Filter exchange imaging with crusher gradient modelling detects increased blood-brain barrier water permeability in response to mild lung infection.

Blood-brain barrier (BBB) dysfunction occurs in many brain diseases, and there is increasing evidence to suggest that it is an early process in dementia which may be exacerbated by peripheral infection. Filter-exchange imaging (FEXI) is an MRI technique for measuring trans-membrane water exchange. FEXI data is typically analysed using the apparent exchange rate (AXR) model, yielding estimates of the AXR. Crusher gradients are commonly used to remove unwanted coherence pathways arising from longitudinal storage pulses during the mixing period. We first demonstrate that when using thin slices, as is needed for imaging the rodent brain, crusher gradients result in underestimation of the AXR. To address this, we propose an extended crusher-compensated exchange rate (CCXR) model to account for diffusion-weighting introduced by the crusher gradients, which is able to recover ground truth values of BBB water exchange (kin) in simulated data. When applied to the rat brain, kin estimates obtained using the CCXR model were 3.10 s-1 and 3.49 s-1 compared to AXR estimates of 1.24 s-1 and 0.49 s-1 for slice thicknesses of 4.0 mm and 2.5 mm respectively. We then validated our approach using a clinically relevant Streptococcus pneumoniae lung infection. We observed a significant 70 ± 10% increase in BBB water exchange in rats during active infection (kin = 3.78 ± 0.42 s-1) compared to before infection (kin = 2.72 ± 0.30 s-1; p = 0.02). The BBB water exchange rate during infection was associated with higher levels of plasma von Willebrand factor (VWF), a marker of acute vascular inflammation. We also observed 42% higher expression of perivascular aquaporin-4 (AQP4) in infected animals compared to non-infected controls, while levels of tight junction proteins remain consistent between groups. In summary, we propose a modelling approach for FEXI data which removes the bias in estimated water-exchange rates associated with the use of crusher gradients. Using this approach, we demonstrate the impact of peripheral infection on BBB water exchange, which appears to be mediated by endothelial dysfunction and associated with an increase in perivascular AQP4.

Targeting firing rate neuronal homeostasis can prevent seizures.

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.

Selective brain entry of lipid nanoparticles in haemorrhagic stroke is linked to biphasic blood-brain barrier disruption.

Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site. Methods: Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI. Results: Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (Ktrans [min-1]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI. Discussion: Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules.

Robust thrombolytic and anti-inflammatory action of a constitutively active ADAMTS13 variant in murine stroke models.

Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.

Characterisation of microvessel blood velocity and segment length in the brain using multi-diffusion-time diffusion-weighted MRI.

Multi-diffusion-time diffusion-weighted MRI can probe tissue microstructure, but the method has not been widely applied to the microvasculature. At long diffusion-times, blood flow in capillaries is in the diffusive regime, and signal attenuation is dependent on blood velocity (v) and capillary segment length (l). It is described by the pseudo-diffusion coefficient (D*=vl/6) of intravoxel incoherent motion (IVIM). At shorter diffusion-times, blood flow is in the ballistic regime, and signal attenuation depends on v, and not l. In theory, l could be estimated using D* and v. In this study, we compare the accuracy and repeatability of three approaches to estimating v, and therefore l: the IVIM ballistic model, the velocity autocorrelation model, and the ballistic approximation to the velocity autocorrelation model. Twenty-nine rat datasets from two strains were acquired at 7 T, with b-values between 0 and 1000 smm-2 and diffusion times between 11.6 and 50 ms. Five rats were scanned twice to assess scan-rescan repeatability. Measurements of l were validated using corrosion casting and micro-CT imaging. The ballistic approximation of the velocity autocorrelation model had lowest bias relative to corrosion cast estimates of l, and had highest repeatability.

Selective Liposomal Transport through Blood Brain Barrier Disruption in Ischemic Stroke Reveals Two Distinct Therapeutic Opportunities.

The development of effective therapies for stroke continues to face repeated translational failures. Brain endothelial cells form paracellular and transcellular barriers to many blood-borne therapies, and the development of efficient delivery strategies is highly warranted. Here, in a mouse model of stroke, we show selective recruitment of clinically used liposomes into the ischemic brain that correlates with biphasic blood brain barrier (BBB) breakdown. Intravenous administration of liposomes into mice exposed to transient middle cerebral artery occlusion took place at early (0.5 and 4 h) and delayed (24 and 48 h) time points, covering different phases of BBB disruption after stroke. Using a combination of in vivo real-time imaging and histological analysis we show that selective liposomal brain accumulation coincides with biphasic enhancement in transcellular transport followed by a delayed impairment to the paracellular barrier. This process precedes neurological damage in the acute phase and maintains long-term liposomal colocalization within the neurovascular unit, which could have great potential for neuroprotection. Levels of liposomal uptake by glial cells are similarly selectively enhanced in the ischemic region late after experimental stroke (2-3 days), highlighting their potential for blocking delayed inflammatory responses or shifting the polarization of microglia/macrophages toward brain repair. These findings demonstrate the capability of liposomes to maximize selective translocation into the brain after stroke and identify two windows for therapeutic manipulation. This emphasizes the benefits of selective drug delivery for efficient tailoring of stroke treatments.

Extent of Ischemic Brain Injury After Thrombotic Stroke Is Independent of the NLRP3 (NACHT, LRR and PYD Domains-Containing Protein 3) Inflammasome.

Background and Purpose- A major process contributing to cell death in the ischemic brain is inflammation. Inflammasomes are multimolecular protein complexes that drive inflammation through activation of proinflammatory cytokines, such as IL (interleukin)-1ß. Preclinical evidence suggests that IL-1ß contributes to a worsening of ischemic brain injury. Methods- Using a mouse middle cerebral artery thrombosis model, we examined the inflammatory response after stroke and the contribution of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome to ischemic injury. Results- There was a marked inflammatory response after stroke characterized by increased expression of proinflammatory cytokines and NLRP3 and by recruitment of leukocytes to the injured tissue. Targeting NLRP3 with the inhibitor MCC950, or using mice in which NLRP3 was knocked out, had no effect on the extent of injury caused by stroke. Conclusions- These data suggest that the NLRP3 pathway does not contribute to the inflammation exacerbating ischemic brain damage, contradicting several recent reports to the contrary.

Interleukin-1 mediates ischaemic brain injury via distinct actions on endothelial cells and cholinergic neurons.

The cytokine interleukin-1 (IL-1) is a key contributor to neuroinflammation and brain injury, yet mechanisms by which IL-1 triggers neuronal injury remain unknown. Here we induced conditional deletion of IL-1R1 in brain endothelial cells, neurons and blood cells to assess site-specific IL-1 actions in a model of cerebral ischaemia in mice. Tamoxifen treatment of IL-1R1 floxed (fl/fl) mice crossed with mice expressing tamoxifen-inducible Cre-recombinase under the Slco1c1 promoter resulted in brain endothelium-specific deletion of IL-1R1 and a significant decrease in infarct size (29%), blood-brain barrier (BBB) breakdown (53%) and neurological deficit (40%) compared to vehicle-treated or control (IL-1R1fl/fl) mice. Absence of brain endothelial IL-1 signalling improved cerebral blood flow, followed by reduced neutrophil infiltration and vascular activation 24 h after brain injury. Conditional IL-1R1 deletion in neurons using tamoxifen inducible nestin-Cre mice resulted in reduced neuronal injury (25%) and altered microglia-neuron interactions, without affecting cerebral perfusion or vascular activation. Deletion of IL-1R1 specifically in cholinergic neurons reduced infarct size, brain oedema and improved functional outcome. Ubiquitous deletion of IL-1R1 had no effect on brain injury, suggesting beneficial compensatory mechanisms on other cells against the detrimental effects of IL-1 on endothelial cells and neurons. We also show that IL-1R1 signalling deletion in platelets or myeloid cells does not contribute to brain injury after experimental stroke. Thus, brain endothelial and neuronal (cholinergic) IL-1R1 mediate detrimental actions of IL-1 in the brain in ischaemic stroke. Cell-specific targeting of IL-1R1 in the brain could therefore have therapeutic benefits in stroke and other cerebrovascular diseases.

Neutrophil infiltration to the brain is platelet-dependent, and is reversed by blockade of platelet GPIbα.

Neutrophils are key components of the innate immune response, providing host defence against infection and being recruited to non-microbial injury sites. Platelets act as a trigger for neutrophil extravasation to inflammatory sites but mechanisms and tissue-specific aspects of these interactions are currently unclear. Here, we use bacterial endotoxin in mice to trigger an innate inflammatory response in different tissues and measure neutrophil invasion with or without platelet reduction. We show that platelets are essential for neutrophil infiltration to the brain, peritoneum and skin. Neutrophil numbers do not rise above basal levels in the peritoneum and skin and are decreased (~60%) in the brain when platelet numbers are reduced. In contrast neutrophil infiltration in the lung is unaffected by platelet reduction, up-regulation of CXCL-1 (2·4-fold) and CCL5 (1·4-fold) acting as a compensatory mechanism in platelet-reduced mice during lung inflammation. In brain inflammation targeting platelet receptor GPIbα results in a significant decrease (44%) in platelet-mediated neutrophil invasion, while maintaining platelet numbers in the circulation. These results suggest that therapeutic blockade of platelet GPIbα could limit the harmful effects of excessive inflammation while minimizing haemorrhagic complications of platelet reduction in the brain. The data also demonstrate the ability to target damaging brain inflammation in stroke and related disorders without compromising lung immunity and hence risk of pneumonia, a major complication post stroke. In summary, our data reveal an important role for platelets in neutrophil infiltration to various tissues, including the brain, and so implicate platelets as a key, targetable component of cerebrovascular inflammatory disease or injury.

Reparative effects of interleukin-1 receptor antagonist in young and aged/co-morbid rodents after cerebral ischemia.

Neuroprotective strategies for ischemic stroke have failed to translate from bench to bedside, possibly due to the lack of consideration of key clinical co-morbidities. Stroke and co-morbidities are associated with raised levels of the pro-inflammatory cytokine interleukin-1 (IL-1). Inhibition of IL-1 by the administration of interleukin-1 receptor antagonist (IL-1Ra) has shown to be neuroprotective after experimental cerebral ischemia. Stroke can also trigger a robust neuroreparative response following injury, yet many of these new born neurons fail to survive or integrate into pre-existing circuits. Thus, we explore here effects of IL-1Ra on post-stroke neurogenesis in young and aged/co-morbid rats. Aged lean, aged Corpulent (a model of atherosclerosis, obesity and insulin resistance) and young Wistar male rats were exposed to transient cerebral ischemia, received subcutaneous IL-1Ra 3 and 6h during reperfusion, and effects on stroke outcome and neurogenesis were analyzed. Our results show that administration of IL-1Ra improves stroke outcome in both young and aged/co-morbid rats. Furthermore, IL-1Ra not only increases stem cell proliferation, but also significantly enhances neuroblast migration and the number of newly born neurons after cerebral ischemia. Overall, our data demonstrate that systemic administration of IL-1Ra improves outcome and promotes neurogenesis after experimental stroke, further highlighting the therapeutic potential of this clinically approved drug.

IL-1α and inflammasome-independent IL-1ß promote neutrophil infiltration following alum vaccination.

Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1α following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1α and IL-1ß are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1ß at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1ß secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1α and IL-1ß are both necessary for alum-induced neutrophil influx in vivo.

AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3.

Inflammation that contributes to acute cerebrovascular disease is driven by the proinflammatory cytokine interleukin-1 and is known to exacerbate resulting injury. The activity of interleukin-1 is regulated by multimolecular protein complexes called inflammasomes. There are multiple potential inflammasomes activated in diverse diseases, yet the nature of the inflammasomes involved in brain injury is currently unknown. Here, using a rodent model of stroke, we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to brain injury. We also show that acute ischemic brain injury is regulated by mechanisms that require ASC (apoptosis-associated speck-like protein containing a CARD), a common adaptor protein for several inflammasomes, and that the NLRP3 (NLR family, pyrin domain containing 3) inflammasome is not involved in this process. These discoveries identify the NLRC4 and AIM2 inflammasomes as potential therapeutic targets for stroke and provide new insights into how the inflammatory response is regulated after an acute injury to the brain.

Requirement for interleukin-1 to drive brain inflammation reveals tissue-specific mechanisms of innate immunity.

The immune system is implicated in a wide range of disorders affecting the brain and is, therefore, an attractive target for therapy. Interleukin-1 (IL-1) is a potent regulator of the innate immune system important for host defense but is also associated with injury and disease in the brain. Here, we show that IL-1 is a key mediator driving an innate immune response to inflammatory challenge in the mouse brain but is dispensable in extracerebral tissues including the lung and peritoneum. We also demonstrate that IL-1α is an important ligand contributing to the CNS dependence on IL-1 and that IL-1 derived from the CNS compartment (most likely microglia) is the major source driving this effect. These data reveal previously unknown tissue-specific requirements for IL-1 in driving innate immunity and suggest that IL-1-mediated inflammation in the brain could be selectively targeted without compromising systemic innate immune responses that are important for resistance to infection. This property could be exploited to mitigate injury- and disease-associated inflammation in the brain without increasing susceptibility to systemic infection, an important complication in several neurological disorders.

Effects of TWIN-OF-EYELESS on Clock Gene Expression and Central-Pacemaker Neuron Development in Drosophila.

Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LNvs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LNvs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs.

Recombinant tissue plasminogen activator enhances microglial cell recruitment after stroke in mice.

The effect of recombinant human tissue plasminogen activator (rtPA) on neuroinflammation after stroke remains largely unknown. Here, we tested the effect of rtPA on expression of cellular adhesion molecules, chemokines, and cytokines, and compared those with levels of inflammatory cell recruitment, brain injury, and mortality over 3 days after transient middle cerebral artery occlusion (MCAO) in mice. Mortality was dramatically increased after rtPA treatment compared with saline treatment during the first day of reperfusion. Among the animals that survived, rtPA significantly increased CCL3 expression, microglia recruitment, and cerebral infarction 6 hours after MCAO. In contrast, the extent of neutrophils and macrophages infiltration in the brain was similar in both saline- and rtPA-treated animals. Recombinant human tissue plasminogen activator induced Il1b and Tnf expression, 6 and 72 hours after MCAO, respectively, and dramatically reduced interleukin 6 (IL-6) level 24 hours after reperfusion. A dose response study confirmed the effect of rtPA on CCL3 and Il1b expressions. The effect was similar at the doses of 1 and 10 mg/kg. In conclusion, we report for the first time that rtPA amplified microglia recruitment early after stroke in association with a rapid CCL3 production. This early response may take part in the higher susceptibility of rtPA-treated animals to reperfusion injury.

Endogenous oils derived from human adipocytes are potent adjuvants that promote IL-1α-dependent inflammation.

Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological "danger signal." Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund's incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α-dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α-dependent proinflammatory responses in vivo.

Analysis of the Drosophila Clock promoter reveals heterogeneity in expression between subgroups of central oscillator cells and identifies a novel enhancer region.

The CLOCK-CYCLE (CLK-CYC) heterodimer lies at the heart of the circadian oscillator mechanism in Drosophila, yet little is known about the identity of transcription factors that regulate the expression of Clk and/or cyc. Here, the authors have used a transgenic approach to isolate regions of the Clk locus that are necessary for expression in central oscillator neurons in the adult fly brain. This analysis shows that central clock cells can be subdivided into 2 distinct groups based on Clk gene regulation. Expression in the lateral neuron (LN), dorsal neuron 1 anterior (DN1a) and 2 (DN2) clusters requires cis-elements located in a 122 base-pair (bp) region (-206 to -84) of the Clk promoter. Expression in the remaining dorsal neurons, 1 posterior (DN1p) and 3 (DN3) and the lateral posterior neurons (LPN), requires regulatory elements located in the -856 to -206 region. In addition, expression in photoreceptors of the compound eye is enhanced by cis-elements located in a 3rd region of the Clk locus (-1982 to -856). This region also enhances expression in nonoscillator cells in the brain including the Kenyon cells, but expression in these neurons is suppressed by regulatory sites located further upstream of -1982. The authors' analysis reveals clear heterogeneity in Clk gene expression in the adult brain and provides a necessary focus to isolate novel transcription factors that bind at the Clk locus to regulate expression in different oscillator neuron subgroups. These results also suggest that the DN1a/DN2 neurons may have more molecular commonality with the LNs than they do with the DN1p/DN3/LPN neurons. Finally, this analysis has generated new transgenic lines that will enable genes to be misexpressed in subgroups of central oscillator cells that have previously been resistant to discrete genetic manipulation. Hence, these lines provide important new tools to facilitate a more complete dissection of the neural network that regulates output rhythms in physiology and behavior.

Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus.

Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.

Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry.

The Amt family of high-affinity ammonium transporters is a family of integral membrane proteins that are found in archaea, bacteria, fungi, plants and animals. Furthermore, the family has recently been extended to humans with the recognition that both the erythroid and non-erythroid Rhesus proteins are also ammonium transporters. The Escherichia coli AmtB protein offers a good model system for the Amt family and in order to address questions relating to both its structure and function we have overproduced a histidine-tagged form of the protein (AmtB6H) and purified it to homogeneity. We examined the quaternary structure of AmtB6H (which is active in vivo) by SDS/PAGE, gel-filtration chromatography, dynamic light scattering and sedimentation ultracentrifugation. The protein was resistant to dissociation by SDS and behaved as a stable oligomer on SDS/PAGE. By equilibrium desorption chromatography we determined the mass ratio of dodecyl beta-D-maltoside to AmtB in the detergent-solubilized complex to be 1.03+/-0.03, and this allowed us to calculate, from analytical-ultracentrifugation data, that AmtB purifies as a trimer.

Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB.

The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya. In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P(II) signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status. We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB-dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB. Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen-sufficient situations. The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status. The results also offer new insights into the evolution of P(II) proteins and a rationale for their trigonal symmetry.