A Genomic and Proteomic Investigation Into the <i>Clostridium botulinum</i> Neurotoxin Complex

Clostridium botulinum and some strains of C. baratii and C. butyricum produce one of the most potent toxins known to man, botulinum neurotoxin, and are responsible for the disease botulism. This severe neuroparalytic disease is the result of botulinum neurotoxin negotiating a complex path to the cholinergic nerve endings. There, it interferes with the release of excitatory neurotransmitters resulting in flaccid paralysis and if untreated, death. The neurotoxin, itself a multi-faceted protein, does not act alone but is produced as part of a large, hetero-multimeric complex with the associated non-toxic proteins. This complex, known to protect the toxin from acids and proteases in the gut, has recently been suggested to play a more active role in toxicity. Here, the proteins from the lesser-studied toxin complex type (the OrfX type) are shown for the first time to share sequence similarity and synteny with clusters of proteins that are co-localised with various putative toxin genes in diverse other species. The extracellular supernatant proteome of C. butyricum is characterised and mined for potential novel virulence factors, with metabolic cost of extracellular protein being highlighted as a potential marker of virulence associated extracellular proteins. The supernatant of 22 clinical strains of C. butyricum were investigated for the presence of the toxin complex; all toxin complex components were identified in the majority of strains indicating the importance of these proteins in the causation of botulism. A relationship between OrfX-encoding strains and infant botulism was also uncovered in clinical strains from the UK. Hypotheses to explain this association are explored. Finally, the transcriptome of C. butyricum was investigated using RNA-sequencing. This uncovered a complex and diverse picture of transcription in C. butyricum and raised questions as to the role of the alternative sigma factor BotR in the regulation of bont

A Versatile Wireless Network Protocol for Spectrum Sharing with Passive Radio Services

With the proliferation of wideband active services in bands shared with passive receivers for remote sensing and radio astronomy, new methods are needed for deconflicting active and passive users. We have developed a technique for active/passive user coordination that is compatible with essentially any existing wireless communications protocol. The passive user transmits an on-off keying modulated signal that can be detected by active radios using simple channel power estimates. Using off-the-shelf WiFi and LoRa hardware and on a software defined radio implementation of LTE, we show that Dynamic Passive to Active Spectrum Sharing (DPASS) is effective on a wide range of frequencies and physical layer implementations. We validate the protocol using these three technologies by demonstrating that each device receives a DPASS packet and dynamically takes an appropriate spectrum coordination action, including shutting off transmissions or switching frequencies.Comment: 9 pages, 8 figure

Whole-genome sequencing for national surveillance of Shiga toxin–producing Escherichia coli O157

Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations