Molecular investigation of Bdellovibrio bacteriovorus envelope processes associated with bacterial predation

Antibiotics have been used to treat bacterial infections for nearly 80 years, however, the increasing emergence of antibiotic-resistant bacteria has now led to a significant global burden of antimicrobial resistance (AMR) and a worldwide healthcare crisis. Combatting AMR requires the complementary development of novel therapeutics. Bdellovibrio bacteriovorus is a small predatory bacterium that invades Gram-negative prey, replicates within the periplasm and then lyses the host cell. This predatory ability presents B. bacteriovorus as a potential novel antimicrobial therapeutic. In this thesis, I describe two short research projects which investigate the predatory envelope processes of gliding motility and cell wall-modification in B. bacteriovorus. B. bacteriovorus uses gliding motility on surfaces to both scout for prey and to ultimately facilitate exit from the dead host. Cyclic-di-GMP (c-di-GMP) positively regulates gliding motility in B. bacteriovorus and c-di-GMP signals are transduced through PilZ domains of receptor proteins. Some B. bacteriovorus PilZ proteins contain an additional putative GYF domain. We hypothesised that PilZ: GYF hybrid proteins may bind c-di-GMP (via the PilZ domain) and interact with the gliding motor (via the GYF domain) to regulate gliding motility. We aimed to test this hypothesis by crystallising the PilZ: GYF protein Bd1996. Bd1996 homologues from two different B. bacteriovorus strains were successfully expressed and purified. Bd1996 bound c-di-GMP in vitro, however, neither protein homologue could be crystallised. Modification of predator and prey cell walls is an important predatory process that involves a repertoire of different enzymes. In my second project, I investigated the function of the two cell wall-modifying enzymes Bd1402 and Bd1075. Bd1402 was secreted into the periplasm of the prey in which it may modify the prey cell wall. In contrast, Bd1075 localised to the B. bacteriovorus predator itself and an unmarked deletion of bd1075 resulted in the formation of predator cells that were straight rods, in comparison to vibroid wild-type cells. Together, these data suggest that Bd1075 is the curvature-determinant of B. bacteriovorus HD100

Catchment-scale assessments of the effects of abandoned metal mines on groundwater quality and stream ecology

This paper presents an overview of a British Geological Survey catchment-scale research project designed to quantify catchment-derived metal loading on surface water quality. This work is focused on the Rookhope Burn, a tributary of the River Wear in the North Pennines, UK. The river has been identified in the Water Framework Directive (WFD) River Basin Management report as being at risk of failing to achieve Good Status due to mines and minewater pressures. Although geologically relatively simple, the catchment is hydrogeologically complex in that it comprises an area of entrenched karst, characterised by Lower Carboniferous Limestone exposed in the base of valleys overlain by Namurian strata, comprising interbedded shales, sandstones and limestones, which are capped by drained blanket peat. Metal loadings in this catchment result from lead and zinc mineralization and its historic exploitation and processing, which have resulted in both point source and diffuse impacts within the catchment. There have been three main phases of research: (i) collection of hydrological and water chemistry data to enable loading and mass balance calculations to be undertaken; (ii) development of a conceptual understanding of the hydrology and hydrogeology of the catchment, and (iii) application of the hydrological understanding to more recent baseline monitoring of ecological impacts. This work has identified previously unreported mine and groundwater contributions to the catchment, which may have significant implications for the design of remedial measures in the catchment

GPs' experiences of diagnosing and managing childhood eczema:A qualitative study in primary care

BackgroundEczema is common among children, and in the UK the majority are managed by GPs. The most common cause of poor disease control is incorrect use of topical treatments. There is a lack of research into the challenges faced by GPs in diagnosing and managing this condition.AimTo explore the experiences of GPs in assessing and managing children with eczema.Design and settingQualitative study in primary care in England.MethodSemi-structured interviews with 15 GPs were audiorecorded, transcribed verbatim, and analysed thematically using the framework method.ResultsGPs described a paucity of dermatology training. Although most GPs were confident diagnosing uncomplicated eczema, they reported using a trial-and-error approach to prescribing emollients, and were uncertain about quantities of topical treatments to issue. Mild and moderate potency topical corticosteroids (TCS) were commonly used, but most GPs lacked confidence in recommending potent TCS, and viewed parents or carers to be fearful of using all strengths of TCS. GPs perceived adherence to treatments to be low, but provision of information to support self-care was variable. Routine review of medication use or disease control was uncommon, which GPs attributed to service constraints. Participants’ views on the causes and management of eczema were perceived to be at odds with parents and carers, who were said to be overly focused on an underlying cause, such as allergy.ConclusionGP uncertainty in managing eczema, lack of routine information and review, and perceived dissonance with parents around causation and management may be contributing to low concordance with treatments.</jats:sec

Asymmetric peptidoglycan editing generates cell curvature in Bdellovibrio predatory bacteria

Peptidoglycan hydrolases contribute to the generation of helical cell shape in Campylobacter and Helicobacter bacteria, while cytoskeletal or periskeletal proteins determine the curved, vibrioid cell shape of Caulobacter and Vibrio. Here, we identify a peptidoglycan hydrolase in the vibrioid-shaped predatory bacterium Bdellovibrio bacteriovorus which invades and replicates within the periplasm of Gram-negative prey bacteria. The protein, Bd1075, generates cell curvature in B. bacteriovorus by exerting LD-carboxypeptidase activity upon the predator cell wall as it grows inside spherical prey. Bd1075 localizes to the outer convex face of B. bacteriovorus; this asymmetric localization requires a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. We solve the crystal structure of Bd1075, which is monomeric with key differences to other LD-carboxypeptidases. Rod-shaped Δbd1075 mutants invade prey more slowly than curved wild-type predators and stretch invaded prey from within. We therefore propose that the vibrioid shape of B. bacteriovorus contributes to predatory fitness

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis

ACKNOWLEDGMENTS We thank Karl Kuchler for the C. albicans superoxide dismutase mutants used in this study. This work was funded by a Medical Research Council Doctoral Training Program studentship to M.A.C.I.; Wellcome Trust Grants 089930 to J.Q., 080088 to A.J.P.B., and 097377 to J.Q., A.J.P.B., and L.P.E.; Biotechnology and Biological Sciences Research Council Grants BB/K016393/1 to J.Q. and BB/F00513X/1 and BB/K017365/1 to A.J.P.B.; European Research Council STRIFE Advanced Grant ERC-2009-AdG-249793 to A.J.P.B.; and Wellcome Trust and Royal Society Sir Henry Dale Fellowship 098375/Z/12/Z to K.J.W. and E.T. The funders had no role in study design, data collection, or interpretation or the decision to submit the work for publication.Peer reviewedPublisher PD

The Effects of Wildfire on Mortality and Resources for an Arboreal Marsupial: Resilience to Fire Events but Susceptibility to Fire Regime Change

BACKGROUND: Big environmental disturbances have big ecological effects, yet these are not always what we might expect. Understanding the proximate effects of major disturbances, such as severe wildfires, on individuals, populations and habitats will be essential for understanding how predicted future increases in the frequency of such disturbances will affect ecosystems. However, researchers rarely have access to data from immediately before and after such events. Here we report on the effects of a severe and extensive forest wildfire on mortality, reproductive output and availability of key shelter resources for an arboreal marsupial. We also investigated the behavioural response of individuals to changed shelter resource availability in the post-fire environment. METHODOLOGY/PRINCIPAL FINDINGS: We fitted proximity-logging radiotransmitters to mountain brushtail possums (Trichosurus cunninghami) before, during and after the 2009 wildfires in Victoria, Australia. Surprisingly, we detected no mortality associated with the fire, and despite a significant post-fire decrease in the proportion of females carrying pouch young in the burnt area, there was no short-term post-fire population decline. The major consequence of this fire for mountain brushtail possums was the loss of over 80% of hollow-bearing trees. The types of trees preferred as shelter sites (highly decayed dead standing trees) were those most likely to collapse after fire. Individuals adapted to resource decline by being more flexible in resource selection after the fire, but not by increased resource sharing. CONCLUSIONS/SIGNIFICANCE: Despite short-term demographic resilience and behavioural adaptation following this fire, the major loss of decayed hollow trees suggests the increased frequency of stand-replacing wildfires predicted under climate change will pose major challenges for shelter resource availability for hollow-dependent fauna. Hollow-bearing trees are typically biological legacies of previous forest generations in post-fire regrowth forests but will cease to be recruited to future regrowth forests if the interval between severe fires becomes too rapid for hollow formation

Knowledge exchange and research capacity building in urban Aboriginal health

The Centre for Research Excellence (CRE) on urban Aboriginal Child Health is linked to this unique base of information on 1600 children and their parents/ caregivers. The Centre is designed to: (a) increase the translation of knowledge arising from the SEARCH program and additional data collection undertaken as part of the CRE; (b) to enable ACCHSs to effectively use the information emerging from SEARCH to improve the quality of primary care, and; (c) enhance research capacity in primary health care within ACCHSs by increasing their capacity to undertake multidisciplinary research and knowledge translation in primary care. This work will provide evidence to support sustainable and transferable improvements in Aboriginal primary health care and, ultimately, health outcomes.The research reported in this paper is a project of the Australian Primary Health Care Research Institute which is supported by a grant from the Australian Government Department of Health and Ageing under the Primary Health Care Research Evaluation and Development Strategy

Early morbidities following paediatric cardiac surgery: a mixed-methods study

BackgroundOver 5000 paediatric cardiac surgeries are performed in the UK each year and early survival has improved to > 98%.ObjectivesWe aimed to identify the surgical morbidities that present the greatest burden for patients and health services and to develop and pilot routine monitoring and feedback.Design and settingOur multidisciplinary mixed-methods study took place over 52 months across five UK paediatric cardiac surgery centres.ParticipantsThe participants were children aged MethodsWe reviewed existing literature, ran three focus groups and undertook a family online discussion forum moderated by the Children’s Heart Federation. A multidisciplinary group, with patient and carer involvement, then ranked and selected nine key morbidities informed by clinical views on definitions and feasibility of routine monitoring. We validated a new, nurse-administered early warning tool for assessing preoperative and postoperative child development, called the brief developmental assessment, by testing this among 1200 children. We measured morbidity incidence in 3090 consecutive surgical admissions over 21 months and explored risk factors for morbidity. We measured the impact of morbidities on quality of life, clinical burden and costs to the NHS and families over 6 months in 666 children, 340 (51%) of whom had at least one morbidity. We developed and piloted methods suitable for routine monitoring of morbidity by centres and co-developed new patient information about morbidities with parents and user groups.ResultsFamilies and clinicians prioritised overlapping but also different morbidities, leading to a final list of acute neurological event, unplanned reoperation, feeding problems, renal replacement therapy, major adverse events, extracorporeal life support, necrotising enterocolitis, surgical infection and prolonged pleural effusion. The brief developmental assessment was valid in children aged between 4 months and 5 years, but not in the youngest babies or 5- to 17-year-olds. A total of 2415 (78.2%) procedures had no measured morbidity. There was a higher risk of morbidity in neonates, complex congenital heart disease, increased preoperative severity of illness and with prolonged bypass. Patients with any morbidity had a 6-month survival of 81.5% compared with 99.1% with no morbidity. Patients with any morbidity scored 5.2 points lower on their total quality of life score at 6 weeks, but this difference had narrowed by 6 months. Morbidity led to fewer days at home by 6 months and higher costs. Extracorporeal life support patients had the lowest days at home (median: 43 days out of 183 days) and highest costs (£71,051 higher than no morbidity).LimitationsMonitoring of morbidity is more complex than mortality, and hence this requires resources and clinician buy-in.ConclusionsEvaluation of postoperative morbidity provides important information over and above 30-day survival and should become the focus of audit and quality improvement.Future workNational audit of morbidities has been initiated. Further research is needed to understand the implications of feeding problems and renal failure and to evaluate the brief developmental assessment.FundingThis project was funded by the NIHR Health Services and Delivery Research programme and will be published in full in Health Services and Delivery Research; Vol. 8, No. 30. See the NIHR Journals Library website for further project information.Katherine L Brown is a member of the Health Technology Assessment (HTA) Clinical Trials Board (2017–21) and a member of the domain expert group of the National Congenital Heart Diseases Audit (2014–19). David L Barron is a member of the National Congenital Heart Disease Audit Steering Committee (2014–18). Monica Lakhanpaul is part of the following boards or panels: HTA Maternal, Neonatal and Child Health (MNCH) Methods Group, HTA MNCH Panel (2012–17) and Psychological and Community Therapies Panel (2012–15). Steve Morris has been a member of the following boards or panels: Health Services and Delivery Research (HSDR) Board Members (2014–18), HSDR Commissioned Board Members, HSDR Evidence Synthesis Sub Board 2016 and the Public Health Research Research Funding Board (2011–17). Thomas Witter was a member of the National Congenital Heart Disease Audit Steering Committee (2014–18). The research reported in this issue of the journal was funded by the HS&DR programme or one of its preceding programmes as project number 12/5005/06

Menopausal hormone therapy and other breast cancer risk factors in relation to the risk of different histological subtypes of breast cancer: a case-control study

INTRODUCTION: Breast cancers of different histology have different clinical and prognostic features. There are also indications of differences in aetiology. We therefore evaluated the risk of the three most common histological subtypes in relation to menopausal hormone therapy and other breast cancer risk factors. METHODS: We used a population-based case-control study of breast cancer to evaluate menopausal hormone therapy and other breast cancer risk factors for risk by histological subtype. Women aged 50 to 74 years, diagnosed with invasive ductal (n = 1,888), lobular (n = 308) or tubular (n = 93) breast cancer in Sweden in 1993 to 1995 were compared with 3,065 age-frequency matched controls randomly selected from the population. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for ductal, lobular, and tubular cancer. RESULTS: Women who had used medium potency estrogen alone were at increased risks of both ductal and lobular cancer. Medium potency estrogen-progestin was associated with increased risks for all subtypes, but the estimates for lobular and tubular cancer were higher compared with ductal cancer. We found OR 5.6 (95% CI 3.2–9.7) for lobular cancer, OR 6.5 (95% CI 2.8–14.9) for tubular cancer and OR 2.3 (95% CI 1.6–3.3) for ductal cancer with ≥5 years use of medium potency estrogen-progestin therapy. Low potency oral estrogen (mainly estriol) appeared to be associated with an increased risk for lobular cancer, but the association was strongest for short-term use. Reproductive and anthropometric factors, smoking, and past use of oral contraceptives were mostly similarly related to the risks of the three breast cancer subtypes. Recent alcohol consumption of > 10 g alcohol/day was associated with increased risk only for tubular cancer (OR 3.1, 95% CI 1.4–6.8). CONCLUSION: Menopausal hormone therapy was associated with increased risks for breast cancer of both ductal and lobular subtype, and medium potency estrogen-progestin therapy was more strongly associated with lobular compared with ductal cancer. We also found medium potency estrogen-progestin therapy and alcohol to be strongly associated with tubular cancer. With some exceptions, most other risk factors seemed to be similarly associated with the three subtypes of breast cancer