Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein.

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αß barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

The transition from child to adult health services for young adults with intellectual disabilities: An evaluation of a pilot of an online learning resource for Registered Nurses.

AIM: This study formed the third phase of a national study on the experience of transition from child to adult health services for young adults with intellectual disabilities. The aim of this phase was to evaluate the accessibility and acceptability of an on-line learning resource for Registered Nurses. BACKGROUND: The population of young adults with intellectual disabilities and complex needs is increasing. Consequently, more will move from child to adult healthcare, with evidence highlighting that for some their experiences of the transition process is poor. The main study provided contemporary evidence to raise the awareness of Registered Nurses of the needs of young adults with intellectual disabilities and their role in enabling an effective transition from child to adult services. METHODS: The online learning resource was developed and piloted with Registered Nurses involved in the transition from child to adult health services for young adults with intellectual disabilities and complex needs. Data collection involved an online survey and semi-structured interviews. RESULTS: Twelve Registered Nurses from 2 Scottish NHS Boards completed the questionnaire and 3 participated in a follow-up interview. The findings suggest that the mode of on-line delivery and most of the content of the learning resource were both acceptable and accessible to Registered Nurses across a range of areas of nursing practice. The learning resource was further adapted in response to the participant data. CONCLUSION: This on-line learning resources offers the potential for Registered Nurses, and potentially other healthcare professionals to undertake evidence-based, structured further education regarding the effective transitions for young adults with intellectual disabilities and their families. TWEETABLE ABSTRACT: Registered Nurses have key contributions to enable the transition from child to adult healthcare for young adults with intellectual disabilities.

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis.

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

A telephone assessment and advice service within an ED physiotherapy clinic: a single-site quality improvement cohort study.

BACKGROUND: In response to issues with timely access and high non-attendance rates for Emergency Department (ED) physiotherapy, a telephone assessment and advice service was evaluated as part of a quality improvement project. This telehealth option requires minimal resources, with the added benefit of allowing the healthcare professional streamline care. A primary aim was to investigate whether this service model can reduce wait times and non-attendance rates, compared to usual care. A secondary aim was to evaluate service user acceptability. METHODS: This was a single-site quality improvement cohort study that compares data on wait time to first physiotherapy contact, non-attendance rates and participant satisfaction between patients that opted for a service based on initial telephone assessment and advice, versus routine face-to-face appointments. 116 patients were referred for ED physiotherapy over the 3-month pilot at the ED and out-patient physiotherapy department, XMercy University Hospital, Cork, Ireland. 91 patients (78%) opted for the telephone assessment and advice service, with 40% (n=36) contacting the service. 25 patients (22%) opted for the face-to-face service. Data on wait time and non-attendance rates was gathered using the hospital data reporting system. Satisfaction data was collected on discharge using a satisfaction survey adapted from the General Practice Assessment Questionnaire. Independent-samples t-test or Mann Whitney U Test was utilised depending on the distribution of the data. For categorical data, Chi-Square tests were performed. A level of significance of p ≤ 0.05 was set for this study. RESULTS: Those that contacted the telephone assessment and advice service had a significantly reduced wait time (median 6 days; 3-8 days) compared to those that opted for usual care (median 35 days; 19-39 days) (p ≤ 0.05). There was no significant between-group differences for non-attendance rates or satisfaction. CONCLUSION: A telephone assessment and advice service may be useful in minimising delays for advice for those referred to ED Physiotherapy for musculoskeleltal problems. This telehealth option appears to be broadly acceptable and since it can be introduced rapidly, it may be helpful in triaging referrals and minimising face-to-face consultations, in line with COVID-19 recommendations. However, a large scale randomised controlled trial is warranted to confirm these findings.

Distortion of the bilayer and dynamics of the BAM complex in lipid nanodiscs.

The ß-barrel assembly machinery (BAM) catalyses the folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The ß-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.

Styrene maleic-acid lipid particles (SMALPs) into detergent or amphipols: An exchange protocol for membrane protein characterisation.

Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.

Transition from child to adult health services: A qualitative study of the views and experiences of families of young adults with intellectual disabilities.

AIMS AND OBJECTIVES: To explore the experiences of the families of young adults with intellectual disabilities at the point of transition from child to adult health services. BACKGROUND: The population of people with intellectual disabilities is changing rapidly, with young people with increasingly complex needs surviving into adulthood and requiring transition from child to adult health services. DESIGN: An interpretative qualitative design. METHODS: Semi-structured interviews were held with ten family carers of young adults with intellectual disabilities and complex care needs, who were in the process of or had recently completed a transition from child to adult health services in Scotland. Data were analysed using thematic analysis. The COREQ checklist was used. RESULTS: Transition emerged as a highly emotional and challenging period for family carers. Their experiences were captured in five main themes: "a deep sense of loss," "an overwhelming process," "parents making transitions happen," "a shock to the adult healthcare system" and "the unbearable pressure." Nurses were often seen as instrumental to counteracting some of these challenges. CONCLUSIONS: There is an urgent need to respond to the challenges experienced by carers at the point of transition and beyond, by ensuring early and coordinated planning, effective information sharing and communication and clear transition processes and guidelines. A person-centred and family-centred approach is required to minimise negative impact on the health and well-being of the young adult with intellectual disabilities and their carers. RELEVANCE TO CLINICAL PRACTICE: Registered nurses have a key role in providing information and support, along with coordinating care at the time of transition from child to adult health services for young adults with complex intellectual disabilities. It is vital that their input is person-centred and responds effectively to the expert knowledge of family carers, while at the same time ensuring their needs for information and support are also addressed.

Transitions from child to adult health care for young people with intellectual disabilities: A systematic review.

AIMS: To examine the experiences of health transitions for young people with intellectual disabilities and their carers and identify the implications for nursing practice. DESIGN: A systematic review and critical appraisal of qualitative, quantitative, and mixed methods studies. DATA SOURCES: A search of the relevant literature published 2007-2017 was carried out in AMED, ASSIA, CINAHL, MEDLINE, PsycINFO, PubMed, and Science Direct Sociological Abstracts databases. REVIEW METHODS: A total of 12 of 637 papers identified in the search met the inclusion criteria for this review. A narrative review of the papers was undertaken by synthesizing the key findings and grouping them into concepts and emergent themes. RESULTS: Four main themes were identified: (a) becoming an adult; (b) fragmented transition process and care; (c) parents as advocates in emotional turmoil; and (d) making transitions happen. CONCLUSION: The range of issues that have an impact on the transition from child to adult health services for young people with intellectual disabilities and their carers raise important implications for policy development, nursing practice, and education.

The Role of SurA PPIase Domains in Preventing Aggregation of the Outer-Membrane Proteins tOmpA and OmpT.

SurA is a conserved ATP-independent periplasmic chaperone involved in the biogenesis of outer-membrane proteins (OMPs). Escherichia coli SurA has a core domain and two peptidylprolyl isomerase (PPIase) domains, the role(s) of which remain unresolved. Here we show that while SurA homologues in early proteobacteria typically contain one or no PPIase domains, the presence of two PPIase domains is common in SurA in later proteobacteria, implying an evolutionary advantage for this domain architecture. Bioinformatics analysis of >350,000 OMP sequences showed that their length, hydrophobicity and aggregation propensity are similar across the proteobacterial classes, ruling out a simple correlation between SurA domain architecture and these properties of OMP sequences. To investigate the role of the PPIase domains in SurA activity, we deleted one or both PPIase domains from E.coli SurA and investigated the ability of the resulting proteins to bind and prevent the aggregation of tOmpA (19 kDa) and OmpT (33 kDa). The results show that wild-type SurA inhibits the aggregation of both OMPs, as do the cytoplasmic OMP chaperones trigger factor and SecB. However, while the ability of SurA to bind and prevent tOmpA aggregation does not depend on its PPIase domains, deletion of even a single PPIase domain ablates the ability of SurA to prevent OmpT aggregation. The results demonstrate that the core domain of SurA endows its generic chaperone ability, while the presence of PPIase domains enhances its chaperone activity for specific OMPs, suggesting one reason for the conservation of multiple PPIase domains in SurA in proteobacteria.

Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding.

The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the ß-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA ß-barrel domain, but is greater when full-length BamA is present, indicating that both the ß-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro.

Lateral opening in the intact ß-barrel assembly machinery captured by cryo-EM.

The ß-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA-E), which is essential for viability in E. coli. BAM promotes the folding and insertion of ß-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a 'lateral gating' motion of the ß-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.

Age-specific incidence of Helicobacter pylori.

BACKGROUND & AIMS: Helicobacter pylori is most likely acquired in childhood, but the incidence of infection has not been determined prospectively by using an appropriate noninvasive test. The aim of this study was to determine the age-specific incidence of Helicobacter pylori infection in children and the risk factors for infection. METHODS: Three hundred twenty-seven healthy index children between 24 and 48 months of age were enrolled over 15 months. At baseline, the Helicobacter pylori infection status of each index child and his or her older siblings and parents was assessed by using the carbon 13-urea breath test. All noninfected index children were then followed up with an annual carbon 13-urea breath test for 4 years to determine whether they became infected with Helicobacter pylori and, if so, the age at first infection. Information on potential risk factors was collected at baseline and each subsequent visit. RESULTS: At baseline assessment, 28 of 327 (8.6%) index children were infected with Helicobacter pylori. The mean age of the 28 infected children was 32.78 months (SD, 5.14 months). Over the next 4 years, 279 index children not infected at baseline contributed 970 person-years of follow-up to the study. During this time, 20 children became infected with Helicobacter pylori. The rate of infection per 100 person-years of follow-up was highest in the 2-3-year age group (5.05 per 100 person-years of follow-up (95% confidence interval, 1.64-11.78) and declined progressively as children aged. Only 1 child became infected after 5 years of age. Having an infected mother, an infected older sibling, and delayed weaning from a feeding bottle (ie, after 24 months of age) were all risk factors for infection. CONCLUSIONS: Children who become infected with Helicobacter pylori are infected at a very young age, and the risk of infection declines rapidly after 5 years of age. These findings have important implications for studies on the mode of transmission of infection.