A toolkit for the identification of NEAT1_2/paraspeckle modulators.

Paraspeckles are ribonucleoprotein granules assembled by NEAT1_2 lncRNA, an isoform of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysregulation of NEAT1_2/paraspeckles has been linked to multiple human diseases making them an attractive drug target. However currently NEAT1_2/paraspeckle-focused translational research and drug discovery are hindered by a limited toolkit. To fill this gap, we developed and validated a set of tools for the identification of NEAT1_2 binders and modulators comprised of biochemical and cell-based assays. The NEAT1_2 triple helix stability element was utilized as the target in the biochemical assays, and the cellular assay ('ParaQuant') was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture and amenable to multiplexing. Using ParaQuant, we identified dual PI3K/mTOR inhibitors as potent negative modulators of paraspeckles. The tools we describe herein should boost paraspeckle studies and help guide the search, validation and optimization of NEAT1_2/paraspeckle-targeted small molecules.

Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains.

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinase are two serine/threonine protein kinases that regulate the function of ion co-transporters through phosphorylation. The highly conserved C-terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream 'With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. Interestingly, these crystal structures show a highly conserved primary pocket adjacent to a flexible secondary pocket. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to short peptides derived from WNK4 and NKCC1. Together, this work provides a platform that facilitates the design of CCT domain specific small molecule binders that inhibit SPAK- and OSR1-activation by WNK kinases, and these could be useful in treating hypertension and ischemic stroke.

Patient outcomes after hospitalisation with COVID-19 and implications for follow-up: results from a prospective UK cohort.

The longer-term consequences of SARS-CoV-2 infection are uncertain. Consecutive patients hospitalised with COVID-19 were prospectively recruited to this observational study (n=163). At 8-12 weeks postadmission, survivors were invited to a systematic clinical follow-up. Of 131 participants, 110 attended the follow-up clinic. Most (74%) had persistent symptoms (notably breathlessness and excessive fatigue) and limitations in reported physical ability. However, clinically significant abnormalities in chest radiograph, exercise tests, blood tests and spirometry were less frequent (35%), especially in patients not requiring supplementary oxygen during their acute infection (7%). Results suggest that a holistic approach focusing on rehabilitation and general well-being is paramount.

The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

Pleural Fluid suPAR Levels Predict the Need for Invasive Management in Parapneumonic Effusions.

Rationale: Parapneumonic effusions have a wide clinical spectrum. The majority settle with conservative management but some progress to complex collections requiring intervention. For decades, physicians have relied on pleural fluid pH to determine the need for chest tube drainage despite a lack of prospective validation and no ability to predict the requirement for fibrinolytics or thoracic surgery.Objectives: To study the ability of suPAR (soluble urokinase plasminogen activator receptor), a potential biomarker of pleural fluid loculation, to predict the need for invasive management compared with conventional fluid biomarkers (pH, glucose, and lactate dehydrogenase) in parapneumonic effusions.Methods: Patients presenting with pleural effusions were prospectively recruited to an observational study with biological samples stored at presentation. Pleural fluid and serum suPAR levels were measured using the suPARnostic double-monoclonal antibody sandwich ELISA on 93 patients with parapneumonic effusions and 47 control subjects (benign and malignant effusions).Measurements and Main Results: Pleural suPAR levels were significantly higher in effusions that were loculated versus nonloculated parapneumonic effusions (median, 132 ng/ml vs. 22 ng/ml; P < 0.001). Pleural suPAR could more accurately predict the subsequent insertion of a chest tube with an area under the curve (AUC) of 0.93 (95% confidence interval, 0.89-0.98) compared with pleural pH (AUC 0.82; 95% confidence interval, 0.73-0.90). suPAR was superior to the combination of conventional pleural biomarkers (pH, glucose, and lactate dehydrogenase) when predicting the referral for intrapleural fibrinolysis or thoracic surgery (AUC 0.92 vs. 0.76).Conclusions: Raised pleural suPAR was predictive of patients receiving more invasive management of parapneumonic effusions and added value to conventional biomarkers. These results need validation in a prospective multicenter trial.

Predicting outcomes of COVID-19 from admission biomarkers: a prospective UK cohort study.

INTRODUCTION: COVID-19 has an unpredictable clinical course, so prognostic biomarkers would be invaluable when triaging patients on admission to hospital. Many biomarkers have been suggested using large observational datasets but sample timing is crucial to ensure prognostic relevance. The DISCOVER study prospectively recruited patients with COVID-19 admitted to a UK hospital and analysed a panel of putative prognostic biomarkers on the admission blood sample to identify markers of poor outcome. METHODS: Consecutive patients admitted to hospital with proven or clinicoradiological suspected COVID-19 were consented. Admission bloods were extracted from the clinical laboratory. A panel of biomarkers (interleukin-6 (IL-6), soluble urokinase plasminogen activator receptor (suPAR), Krebs von den Lungen 6, troponin, ferritin, lactate dehydrogenase, B-type natriuretic peptide, procalcitonin) were performed in addition to routinely performed markers (C reactive protein (CRP), neutrophils, lymphocytes, neutrophil:lymphocyte ratio). Age, National Early Warning Score (NEWS2), CURB-65 and radiographic severity score on initial chest radiograph were included as comparators. All biomarkers were tested in logistic regression against a composite outcome of non-invasive ventilation, intensive care admission or death, with area under the curve (AUC) (figures calculated). RESULTS: 187 patients had 28-day outcomes at the time of analysis. CRP (AUC: 0.69, 95% CI: 0.59 to 0.78), lymphocyte count (AUC: 0.62, 95% CI: 0.53 to 0.72) and other routine markers did not predict the primary outcome. IL-6 (AUC: 0.77, 0.65 to 0.88) and suPAR (AUC: 0.81, 0.72 to 0.88) showed some promise, but simple clinical features alone such as NEWS2 score (AUC: 0.70, 0.60 to 0.79) or age (AUC: 0.70, 0.62 to 0.77) performed nearly as well. DISCUSSION: Admission blood biomarkers have only moderate predictive value for predicting COVID-19 outcomes, while simple clinical features such as age and NEWS2 score outperform many biomarkers. IL-6 and suPAR had the best performance, and further studies should focus on the additive value of these biomarkers to routine care.

Effectiveness of physiotherapy exercise following total knee replacement: systematic review and meta-analysis.

BACKGROUND: Rehabilitation, with an emphasis on physiotherapy and exercise, is widely promoted after total knee replacement. However, provision of services varies in content and duration. The aim of this study is to update the review of Minns Lowe and colleagues 2007 using systematic review and meta-analysis to evaluate the effectiveness of post-discharge physiotherapy exercise in patients with primary total knee replacement. METHODS: We searched MEDLINE, Embase, PsycInfo, CINAHL and Cochrane CENTRAL to October 4(th) 2013 for randomised evaluations of physiotherapy exercise in adults with recent primary knee replacement. Outcomes were: patient-reported pain and function, knee range of motion, and functional performance. Authors were contacted for missing data and outcomes. Risk of bias and heterogeneity were assessed. Data was combined using random effects meta-analysis and reported as standardised mean differences (SMD) or mean differences (MD). RESULTS: Searches identified 18 randomised trials including 1,739 patients with total knee replacement. Interventions compared: physiotherapy exercise and no provision; home and outpatient provision; pool and gym-based provision; walking skills and more general physiotherapy; and general physiotherapy exercise with and without additional balance exercises or ergometer cycling. Compared with controls receiving minimal physiotherapy, patients receiving physiotherapy exercise had improved physical function at 3-4 months, SMD -0.37 (95% CI -0.62, -0.12), and pain, SMD -0.45 (95% CI -0.85, -0.06). Benefit up to 6 months was apparent when considering only higher quality studies. There were no differences for outpatient physiotherapy exercise compared with home-based provision in physical function or pain outcomes. There was a short-term benefit favouring home-based physiotherapy exercise for range of motion flexion. There were no differences in outcomes when the comparator was hydrotherapy, or when additional balancing or cycling components were included. In one study, a walking skills intervention was associated with a long-term improvement in walking performance. However, for all these evaluations studies were under-powered individually and in combination. CONCLUSION: After recent primary total knee replacement, interventions including physiotherapy and exercise show short-term improvements in physical function. However this conclusion is based on meta-analysis of a few small studies and no long-term benefits of physiotherapy exercise interventions were identified. Future research should target improvements to long-term function, pain and performance outcomes in appropriately powered trials.

Local anaesthetic infiltration for peri-operative pain control in total hip and knee replacement: systematic review and meta-analyses of short- and long-term effectiveness.

BACKGROUND: Surgical pain is managed with multi-modal anaesthesia in total hip replacement (THR) and total knee replacement (TKR). It is unclear whether including local anaesthetic infiltration before wound closure provides additional pain control. METHODS: We performed a systematic review of randomised controlled trials of local anaesthetic infiltration in patients receiving THR or TKR. We searched MEDLINE, Embase and Cochrane CENTRAL to December 2012. Two reviewers screened abstracts, extracted data, and contacted authors for unpublished outcomes and data. Outcomes collected were post-operative pain at rest and during activity after 24 and 48 hours, opioid requirement, mobilisation, hospital stay and complications. When feasible, we estimated pooled treatment effects using random effects meta-analyses. RESULTS: In 13 studies including 909 patients undergoing THR, patients receiving local anaesthetic infiltration experienced a greater reduction in pain at 24 hours at rest by standardised mean difference (SMD) -0.61 (95% CI -1.05, -0.16; p = 0.008) and by SMD -0.43 (95% CI -0.78 -0.09; p = 0.014) at 48 hours during activity.In TKR, diverse multi-modal regimens were reported. In 23 studies including 1439 patients undergoing TKR, local anaesthetic infiltration reduced pain on average by SMD -0.40 (95% CI -0.58, -0.22; p < 0.001) at 24 hours at rest and by SMD -0.27 (95% CI -0.50, -0.05; p = 0.018) at 48 hours during activity, compared with patients receiving no infiltration or placebo. There was evidence of a larger reduction in studies delivering additional local anaesthetic after wound closure. There was no evidence of pain control additional to that provided by femoral nerve block.Patients receiving local anaesthetic infiltration spent on average an estimated 0.83 (95% CI 1.54, 0.12; p = 0.022) and 0.87 (95% CI 1.62, 0.11; p = 0.025) fewer days in hospital after THR and TKR respectively, had reduced opioid consumption, earlier mobilisation, and lower incidence of vomiting.Few studies reported long-term outcomes. CONCLUSIONS: Local anaesthetic infiltration is effective in reducing short-term pain and hospital stay in patients receiving THR and TKR. Studies should assess whether local anaesthetic infiltration can prevent long-term pain. Enhanced pain control with additional analgesia through a catheter should be weighed against a possible infection risk.

What is the evidence base to guide surgical treatment of infected hip prostheses? systematic review of longitudinal studies in unselected patients.

BACKGROUND: Prosthetic joint infection is an uncommon but serious complication of hip replacement. There are two main surgical treatment options, with the choice largely based on the preference of the surgeon. Evidence is required regarding the comparative effectiveness of one-stage and two-stage revision to prevent reinfection after prosthetic joint infection. METHODS: We conducted a systematic review to identify randomised controlled trials, systematic reviews and longitudinal studies in unselected patients with infection treated exclusively by one- or two-stage methods or by any method. The Embase, MEDLINE and Cochrane databases were searched up to March 2011. Reference lists were checked, and citations of key articles were identified by using the ISI Web of Science portal. Classification of studies and data extraction were performed independently by two reviewers. The outcome measure studied was reinfection within 2 years. Data were combined to produce pooled random-effects estimates using the Freeman-Tukey arc-sine transformation. RESULTS: We identified 62 relevant studies comprising 4,197 patients. Regardless of treatment, the overall rate of reinfection after any treatment was 10.1% (95% CI = 8.2 to 12.0). In 11 studies comprising 1,225 patients with infected hip prostheses who underwent exclusively one-stage revision, the rate of reinfection was 8.6% (95% CI = 4.5 to 13.9). After two-stage revision exclusively in 28 studies comprising 1,188 patients, the rate of reinfection was 10.2% (95% CI = 7.7 to 12.9). CONCLUSION: Evidence of the relative effectiveness of one- and two-stage revision in preventing reinfection of hip prostheses is largely based on interpretation of longitudinal studies. There is no suggestion in the published studies that one- or two stage methods have different reinfection outcomes. Randomised trials are needed to establish optimum management strategies.

Tyrosine 121 moves revealing a ligandable pocket that couples catalysis to ATP-binding in serine racemase.

Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a 'closed' hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the α-phosphate of ATP. In contrast, in 'open' hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzyme-fragment structures show Tyr121 flipped out of its pocket in the core of the small domain. Data suggest that this ligandable pocket could be targeted by molecules that inhibit enzyme activity.

Evaluation of perturbed iron-homeostasis in a prospective cohort of patients with COVID-19.

Background: Marked reductions in serum iron concentrations are commonly induced during the acute phase of infection. This phenomenon, termed hypoferremia of inflammation, leads to inflammatory anemia, but could also have broader pathophysiological implications. In patients with coronavirus disease 2019 (COVID-19), hypoferremia is associated with disease severity and poorer outcomes, although there are few reported cohorts. Methods: In this study, we leverage a well characterised prospective cohort of hospitalised COVID-19 patients and perform a set of analyses focussing on iron and related biomarkers and both acute severity of COVID-19 and longer-term symptomatology. Results: We observed no associations between acute serum iron and long-term outcomes (including fatigue, breathlessness or quality of life); however, lower haemoglobin was associated with poorer quality of life. We also quantified iron homeostasis associated parameters, demonstrating that among 50 circulating mediators of inflammation IL-6 concentrations were strongly associated with serum iron, consistent with its central role in inflammatory control of iron homeostasis. Surprisingly, we observed no association between serum hepcidin and serum iron concentrations. We also observed elevated erythroferrone concentrations in COVID-19 patients with anaemia of inflammation. Conclusions: These results enhance our understanding of the regulation and pathophysiological consequences of disturbed iron homeostasis during SARS-CoV-2 infection.

Molecular tracking, through processing, of Campylobacter strains colonizing broiler flocks.

Many of the poultry flocks produced in the United Kingdom are colonized with Campylobacter, and the intensive nature of poultry processing usually results in contaminated carcasses. In this study, a previously reported molecular oligonucleotide probe method was used to track a specific flock-colonizing strain(s) on broiler carcasses during processing in two United Kingdom commercial poultry processing plants. Five Campylobacter-positive flocks were sampled at four points along the processing line, postbleed, postpluck, prechill, and postchill, and two Campylobacter-negative flocks processed immediately after positive flocks were sampled prechill. flaA was sequenced from Campylobacter strains isolated from these flocks, and strain-specific probes were synthesized. Skin and cecal samples were plated onto selective agar to give individual colonies, which were transferred onto membranes. These were then hybridized with the strain- and genus-specific probes. For all the 5 positive flocks, there was a significant reduction in campylobacters postbleed compared to postpluck but no subsequent fall on sampling pre- and postchill, and the strain(s) predominating on the carcasses throughout processing came from the flock being processed. This indicates that strains from the abattoir environment were not a significant cause of carcass contamination in flocks with well-established campylobacter colonization. However, negative flocks that were preceded by positive flocks were contaminated by strains that did not generally originate from the predominating strains recovered from the ceca of the previous positive flocks. This suggests that the abattoir environment has a significant role in the contamination of carcasses from negative but not fully colonized flocks.

Antibiotic-induced changes in the human gut microbiota for the most commonly prescribed antibiotics in primary care in the UK: a systematic review.

OBJECTIVE: The gut microbiota influences many aspects of human health. We investigated the magnitude and duration of changes in gut microbiota in response to antibiotics commonly prescribed in UK primary care. METHODS: We searched MEDLINE, EMBASE and AMED, all years up to May 2020 including all study designs, collecting and analysing data on the effect of antibiotics prescribed for respiratory and urinary tract infections. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and Cochrane standard methods. Risk of bias was evaluated using the Critical Appraisal Skills Programme. Narrative synthesis was used to report the themes emerging from the data. MAIN OUTCOME MEASURES: Primary outcomes were antibiotic-induced changes in the composition and/or diversity of the gut microbiota. Secondary outcome was the time for the microbiota to return to baseline. RESULTS: Thirty-one articles with low or unclear risk of bias showed that antibiotics impact the gut microbiota by causing rapid and diminished levels of bacterial diversity and changes in relative abundances. After cessation of treatment, gut bacteria recover, in most individuals, to their baseline state within a few weeks. Some studies suggested longer term effects from 2 to 6 months. Considerable heterogeneity in methodology makes the studies prone to biases and other confounding factors. Doxycycline was associated with a marked short-term decrease in Bifidobacterium diversity. Clarithromycin decreased the populations of Enterobacteria, and the anaerobic bacteria Bifidobacterium sp and Lactobacillus sp in numbers and diversity for up to 5 weeks. Phenoxymethylpenicillin, nitrofurantoin and amoxicillin had very little effect on the gut microbiome. CONCLUSIONS: Despite substantial heterogeneity of the studies and small sample sizes, there is evidence that antibiotics commonly used in primary care influence the composition of the gastrointestinal microbiota. Larger population-based studies are needed to fully understand how antibiotics modulate the microbiota, and to determine if these are associated with (longer term) health consequences. PROSPERO REGISTRATION NUMBER: CRD42017073750.

Development of a strain-specific molecular method for quantitating individual campylobacter strains in mixed populations.

The identification of sites resulting in cross-contamination of poultry flocks in the abattoir and determination of the survival and persistence of campylobacters at these sites are essential for the development of intervention strategies aimed at reducing the microbial burden on poultry at retail. A novel molecule-based method, using strain- and genus-specific oligonucleotide probes, was developed to detect and enumerate specific campylobacter strains in mixed populations. Strain-specific oligonucleotide probes were designed for the short variable regions (SVR) of the flaA gene in individual Campylobacter jejuni strains. A 16S rRNA Campylobacter genus-specific probe was also used. Both types of probes were used to investigate populations of campylobacters by colony lift hybridization. The specificity and proof of principle of the method were tested using strains with closely related SVR sequences and mixtures of these strains. Colony lifts of campylobacters were hybridized sequentially with up to two labeled strain-specific probes, followed by the generic 16S rRNA probe. SVR probes were highly specific, differentiating down to 1 nucleotide in the target sequence, and were sufficiently sensitive to detect colonies of a single strain in a mixed population. The 16S rRNA probe detected all Campylobacter spp. tested but not closely related species, such as Arcobacter skirrowi and Helicobacter pullorum. Preliminary field studies demonstrated the application of this technique to target strains isolated from poultry transport crate wash tank water. This method is quantitative, sensitive, and highly specific and allows the identification and enumeration of selected strains among all of the campylobacters in environmental samples.

Molecular Methods and Protein Synthesis for Definition of Autoantibody Epitopes.

Epitope mapping is the process of experimentally identifying the binding sites, or "epitopes," of antibodies on their target antigens. Understanding the antibody-epitope interaction provides a basis for the rational design of potential preventative vaccines. Islet autoantibodies are currently the best available biomarkers for predicting future type 1 diabetes. These include autoantibodies to the islet beta cell proteins, insulin and the tyrosine phosphatase islet antigen-2 (IA-2) which selectively bind to a small number of dominant epitopes associated with increased risk of disease progression. The major epitope regions of insulin and IA-2 autoantibodies have been identified, but need to be mapped more precisely. In order to characterize these epitopes more accurately, this article describes the methods of cloning and mutagenesis of insulin and IA-2 and subsequent purification of the proteins that can be tested in displacement analysis and used to monitor immune responses, in vivo, to native and mutated proteins in a humanized mouse model carrying the high-risk HLA class II susceptibility haplotype DRB1*04-DQ8.

The core cysteines, (C909) of islet antigen-2 and (C945) of islet antigen-2ß, are crucial to autoantibody binding in type 1 diabetes.

Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2ß also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2ß resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2ß PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions.

Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress.

Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food-borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra-haem c-type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron-sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate-dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed approximately 50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO-inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.

Oxygen reactivity of both respiratory oxidases in Campylobacter jejuni: the cydAB genes encode a cyanide-resistant, low-affinity oxidase that is not of the cytochrome bd type.

The microaerophilic bacterium Campylobacter jejuni is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (cydAB) apparently encoding a cytochrome bd-like oxidase homologous to oxidases in Escherichia coli and Azotobacter vinelandii. However, C. jejuni cells lacked all spectral signals characteristic of the high-spin hemes b and d of these oxidases. Mutation of the cydAB operon of C. jejuni did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that C. jejuni CydAB be renamed CioAB (cyanide-insensitive oxidase), as in Pseudomonas aeruginosa. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K(m) = 0.8 microM) and a V(max) of >20 nmol/mg/s. Expression of cioAB was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative, ccoNOQP-encoded cyanide-sensitive oxidase, expected to encode a cytochrome cb'-type enzyme, plays a major role in the microaerobic respiration of C. jejuni, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a K(m) value of 40 nM and a V(max) of 6 to 9 nmol/mg/s. Low-temperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.

A truncated haemoglobin implicated in oxygen metabolism by the microaerophilic food-borne pathogen Campylobacter jejuni.

Of the three groups of haemoglobins identified in micro-organisms (single-domain globins, flavohaemoglobins and truncated globins), the last group is the least well understood. The function of the truncated haemoglobin (Ctb) encoded by Cj0465c in the microaerophilic food-borne bacterial pathogen Campylobacter jejuni was investigated by constructing a ctb mutant and characterizing its phenotype. The effects of the ctb mutation on the kinetics of terminal oxidase function in C. jejuni were investigated using oxyleghaemoglobin and oxymyoglobin as sensitive reporters of O2 consumption. The Vmax of ctb mutant cells for O2, calculated using either globin, was greater than that of wild-type cells at extracellular O2 concentrations up to approximately 1 microM, suggesting a role for Ctb in moderating O2 supply for reduction by high-affinity terminal oxidases. However, cells mutated in ctb were disadvantaged when grown under conditions of high aeration, as revealed by measurements of growth yields and rates in batch culture. Furthermore, the rate at which ctb mutant cells consumed O2 in an O2 electrode (10-200 microM O2) was approximately half the rate displayed by wild-type cells, reflecting a role for Ctb in respiration at physiologically relevant external O2 concentrations. However, a lack of sensitivity of the mutant to paraquat or H2O2 indicated that increased oxidative stress under such conditions was not the cause of these phenotypes. O2 affinities of cells (Km values of approximately 40 nM and 1 microM) were unaffected by mutation of either Ctb or the full-length C. jejuni globin, Cgb. Although the gene encoding Ctb was found to be upregulated by S-nitrosoglutathione (GSNO) and the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP), a ctb mutant did not display sensitivity to a number of nitrosative stress-generating compounds. The authors conclude that Ctb is involved in moderating O2 flux within C. jejuni.

NssR, a member of the Crp-Fnr superfamily from Campylobacter jejuni, regulates a nitrosative stress-responsive regulon that includes both a single-domain and a truncated haemoglobin.

Consistent with its role as a nitric oxide (NO)-detoxifying globin in Campylobacter jejuni, Cgb (Campylobacter globin) expression is strongly and specifically induced following exposure to nitrosative stress, suggesting a previously unrecognized capacity for NO-related stress sensing in this food-borne pathogen. In this study, Fur and PerR have been eliminated as major regulators of cgb, and NssR (Cj0466), a member of the Crp-Fnr superfamily, has been identified as the major positive regulatory factor that controls nitrosative stress-responsive expression of this gene. Accordingly, disruption of nssR resulted in the abolition of inducible cgb expression, which was restored by a complementing chromosomal insertion of the wild-type gene with its indigenous promoter at a second location. The NssR-deficient mutant was more sensitive to NO-related stress than a cgb mutant and this phenotype most likely arises from the failure of these cells to induce other NO-responsive components in addition to Cgb. Indeed, analysis of global gene expression, by microarray and confirmatory real-time polymerase chain reaction (PCR) in the wild type and nssR mutant, not only confirmed the dependence of inducible cgb expression on NssR, but also revealed for the first time a novel NssR-dependent nitrosative stress-responsive regulon. This regulon of at least four genes includes Cj0465c, a truncated globin. Consistent with NssR being a Crp-Fnr superfamily member, an Fnr-like binding sequence (TTAAC-N(4)-GTTAA) was found upstream of each gene at locations -40.5 to -42.5 relative to the centre of the binding sites and the transcription start point. Site-directed mutagenesis confirmed that this cis-acting motif mediates the nitrosative stress-inducible expression of cgb.