The role of iron uptake in pathogenicity and symbiosis in Photorhabdus luminescens TT01

<p>Abstract</p> <p>Background</p> <p><it>Photorhabdus </it>are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family <it>Heterorhabditis</it>. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe²⁺) and ferric (Fe³⁺) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe³⁺and Fe²⁺iron uptake systems in the ability of <it>Photorhabdus </it>to interact with its invertebrate hosts.</p> <p>Results</p> <p>We constructed targeted deletion mutants of <it>exbD</it>, <it>feoABC </it>and <it>yfeABCD </it>in <it>P. luminescens </it>TT01. The <it>exbD </it>mutant was predicted to be crippled in its ability to obtain Fe³⁺and we show that this mutant does not grow well in iron-limited media. We also show that this mutant was avirulent to the insect but was unaffected in its symbiotic interaction with <it>Heterorhabditis</it>. Furthermore we show that a mutation in <it>feoABC </it>(encoding a predicted Fe²⁺permease) was unaffected in both virulence and symbiosis whilst the divalent cation transporter encoded by <it>yfeABCD </it>is required for virulence in the Tobacco Hornworm, <it>Manduca sexta </it>(Lepidoptera) but not in the Greater Wax Moth, <it>Galleria mellonella </it>(Lepidoptera). Moreover the Yfe transporter also appears to have a role during colonization of the IJ stage of the nematode.</p> <p>Conclusion</p> <p>In this study we show that iron uptake (via the TonB complex and the Yfe transporter) is important for the virulence of <it>P. luminescens </it>to insect larvae. Moreover this study also reveals that the Yfe transporter appears to be involved in Mn²⁺-uptake during growth in the gut lumen of the IJ nematode. Therefore, the Yfe transporter in <it>P. luminescens </it>TT01 is important during colonization of both the insect and nematode and, moreover, the metal ion transported by this pathway is host-dependent.</p

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

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Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Dyshomeostatic modulation of Ca2+-activated K+ channels in a human neuronal model of KCNQ2 encephalopathy

Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons. We find that patient iPSC-derived neurons exhibit faster action potential repolarization, larger post-burst afterhyperpolarization and a functional enhancement of Ca2+-activated K+ channels. These properties, which can be recapitulated by chronic inhibition of M-current in control neurons, facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function leading to alterations in the neurodevelopmental trajectory of patient iPSC-derived neurons

De novo variants in neurodevelopmental disorders with epilepsy

Epilepsy is a frequent feature of neurodevelopmental disorders (NDDs), but little is known about genetic differences between NDDs with and without epilepsy. We analyzed de novo variants (DNVs) in 6,753 parent-offspring trios ascertained to have different NDDs. In the subset of 1,942 individuals with NDDs with epilepsy, we identified 33 genes with a significant excess of DNVs, of which SNAP25 and GABRB2 had previously only limited evidence of disease association. Joint analysis of all individuals with NDDs also implicated CACNA1E as a novel disease-associated gene. Comparing NDDs with and without epilepsy, we found missense DNVs, DNVs in specific genes, age of recruitment, and severity of intellectual disability to be associated with epilepsy. We further demonstrate the extent to which our results affect current genetic testing as well as treatment, emphasizing the benefit of accurate genetic diagnosis in NDDs with epilepsy