macrophages (BMDMs) from LysMCreSOCS3 fl/fl and C57BL/6J mice were cultured in high glucose (HG) medium, and cell activation was evaluated by real-time RT-PCR.
Results: In C57BL/6J diabetic mice the expression of phosphorylated STAT3 (pSTAT3) was increased and SOCS3 was decreased in the retina. Interleukin 6 (IL-6), the main cytokine that stimulates STAT3 activation, was increased in the plamsa in diabetic mice. Although blood glucose levels and Hbac-1 were comparable between LysMCre-SOCS3fl/fl and WT mice after STZ injection, the LysMCreSOCS3 fl/fl diabetic mice developed severe retinal vasculopathy, including increased leukostasis and microglial activation at one month and enhanced acellular capillary formation at 6 months after diabetes induction. 
Conclusions: our study suggests that the JAK/STAT3 pathway is involved in the initiation and progression of DR, and uncontrolled STAT3 activation results in accelerated DR progression. Targeting the STAT3 pathway may be a novel approach for the management of DR.

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BACKGROUND: Bdellovibrio bacteriovorus HD100 must regulate genes in response to a variety of environmental conditions as it enters, preys upon and leaves other bacteria, or grows axenically without prey. In addition to "housekeeping" sigma factors, its genome encodes several alternate sigma factors, including 2 Group IV-RpoE-like proteins, which may be involved in the complex regulation of its predatory lifestyle.

RESULTS: We find that one sigma factor gene, bd3314, cannot be deleted from Bdellovibrio in either predatory or prey-independent growth states, and is therefore possibly essential, likely being an alternate sigma 70. Deletion of one of two Group IV-like sigma factor genes, bd0881, affects flagellar gene regulation and results in less efficient predation, although not due to motility changes; deletion of the second, bd0743, showed that it normally represses chaperone gene expression and intriguingly we find an alternative groES gene is expressed at timepoints in the predatory cycle where intensive protein synthesis at Bdellovibrio septation, prior to prey lysis, will be occurring.

CONCLUSIONS: We have taken the first step in understanding how alternate sigma factors regulate different processes in the predatory lifecycle of Bdellovibrio and discovered that alternate chaperones regulated by one of them are expressed at different stages of the lifecycle.

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Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

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Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

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The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

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Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5.

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Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.

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The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.