Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii

BACKGROUND: The ability to introduce site-specific mutations in bacterial pathogens is essential towards understanding their molecular mechanisms of pathogenicity. This has been greatly facilitated by the genetic engineering technique of recombineering. In recombineering, linear double- or single-stranded DNA molecules with two terminal homology arms are electroporated into hyperrecombinogenic bacteria that express a phage-encoded recombinase. The recombinase catalyzes the replacement of the endogenous allele with the exogenous allele to generate selectable or screenable recombinants. In particular, lambda red recombinase has been instrumental in engineering mutations to characterize the virulence arsenal of the attaching and effacing (A/E) pathogens enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. Escherichia albertii is another member of this taxon; however, the virulence of E. albertii remains cryptic despite accumulating evidence that E. albertii is an emerging pathogen. Multiple retrospective studies have reported that a substantial number of EPEC and EHEC isolates (~15 %) that were previously incriminated in human outbreaks actually belong to the E. albertii lineage. Thus, there is increased urgency to reliably identify and rapidly engineer mutations in E. albertii to systematically characterize its virulence determinants. To the best of our knowledge not a single chromosomal gene has been altered by targeted mutagenesis in E. albertii since it was first isolated almost 25 years ago. This is disconcerting because an E. albertii outbreak could cause significant morbidity and mortality owing to our inadequate understanding of its virulence program. RESULTS: In this report we describe a modified lambda red recombineering protocol to mutagenize E. albertii. As proof of principle, we successfully deleted three distinct virulence-associated genetic loci - ler, grlRA, and hfq - and replaced each wild type allele by a mutant allele with an encodable drug resistance cassette bracketed by FRT sites. Subsequently, the FRT-site flanked drug resistance marker was evicted by FLP-dependent site-specific recombination to generate excisants containing a solitary FRT site. CONCLUSIONS: Our protocol will enable researchers to construct marked and unmarked genome-wide mutations in E. albertii, which, in turn, will illuminate its molecular mechanisms of pathogenicity and aid in developing appropriate preventative and therapeutic approaches to combat E. albertii outbreaks

Concussion, Microvascular Injury, and Early Tauopathy in Young Athletes After Impact Head Injury and an Impact Concussion Mouse Model

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001

Gβγ and the C Terminus of SNAP-25 Are Necessary for Long-Term Depression of Transmitter Release

Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca(2+)]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD

Cross-sectional area and echo intensity values of peripheral nerves: Ultrasonographic and cadaveric correlation

Ultrasonography allows high-resolution visualisation of the peripheral nerves for quantitative and qualitative analyses. We report cross-sectional area values (quantitative measure) and echo intensity values (qualitative measure) for 46 peripheral nerve sites in upper and lower extremities in cadaveric specimens

Botulinum Toxin type A pretreatment markedly reduces induction of LTD, but not LTP, at Schaffer collateral-CA1 synapses.

<p><b>A</b>: Time course of reduction in field excitatory postsynaptic potential (fEPSP) slopes recorded in CA1 <i>stratum radiatum</i> and induced by bath application of BotoxA (200 ng/ml, 90 min; grey bar), plotting normalized fEPSP slopes (<i>n</i> = 9 slices). <b>B</b>: Time course of the effect of low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) on fEPSP slopes in field CA1 of slices (<i>n</i> = 6) pretreated for 90 min with BotoxA (200 ng/ml), which eliminated LTD (<i>P</i><0.01; Student’s t-test compared to untreated slice LTD). <b>C</b>: Time course of LTP elicited in field CA1 by four trains of theta burst Schaffer collateral stimulation (arrow; each train 10 bursts of 4 stimuli at 100 Hz frequency, 200 ms interburst interval) in slices (<i>n</i> = 6) pretreated for 90 min with BotoxA (200 ng/ml). <b>D</b>: Time course of the magnitude of depotentiation, where BotoxA (200 ng/ml, grey bar) was bath applied for 90 min prior to induction of LTP by high-frequency theta burst stimulation (arrow; 4 trains of 10 4×100 Hz bursts, 200 ms interburst interval). After LTP was established for 30 min, low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) elicited significant depotentation reversal of LTP (<i>n</i> = 6). Each point mean ± SEM of <i>n</i> slices. Representative fEPSP waveforms insets recorded at times indicated by numbers on traces and time course.</p

BotoxA reduces release probability by a mechanism distinct from that of elevating [cyclic GMP].

<p>Time course of the additive effects of bath application of BotoxA (200 ng/ml, light gray bar), followed by the type V phosphodiesterase inhibitor zaprinast (20 µM, dark gray bar), on Schaffer collateral-evoked fEPSP slopes in field CA1. Each point is mean ± SEM fEPSP slope from 5 slices. Insets show representative fEPSPs recorded in CA1 <i>stratum radiatum</i> at times indicated by corresponding numbers on traces and time course.</p

Saturating LTD occludes BoNT/A actions, while reversing LTD with LTP restores BoNT/A efficacy.

<p><b>A</b>: Time course of the lack of effect of BoNT/A (200 ng/ml; solid bar) on fEPSP slopes (each point mean ± SEM of 8 slices) after saturation of LTD by four 2 Hz/5 min low frequency Schaffer collateral stimulus trains spaced 15 minutes apart. <b>B</b>: Time course of the depression of fEPSPs elicited by BoNT/A (200 ng/ml) after bath application of Cd²⁺ (5 µM) to reduce Pr by ∼50% in 8 slices. <b>C</b>: Time course in 8 slices of depression of fEPSPs by BoNT/A (200 ng/ml, bar) elicited after saturating LTD (4×2 Hz/5 min trains), then reversing it with induction of LTP by high-frequency theta-burst stimulation (TBS; 4x trains of 10 bursts of 5pulse/100 Hz each, 200 ms interburst interval).</p

Selective filling of presynaptic CA3 pyramidal neurons using Electroporation.

<p>Schematic of a hippocampal slice illustrating in red the region where peptides were infused by electroporation into somata of multiple CA3 pyramidal neurons, and in pink the region where Schaffer collateral presynaptic terminals were imaged to confirm successful fill. Blunt patch electrodes were filled with 1 mM AlexaFluoro-594, and trains of 10 square current pulses (30 V/200 ms, 0.5 Hz) were applied at 50, 100, and 150 µm depth, after which the electrode was removed from the slice, shift laterally 20 µM, and the depth electroporations were repeated. One hour after electroporation, two-photon laser scanning microscopy (63x objective) was used to acquire the attached images of CA3 pyramidal neuron cell bodies (lower left) and Schaffer collateral terminals (upper left) to confirm successful presynaptic infusion.</p

Altering extracellular [Ca^2<b>+</b>]o shows that BotoxA reduction in LTD is not due to reduced transmitter release probability.

<p><b>A</b>: Time course of the increase in normalized fEPSP slope (each point mean ± SEM of 8 slices) elicited by raising extracellular [Ca²⁺]o from 2.6 mM (light gray bar) to 4 mM (dark gray bar). After responses had plateaued in high [Ca²⁺]o, a low-frequency stimulus train (2 Hz/10 min, black bar) elicited marked LTD. <b>B</b>: Time course of the effect on fEPSP slope of raising extracellular [Ca²⁺]o to 4 mM (dark gray bar), and of LTD elicited by LFS (2 Hz/10 min, black bar), in slices (<i>n</i> = 4) pretreated for 90 min with BotoxA (200 ng/ml). Significantly less LTD was evoked (<i>P</i><0.05, Student’s t-test compared to untreated control slices in A). <b>C</b>: Time course of the effect of lowering [Ca²⁺]o from 2.6 mM (dark gray bar) to 1.3 mM (light gray bar), and of LTD elicited by LFS (2 Hz/10 min, black bar, <i>n</i> = 6). <b>D</b>: Mean ± SEM % LTD of Schaffer collateral-evoked fEPSP in <i>stratum radiatum</i> of field CA1 in slices treated with 4, 2.6 or 1.3 mM [Ca²⁺]o (hatched bars), compared to slices pre-treated with BoNT/A in 4 or 2.6 mM [Ca²⁺]o (open bars). BoNT/A-treated slices exhibited significantly less LTD than slices in either 4 mM [Ca²⁺]o (*, <i>P</i><0.01; Student’s t-test) or 2.6 mM [Ca²⁺]o (*, <i>P</i><0.05, Student’s t-test).</p