Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group have shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-on-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening, that of metabolism and transport (32% of the mutants), and of enveloperelated genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression which in turn underlined the NF- κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS Opolysaccharide and T2SS mutants-induced responses were dependent on TLR2-TLR4- MyD88 activation suggested that LPS Opolysaccharide and PulA perturbed TLRdependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS Opolysaccharide and PulA T2SS could be new targets for designing new antimicrobials. Increasing TLR-governed defence responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth Galleria mellonella

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae.

The Role and Effectiveness of Public – Private Partnerships (NHS LIFT) in the Development of Enhanced Primary Care Premises and Services:Report for the National Institute for Health Research Service Delivery and Organisation programme

The research project analysed the role and effectiveness of LIFT via a multi-method study which included semi-structured interviews with policy elites and users, as well as case studies and an exploratory analysis of the financial characteristics of three LIFT Companies. While the team felt that it was able to identify key aspects relating to the advantages and drawbacks surrounding LIFT, some aspects relating to the representativeness of the study was adversely affected by a reluctance of PCTs to participate in the case study analysis and commercial confidentiality restrictions. The study was nonetheless able to identify important issues in relation to the funding and procurement of primary care premises and services.

A stacked Late Quaternary fluvio-periglacial sequence from the Axe valley, southern England with implications for landscape evolution and Palaeolithic archaeology

The current model of mid-latitude late Quaternary terrace sequences, is that they are uplift-driven but climatically controlled terrace staircases, relating to both regional-scale crustal and tectonic factors, and palaeohydrological variations forced by quasi-cyclic climatic conditions in the 100 K world (post Mid Pleistocene Transition). This model appears to hold for the majority of the river valleys draining into the English Channel which exhibit 8–15 terrace levels over approximately 60–100 m of altitudinal elevation. However, one valley, the Axe, has only one major morphological terrace and has long-been regarded as anomalous. This paper uses both conventional and novel stratigraphical methods (digital granulometry and terrestrial laser scanning) to show that this terrace is a stacked sedimentary sequence of 20–30 m thickness with a quasi-continuous (i.e. with hiatuses) pulsed, record of fluvial and periglacial sedimentation over at least the last 300–400 K yrs as determined principally by OSL dating of the upper two thirds of the sequence. Since uplift has been regional, there is no evidence of anomalous neotectonics, and climatic history must be comparable to the adjacent catchments (both of which have staircase sequences) a catchment-specific mechanism is required. The Axe is the only valley in North West Europe incised entirely into the near-horizontally bedded chert (crypto-crystalline quartz) and sand-rich Lower Cretaceous rocks creating a buried valley. Mapping of the valley slopes has identified many large landslide scars associated with past and present springs. It is proposed that these are thaw-slump scars and represent large hill-slope failures caused by Vauclausian water pressures and hydraulic fracturing of the chert during rapid permafrost melting. A simple 1D model of this thermokarstic process is used to explore this mechanism, and it is proposed that the resultant anomalously high input of chert and sand into the valley during terminations caused pulsed aggradation until the last termination. It is also proposed that interglacial and interstadial incision may have been prevented by the over-sized and interlocking nature of the sub-angular chert clasts until the Lateglacial when confinement of the river overcame this immobility threshold. One result of this hydrogeologically mediated valley evolution was to provide a sequence of proximal Palaeolithic archaeology over two MIS cycles. This study demonstrates that uplift tectonics and climate alone do not fully determine Quaternary valley evolution and that lithological and hydrogeological conditions are a fundamental cause of variation in terrestrial Quaternary records and landform evolution.

The geoarchaeology of Paleolithic rivers of southwest Britain

In Britain, the majority of Lower and Middle Paleolithic archaeological finds come from river terrace deposits. The impressive “staircase” terrace sequences of southeast England, and research facilitated by aggregate extraction have provided a considerable body of knowledge about the terrace chronology and associated archaeology in that area. Such research has been essential in considering rates of uplift, climatic cycles, archaeological chronologies, and the landscapes in which hominins lived. It has also promoted the view that southeast England was a major hominin route into Britain. By contrast, the terrace deposits of the southwest have been little studied. The Palaeolithic Rivers of South West Britain (PRoSWEB) project employed a range of geoarchaeological methodologies to address similar questions at different scales, focusing on the rivers Exe, Axe, Otter, and the paleo-Doniford, all of which were located south of the maximum Pleistocene glacial limit (marine oxygen isotope stage [MIS] 4–2). Preliminary analysis of the fieldwork results suggests that although the evolution of these catchments is complex, most conform to a standard staircase-type model, with the exception of the Axe, and, to a lesser extent, the paleo-Doniford, which are anomalous. Although the terrace deposits are less extensive than in southeast Britain, differentiation between terraces does exist, and new dates show that some of these terraces are of great antiquity (MIS 10+). The project also reexamined the distribution of artifacts in the region and confirms the distributional bias to the river valleys, and particularly the rivers draining southward to the paleo–Channel River system. This distribution is consistent with a model of periodic occupation of the British peninsula along and up the major river valleys from the paleo–Channel River corridor. These data have a direct impact on our understanding of the paleolandscapes of the southwest region, and therefore our interpretations of the Paleolithic occupation of the edge of the continental landmass.

Towards a budget approach to Pleistocene terraces: preliminary studies using the River Exe in South West England, UK

This paper presents a first approach to using a sediment budget methodology for paired terrace staircase sediments in SW England. Although a budget approach has become firmly established in Holocene fluvial studies, it has not been used in Pleistocene sequences due to the problems of temporal resolution, catchment changes and downstream loss from the system. However, this paper uses a budget approach in a paired non-glaciated basin, primarily as a method of interrogating the terrace record concerning the degree of reworking and new sediment input required to produce the reconstructed terrace sequences. In order to apply a budget approach a number of assumptions have to be made and these are justified in the paper. The results suggest that the Exe system can most parsimoniously be explained principally by the reworking of a Middle Pleistocene floodplain system with relatively little input of new resistant clasts required and a cascade-type model in geomorphological terms. Whilst this maybe partially a result of the specific geology of the catchment, it is likely to be representative of many Pleistocene terrace systems in NW Europe due to their litho-tectonic similarities. This cascade-type model of terrace formation has archaeological implications and sets the context for the Palaeolithic terrace record in the UK. Future work will involve the testing of this and similar budget models using a combination of landscape modelling and chronometric dating. ?? 2009 The Geologists' Association.

New lower Palaeolithic finds from the Axe valley Dorset

The Axe Valley has long been known for its Palaeolithic finds, particularly from the site at Broom. While research has continued at Broom, other sites have also been investigated in the valley as part of the English Heritage-managed and ALSFfunded project ?Palaeolithic Rivers of South West Britain? (PRoSWeB). This project was completed in March 2007. Since then, research focussing on the Quaternary geology and Palaeolithic archaeology of the southwest region has been continued at selected locations by Prof. Tony Brown (University of Southampton), Dr Laura Basell (University of Oxford) and Dr Phil Toms (University of Gloucestershire), with assistance from Dr Ramues Gallois and Dr Richard Scrivener (formerly British Geological Survey).