Burkholderia pseudomallei modulates host iron homeostasis to facilitate iron availability and intracellular survival.

BACKGROUND: The control over iron homeostasis is critical in host-pathogen-interaction. Iron plays not only multiple roles for bacterial growth and pathogenicity, but also for modulation of innate immune responses. Hepcidin is a key regulator of host iron metabolism triggering degradation of the iron exporter ferroportin. Although iron overload in humans is known to increase susceptibility to Burkholderia pseudomallei, it is unclear how the pathogen competes with the host for the metal during infection. This study aimed to investigate whether B. pseudomallei, the causative agent of melioidosis, modulates iron balance and how regulation of host cell iron content affects intracellular bacterial proliferation. PRINCIPAL FINDINGS: Upon infection of primary macrophages with B. pseudomallei, expression of ferroportin was downregulated resulting in higher iron availability within macrophages. Exogenous modification of iron export function by hepcidin or iron supplementation by ferric ammonium citrate led to increased intracellular iron pool stimulating B. pseudomallei growth, whereas the iron chelator deferoxamine reduced bacterial survival. Iron-loaded macrophages exhibited a lower expression of NADPH oxidase, iNOS, lipocalin 2, cytokines and activation of caspase-1. Infection of mice with the pathogen caused a diminished hepatic ferroportin expression, higher iron retention in the liver and lower iron levels in the serum (hypoferremia). In vivo administration of ferric ammonium citrate tended to promote the bacterial growth and inflammatory response, whereas limitation of iron availability significantly ameliorated bacterial clearance, attenuated serum cytokine levels and improved survival of infected mice. CONCLUSIONS: Our data indicate that modulation of the cellular iron balance is likely to be a strategy of B. pseudomallei to improve iron acquisition and to restrict antibacterial immune effector mechanisms and thereby to promote its intracellular growth. Moreover, we provide evidence that changes in host iron homeostasis can influence susceptibility to melioidosis, and suggest that iron chelating drugs might be an additional therapeutic option.

Genome Sequence of Escherichia coli E28, a Multidrug-Resistant Strain Isolated from a Chicken Carcass, and Its Spontaneously Inducible Prophage.

In this study, we sequenced the complete genome of the multidrug-resistant Escherichia coli strain E28, which was used as an indicator strain for phage therapy in vivo We used a combination of single-molecule real-time and Illumina sequencing technology to reveal the presence of a spontaneously inducible prophage.

Heme Oxygenase-1 and Carbon Monoxide Promote Burkholderia pseudomallei Infection.

The environmental bacterium and potential biothreat agent Burkholderia pseudomallei causes melioidosis, an often fatal infectious disease. Increased serum bilirubin has been shown to be a negative predictive factor in melioidosis patients. We therefore investigated the role of heme oxygenase-1 (HO-1), which catalyzes the degradation of heme into the bilirubin precursor biliverdin, ferrous iron, and CO during B. pseudomallei infection. We found that infection of murine macrophages induces HO-1 expression, involving activation of several protein kinases and the transcription factor nuclear erythroid-related factor 2 (Nrf2). Deficiency of Nrf2 improved B. pseudomallei clearance by macrophages, whereas Nrf2 activation by sulforaphane and tert-butylhydroquinone with subsequent HO-1 induction enhanced intracellular bacterial growth. The HO-1 inducer cobalt protoporphyrin IX diminished proinflammatory cytokine levels, leading to an increased bacterial burden in macrophages. In contrast, HO-1 gene knockdown reduced the survival of intramacrophage B. pseudomallei Pharmacological administration of cobalt protoporphyrin IX to mice resulted in an enhanced bacterial load in various organs and was associated with higher mortality of intranasally infected mice. The unfavorable outcome of B. pseudomallei infection after HO-1 induction was associated with higher serum IL-6, TNF-α, and MCP-1 levels but decreased secretion of IFN-γ. Finally, we demonstrate that the CO-releasing molecule CORM-2 increases the B. pseudomallei load in macrophages and mice. Thus, our data suggest that the B. pseudomallei-mediated induction of HO-1 and the release of its metabolite CO impair bacterial clearance in macrophages and during murine melioidosis.

Glia ECM interactions are required to shape the Drosophila nervous system.

Organs are characterized by a specific shape that is often remodeled during development. The dynamics of organ shape is in particular evident during the formation of the Drosophila nervous system. During embryonic stages the central nervous system compacts, whereas selective growth occurs during larval stages. The nervous system is covered by a layer of surface glial cells that form the blood brain barrier and a thick extracellular matrix called neural lamella. The size of the neural lamella is dynamically adjusted to the growing nervous system and we show here that perineurial glial cells secrete proteases to remodel this matrix. Moreover, an imbalance in proteolytic activity results in an abnormal shape of the nervous system. To identify further components controlling nervous system shape we performed an RNAi based screen and identified the gene nolo, which encodes an ADAMTS-like protein. We generated loss of function alleles and demonstrate a requirement in glial cells. Mutant nolo larvae, however, do not show an abnormal nervous system shape. The only predicted off-target of the nolo(dsRNA) is Oatp30B, which encodes an organic anion transporting protein characterized by an extracellular protease inhibitor domain. Loss of function mutants were generated and double mutant analyses demonstrate a genetic interaction between nolo and Oatp30B which prevented the generation of maternal zygotic mutant larvae.

Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1ß and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1ß secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1ß, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.

Kinesin heavy chain function in Drosophila glial cells controls neuronal activity.

Kinesin heavy chain (Khc) is crucially required for axonal transport and khc mutants show axonal swellings and paralysis. Here, we demonstrate that in Drosophila khc is equally important in glial cells. Glial-specific downregulation of khc by RNA interference suppresses neuronal excitability and results in spastic flies. The specificity of the phenotype was verified by interspecies rescue experiments and further mutant analyses. Khc is mostly required in the subperineurial glia forming the blood-brain barrier. Following glial-specific knockdown, peripheral nerves are swollen with maldistributed mitochondria. To better understand khc function, we determined Khc-dependent Rab proteins in glia and present evidence that Neurexin IV, a well known blood-brain barrier constituent, is one of the relevant cargo proteins. Our work shows that the role of Khc for neuronal excitability must be considered in the light of its necessity for directed transport in glia.

Transcriptional regulation of peripheral glial cell differentiation in the embryonic nervous system of Drosophila.

The Drosophila nervous system is ideally suited to study glial cell development and function, because it harbors only relatively few glial cells, and nervous system development is very well conserved during evolution. In the past, enhancer trap studies provided tools allowing to study glial cells with a single-cell resolution and, moreover, disclosed a surprising molecular heterogeneity among the different glial cells. The peripheral nervous system in the embryo comprises only 12 glial cells in one hemisegment and thus offers a unique opportunity to decipher the mechanisms directing glial development. Here, we focus on transcriptional regulators that have been reported to function during gliogenesis. To uncover additional regulators, we have conducted a genetic screen and report the identification of two additional transcriptional regulators involved in the control of peripheral glial migration: nejire and tango.

Comparing peripheral glial cell differentiation in Drosophila and vertebrates.

In all complex organisms, the peripheral nerves ensure the portage of information from the periphery to central computing and back again. Axons are in part amazingly long and are accompanied by several different glial cell types. These peripheral glial cells ensure electrical conductance, most likely nature the long axon, and establish and maintain a barrier towards extracellular body fluids. Recent work has revealed a surprisingly similar organization of peripheral nerves of vertebrates and Drosophila. Thus, the genetic dissection of glial differentiation in Drosophila may also advance our understanding of basic principles underlying the development of peripheral nerves in vertebrates.

Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei.

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1ß, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.

Switch in FGF signalling initiates glial differentiation in the Drosophila eye.

The formation of a complex nervous system requires the intricate interaction of neurons and glial cells. Glial cells generally migrate over long distances before they initiate their differentiation, which leads to wrapping and insulation of axonal processes. The molecular pathways coordinating the switch from glial migration to glial differentiation are largely unknown. Here we demonstrate that, within the Drosophila eye imaginal disc, fibroblast growth factor (FGF) signalling coordinates glial proliferation, migration and subsequent axonal wrapping. Glial differentiation in the Drosophila eye disc requires a succession from glia-glia interaction to glia-neuron interaction. The neuronal component of the fly eye develops in the peripheral nervous system within the eye-antennal imaginal disc, whereas glial cells originate from a pool of central-nervous-system-derived progenitors and migrate onto the eye imaginal disc. Initially, glial-derived Pyramus, an FGF8-like ligand, modulates glial cell number and motility. A switch to neuronally expressed Thisbe, a second FGF8-like ligand, then induces glial differentiation. This switch is accompanied by an alteration in the intracellular signalling pathway through which the FGF receptor channels information into the cell. Our findings reveal how a switch from glia-glia interactions to glia-neuron interactions can trigger formation of glial membrane around axonal trajectories. These results disclose an evolutionarily conserved control mechanism of axonal wrapping, indicating that Drosophila might serve as a model to understand glial disorders in humans.