Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity

C. elegans SWAN-1 Binds to EGL-9 and Regulates HIF-1-Mediated Resistance to the Bacterial Pathogen Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect the nematode C. elegans from P. aeruginosa PAO1 fast killing. EGL-9 inhibits the HIF-1 transcription factor via two pathways. First, EGL-9 is the enzyme that targets HIF-1 for oxygen-dependent degradation via the VHL-1 E3 ligase. Second, EGL-9 inhibits HIF-1-mediated gene expression through a VHL-1-independent mechanism. Here, we show that a loss-of-function mutation in hif-1 suppresses P. aeruginosa PAO1 resistance in egl-9 mutants. Importantly, we find stabilization of HIF-1 protein is not sufficient to protect C. elegans from P. aeruginosa PAO1 fast killing. However, mutations that inhibit both EGL-9 pathways result in higher levels of HIF-1 activity and confer resistance to the pathogen. Using forward genetic screens, we identify additional mutations that confer resistance to P. aeruginosa. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. SWAN-1 is an evolutionarily conserved WD-repeat protein belonging to the AN11 family. Yeast two-hybrid and co-immunoprecipitation assays show that EGL-9 forms a complex with SWAN-1. Additionally, we present genetic evidence that the DYRK kinase MBK-1 acts downstream of SWAN-1 to promote HIF-1-mediated transcription and to increase resistance to P. aeruginosa. These data support a model in which SWAN-1, MBK-1 and EGL-9 regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing

Behavioral and Immune Responses to Infection Require Gαq- RhoA Signaling in C. elegans

Following pathogen infection the hosts' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection

Pseudomonas aeruginosa Suppresses Host Immunity by Activating the DAF-2 Insulin-Like Signaling Pathway in Caenorhabditis elegans

Some pathogens have evolved mechanisms to overcome host immune defenses by inhibiting host defense signaling pathways and suppressing the expression of host defense effectors. We present evidence that Pseudomonas aeruginosa is able to suppress the expression of a subset of immune defense genes in the animal host Caenorhabditis elegans by activating the DAF-2/DAF-16 insulin-like signaling pathway. The DAF-2/DAF-16 pathway is important for the regulation of many aspects of organismal physiology, including metabolism, stress response, longevity, and immune function. We show that intestinal expression of DAF-16 is required for resistance to P. aeruginosa and that the suppression of immune defense genes is dependent on the insulin-like receptor DAF-2 and the FOXO transcription factor DAF-16. By visualizing the subcellular localization of DAF-16::GFP fusion protein in live animals during infection, we show that P. aeruginosa–mediated downregulation of a subset of immune genes is associated with the ability to translocate DAF-16 from the nuclei of intestinal cells. Suppression of DAF-16 is mediated by an insulin-like peptide, INS-7, which functions upstream of DAF-2. Both the inhibition of DAF-16 and downregulation of DAF-16–regulated genes, such as thn-2, lys-7, and spp-1, require the P. aeruginosa two-component response regulator GacA and the quorum-sensing regulators LasR and RhlR and are not observed during infection with Salmonella typhimurium or Enterococcus faecalis. Our results reveal a new mechanism by which P. aeruginosa suppresses host immune defense

Pratos e mais pratos: louças domésticas, divisões culturais e limites sociais no Rio de Janeiro, século XIX

Reply to ten comments on a paper published in the last issue of this journal. The discussion follows along six main lines: History museums, identity, ideology and the category of nation; the need of material collections and their modalities: patrimonial, operational, virtual; theater versus laboratory; visitors and their ambiguities; Public History: the museum and the academy.Resposta aos comentários de dez especialistas que contribuíram no debate de texto publicado no último número desta revista. A discussão orientou-se segundo seis tópicos principais: museus históricos, identidade, ideologia e a categoria de nação; a necessidade de acervos materiais e suas modalidades: acervo patrimonial, operacional, virtual; teatro versus laboratório; o público e suas ambigüidades; História Pública: o museu e a Academia

Long-term cognitive outcome following aneurysmal subarachnoid haemorrhage

Objectives: Survivors of aneurysmal subarachnoid haemorrhage (aSAH) frequently suffer from cognitive dysfunction. The aim of this study was to assess, in a large sample size with long term follow-up, the characteristics of cognitive dysfunction following aSAH and explore whether cognitive deficits mediate employment outcome. Materials and methods: In this retrospective case-controlled study, aSAH survivors (n=884) were identified from the UK Biobank and compared to matched controls (n=3536). Controls were propensity score matched according to age, sex, Townsend deprivation score, educational status and relevant medications known to influence cognition. Cognitive outcomes and employment status were compared between cases and controls using group comparison and cross-tabulation tests. A regression-based mediation analysis was performed to assess whether cognitive deficits mediate employment status following aSAH. Results: Psychomotor reaction time and employment status significantly differed between aSAH cases and controls with slower reaction times (p <0.001) and more unemployment or inability to work due to illness (p <0.001) in the aSAH cohort at a mean follow-up of 125 months. Psychomotor slowing was estimated to mediate a significant proportion (6.59%) of the effect of aSAH on employment status. Conclusions: Psychomotor reaction time and employment status differed significantly between aSAH cases and control matched individuals in the UK Biobank. Psychomotor slowing following aSAH had a discernible impact on employment status. Psychomotor reaction time and employment status are practical to acquire and can be used as surrogate measures of outcome in future studies of aSAH survivors

An evaluation of commonly used external ventricular drain securement methods in a porcine model: Recommendations to improve practice

Background: External ventricular drain (EVD) dislodgement is common and leads to significant morbidity and mortality. Many securement techniques to prevent this are described. There are, however, no objective studies comparing them. This study aimed to determine the most secure method of securing an EVD. Methods: A survey was distributed through the British Neurosurgical Trainee Research Collaborative to determine common EVD securement methods and select techniques for testing. Securement methods were tested in a pig cadaver model. Peak pull force before EVD failure was measured. Failure was defined as catheter displacement 1 cm from the insertion site, catheter fracture, or suture fracture. Results: Twenty-three neurosurgical units responded. Five basic EVD securement methods were in common use. These were tested in isolation and in combination so that in total 15 common methods were tested. The most secure method was a triple construct, consisting of an anchoring suture, sutures around a coil of the catheter, and either a soft plastic flange (25.85 N, 95% confidence interval 24.95 N–26.75 N) or a hard plastic flange (29.05 N, 95% confidence interval 25.69 N–32.41 N). Of the individual methods, single anchoring sutures, soft flanges, VentriFix, and staples were found to be the least secure, whereas multiple sutures and hard flanges were the most secure. Conclusions: An anchoring suture followed by a coil of the catheter and finally a flange is the most secure method for securing EVDs. This simple technique can withstand up to 8.2 times the force of a single anchoring suture, is easily used, and decreases the likelihood of EVD dislodgement and associated complications